UNITED NATIONS RELIEF & WORKS AGENCY

FOR PALESTINE REFUGEES IN THE NEAR EAST

(UNRWA)

MANUAL ON
BASIC LABORATORY TECHNIQUES

Health Department Fourth Edition

Headquarters, Amman March 2005
 FORWARD

The third edition of this manual was issued in 1999. Since then several changes
and improvements have been introduced to the Agency’s laboratory health services,
including upgrading of equipment, adoption of modern techniques, and expansion of
bacteriological services.

It had therefore become necessary to revise and update the manual, in order to
respond to programme needs.

The fourth edition of the “Manual on Basic Laboratory Techniques” was prepared by
Mr. Ahmad Al-Natour, Senior Laboratory Services Officer, Headquarters, in close
consultation with the Laboratory Superintendents in the five Fields of the Agency’s area of
operations, whose efforts are readily acknowledged and appreciated.

Not only that this manual should serve as a technical guide for quality assurance
through all the steps of the laboratory analytical process, but it should also serve as a
guide for training of laboratory personnel.

Dr. Fathi Mousa

Director of Health

March 2005
 CONTENTS
Page

Forward i
Contents ii

Part I GENERAL

I. Introduction 3

II. Standard Laboratory Equipment 3

III. Standard Laboratory Tests and Methods 4

IV. Laboratory Safety 6

V. Quality Control 15

VI. Preparation of Solutions 28

VII. International System of Units (SI. Units) 31

VIII. Care of Equipment 33

Part II EXAMINATIONS FOR PARASITES

I. Stool Specimens 47

1. Collection of stool specimen 47

2. Macroscopic Examination 48
3. Microscopic Examination 48

3.1 Direct Method 48

3.2 Concentration Method 49

4. Bench Aid for the Diagnosis of Intestinal Parasites 51

5. Various Structures Seen in Stool Preparations 71
6. Occult Blood 72

II. Urine Specimen 72

III. Vaginal and Urithral Materials 74

IV. Blood Specimens 75

V. Skin Specimens 82

VI. References 83

Part III EXAMINATIONS ON URINE

I. Collection of Urine Specimen 87

II. Physical Characteristics 87

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 CONTENTS
Page

III. Strips for Urine Testing 89

IV. Microscopic Examination of Deposits 95

V. Illustrations of Urine Deposits 98

VI. Pregnancy Test 104

VII. References 104

Part IV HAEMATOLOGY AND BLOOD BANK

I. Blood Collection 109

1. Capillary Blood 109

2. Venous Blood 109
3. Use of Anticoagulants 110

II. Blood Cell Counting 111

1. Red Blood Cells (RBCs) Counts 111

2. White Blood Cells (WBCs) Counts 115
3. Platelets Count 117
4. Reticulocytes Count 118

III. Blood Film Examination 119

1. Preparation of a Thin Blood Film 119

2. Preparation of aThick Blood Film 120
3. Staining of Thin Blood Film with Wright's Stain 120

IV. Haemoglobin Determination 124

1. Cyanmethaemoglobin Method 124

2. Haemoglobinometer 126

V. Haematocrit 127

VI. Erythrocyte Sedimentation Rate (ESR) 128

VII. Clotting Time 130

VIII. Bleeding Time 132

IX. Sickle Cell Test 132

X. ABO and Rh Grouping 133

XI. Direct Antiglobulin Test (Direct Coomb's Test) 135

XII. Indirect Antiglobulin Test (Indirect Coomb's Test) 135

XIII. References 136

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 CONTENTS
Page

Part V BIOCHEMISTRY

I. Glucose 141

1. Glucose by Blood Glucose Meter 141

2. Glucose by GOD – PAP Method 141
3. Oral Glucose Tolerance Test (OGTT) 143

II. Urea 144

III. Creatinine (Jaffe Method with Deproteinization) 145

IV. Creatinine (Without Deproteinization) 147

V. Uric Acid 150

VI. Cholesterol 151

VII. HDL-Cholesterol (Precipitation Method) 153

VIII. DHL-Cholesterol (Direct Enzymatic Colorimetric Method) 154

IX. Triglycerides 157

X. Bilirubin Direct & Total 159

XI. Total Protein 161

XII. Albumin 163

XIII. AST (SGOT) 164

IVX. ALT (SGPT) 166

VX. Alkaline Phosphatase 167

Part VI SEROLOGY

I. Antigen-Antibody REactions 173

II. Brucella Test 176

1. Slide Method 177

2. Tube Method 177

III. C-Reactive Protein (CRP) 178

IV. Anti Streptolysin O (ASO) 180

V. Rheumatoid Factor (RF) 182

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 CONTENTS
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Part VII BACTERIOLOGY

I. Classification of Bacteria 187

II. Grouth of Bacteria 187

III. Pathogenicity of Bacteria 188

IV. Sterilization 189

V. Decontamination 190

VI. Safety Measures 190

VII. Artificial Culture Media 191

VIII. Identification of Common Bacterial Pathogens 196

IX. Bacteriological Examinations 202

X. Sensitivity Tests 223

XI. Gram Stain 231

XII. Ziehl Neelsen Stain 232

XIII. Direct Microscopic Examination for Fungi 233

Part VIII REFERENCE VALUES

I. Reference Values 237

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 PART I - GENERAL

I. Introduction

II. Standard Laboratory Equipment

III. Standard Laboratory Tests and Methods

IV. Laboratory Safety

V. Quality Control

VI. Preparation of Solutions

VII. International System of Units (SI. Units)

VIII. Care of Equipment

1
2
I. Introduction:

The biochemical tests described in this chapter are based on the established procedures and
adopted recommendations for use in laboratory diagnosis.

The Laboratory methods described satisfy the criteria for accuracy, precision and specificity, and
also for a moderate reagent cost and simple equipment requirements.

Description of the methods includes: Principles, Procedures, Calculations, Reference values and
Literature.

II. Standard Laboratory Equipment:

1. Health Centre (Clinical) Laboratory:

a) Refrigerator

b) Centrifuge

c) Spectrophotometer

d) Binocular Microscope

e) Deioniser/Still water

f) Balance

g) Automatic Pipettes

h) Refractometer

i) Vortex Mixer

j) Oven

k) Water Bath

l) Haematology Cell Counter

m) Chemistry Analyzer

2 Central Laboratory and at Area Level

All above (plus):

a. Autoclave

b. Incubator

3
III. Standard Laboratory Tests and Methods:

(Table I.1)
Laboratory Test Method
1. Health Centre II) Examination for parasites:
(Clinical) Laboratory
a. Stool & occult blood - Microscopy and strip.

b. Urine specimen - Microscopy.

c. Vaginal and urethral materials - Wet mount preparation.

d. Blood specimens - Stained blood film

e. Skin specimens - Stained smears

III) Examination of Urine:

a. Chemical Examination - Test strips (Combur 3 & 9).

b. Specific gravity - Refractometer.

c. Deposits - Microscopy.

d. Pregnancy test - Test strips.

IV) Haematology:

a. Haemoglobin - Cyanmethaemoglobin / cell

counter.

b. Haematocrit (PVC) - Haematocrit Centrifuge / cell

counter.

c. Leucocyte Count (WBC) - Counting Chamber / cell counter.

d. Erythrocyte Count (RBC) - Counting Chamber / cell counter.

e. Platelets Count - Counting Chamber / cell counter.

f. ESR - Westergren method

g. Differential Count - Stained blood film.

h. Bleeding time - Blotting paper.

i. Clotting time - Lee and white method.

j. Sickling test - Sodium-metabisulfite.

k. Reticulocyte count - Brilliant crysl-blue.

l. Grouping and Rh - Slide and Tube-methods.

4
 V) Biochemistry:

a. Glucose - Enzymatic & Glucose meter.

b. Urea - Enzymatic.

c. Creatinine - Jaffe method & Enz.

d. Uric Acid - Enzymatic-colorimetric.

e. Cholesterol - Enzymatic-colorimetric.

f. HDL-Cholesterol - Enzymatic-colorimetric.

g. Triglycerides - Enzymatic-colorimetric.

h. Bilirubin Dir.&Tot. - Photo.-colorimetric method.

i. Total Protein - Biuret method.

j. Albumin - BCG-method.

k. ALT(SGPT) - Enzymatic.

l. AST(SGOT) - Enzymatic.

m. Alkaline phosphatase - Enzymatic.

VI) Serology:

a. Brucella - Agglutination Test.

b. C-Reactive Protein - Agglutination Test.

c. Anti-Streptolysin-O - Agglutination Test.

d. Rheumatoid Factor - Agglutination Test.

VII) Microbiology:

a. Gram's Stain

b. Ziel Nelsen Stain

c. Test for Fungus - Potassium Hydroxide.

3. Hospital Laboratory All Above (+)

1. Sodium and Potassium - Flame Photometer.

2. Blood Gases, pH, and - Blood Gas Analyzer.

Bicarbonate

3. Blood Banking - Elisa and cross matching

5
IV. Laboratory Safety:

In general, safety rules are similar from one laboratory to another, and should be learned
early in the training of technicians. Future practices in a laboratory are then governed
almost automatically by these safety habits.

1. General Safety Guidelines:

1.1 Accidents should be avoided by preventive actions:

a) Choose methods without hazards.

b) Avoid flammable materials in method selection.
c) Avoid carcinogenic and other toxic substances.
d) Indicate clearly all hazards in method documentation.

1.2 All staff are to be issued with and made aware of these laboratory safety
regulations upon appointment.

1.3 All specimens must be regarded as potentially hazardous or contagious.

1.4 Admittance to the laboratory should be restricted.

2. Safety of Laboratory Staff:

2.1 Potential hazards (use of needles, syringes and other sharp instruments or
objectives, etc) should be avoided whenever possible.

The used needles and syringes should be collected in a puncture-resistant

container before disposal. Needles should neither be recapped nor be removed
from syringes.

2.2 Technical procedures should be performed in a way that minimizes the risk of
creating aerosols, droplets, splashes, or spills.

2.3 Regulations which apply to the materials being used should be known and followed.

2.4 Gowns or protective coats:

a) Must be worn when working in the laboratory.

b) Must not be worn outside the laboratory, if necessary use a separate coat.

2.5 Protective gloves:

a) Should be worn when handling infectious materials or when there is a

possibility of exposure to blood or other body fluids.
b) Should be removed before handling telephones, keyboards … etc., or
touching eyes, nose, skin … etc.
c) Should be discarded whenever they are thought to have become
contaminated.

6
 2.6 Pipetting by mouth is not allowed.

2.7 Automatic samplers and diluters should be used whenever possible.

2.8 A rubber bulb system or equivalent must be used if a glass volumetric pipette is
required.

2.9 Eating, drinking, or smoking is not permitted in the laboratory.

2.10 Drinks and foodstuffs must be kept only in a refrigerator set aside uniquely for
this purpose.

2.11 Lipstick or cosmetics should not be applied in the laboratory.

2.12 Labels must not be licked. Pencils and pens must not be placed in the mouth.

2.13 Instruments or machines connected to power and water supplies should not be
touched or turned off except by those authorized to do so.

2.14 Specific instructions for packaging and transportation of biological material

should be observed.

2.15 It is recommended that all laboratory personnel receive protective immunization

against the following diseases: diphtheria, hepatitis B, measles, mumps,
poliomyelitis, rubella, tetanus, tuberculosis, typhoid fever. Some workers may
have been immunized during childhood but documentary evidence of protection
should be obtained.

2.16 Training Programmes:

Continuous, on-the-job safety training programme is essential to maintain safety

awareness among the laboratory and support staff. Laboratory supervisors, with
the assistance of the bio-safety officer and other resource persons, play the key
role in staff training. A basic course on good laboratory practice that can be
modified to suite most laboratories is offered in the Biosafety Manual for UNRWA
Laboratory Personnel, First edition, 2000.

3. Laboratory Safety:

3.1 Special care should be taken to ensure protection of staff and patients by:

a) Identifying hazardous materials and work area.

b) Proper handling of hazardous materials and reagents (chemical and

biological hazards).

c) Using sterile instruments and equipment for sampling.

d) Using protective measures (gloves, coats and glasses) whenever handling

unknown samples, patients, or hazardous materials and reagents.

7
 3.2 Instructions for prevention of use of aerosols should be available.

3.3 Hoods and forced ventilation should be in place, with instructions for their used.

3.4 Toxic substances and chemicals:

a) All chemicals are to be assumed toxic.

b) International hazard symbols on labels should be recognized, and separate

storage area provided for materials which are explosive, flammable, toxic,
corrosive or radioactive.

c) Storage should be indicated with the hazard symbol.

d) Chemical that must not be stored together should be indicated.

3.5 Laboratory should be kept clean, neat, and free from extraneous materials and
equipment.

3.6 Work surfaces should be disinfected when procedures are completed and at the
end of each working day. An effective all-purpose disinfectant is a hypo-chlorite
solution with a concentration of 0.1% available chlorine.

3.7 An effective insect and rodent control programme should be available.

4. Safe Laboratory Techniques:

Human error, poor techniques and misuse of equipment cause the majority of
laboratory accidents and related infections. This section provides a compendium of
technical methods that are designed to avoid or minimize the most commonly reported
accidents caused by these factors. Detailed information about each method is offered
in the Biosafety Manual for UNRWA Laboratory Personnel, First edition, 2000.

a) Techniques for the safe handling of specimens in the laboratory.

b) Techniques for the safe use of pipettes and pipetting aids.

c) Techniques for avoiding the dispersal of infectious materials.

d) Techniques for avoiding ingestion of infectious materials and their contact with
skin and eyes.

e) Techniques for avoiding injection of infectious materials.

f) Techniques for the safe use of centrifuges.

g) Techniques for the care and use of refrigerators and freezers.

h) Special precautions with blood and other body fluids

8
5. Safe Transport of Infectious Substances and Diagnostic Materials:

Although all specimens are potentially hazardous, high-risk samples should be

identified:

5.1 Diseases of particular risk should be listed, (lymphadenopathy syndrome and

AIDS, hepatitis B and C, tuberculosis, anthrax, shigella, salmonella - including
typhoid, plague, psittacosis, Creutzfeld-Jacob disease, slow virus disease,
brucellosis, … etc.).

5.2 Instructions for handling high-risk specimens should be communicated to all

relevant staff as per the Biosafety Manual for UNRWA Laboratory Personnel, First
edition, 2000.

6. Spillage and contamination:

6.1 If spillage occurs:

a) Use disinfectants for cleaning blood, urine, or other biological substances:

A disinfectant should be poured around the spill area and then over the
absorbent material, and left for 10 minutes. The standard disinfectant
recommended for cleaning contaminated surfaces is a hypochlorite solution
with a concentration of 0.5% available chlorine (5 g/liter, 5000 ppm).

However, for laboratories working with HIV cultures and virus preparations,
a higher concentration of available chlorine (1.0%) is recommended. The
mixture of disinfectant and spilt material should be cleaned up with
absorbent material, which should be placed in the contaminated waste
container. The surface should then be wiped again with disinfectant. Gloves
should be worn throughout the procedure, and direct contact between
gloved hands and the disinfected spilt material should be avoided. Broken
glass or plastic should be swept up with a dustpan and brush. Needle-stick
or other puncture wounds, cuts, and skin contaminated by spills or splashes
of specimen material should be thoroughly washed with soap and water.
Bleeding from any wound should be encouraged.

b) Chemical spills should not be cleaned up until appropriate scientific advice

has been sought.

c) All spills, accidents, and overt or potential exposure to infectious material

should be reported immediately to the laboratory supervisor. A written
record should be kept of all such incidents. Appropriate medical evaluation,
surveillance, treatment, and if necessary, counseling should be provided.

6.2 An emergency eye wash facility should be available and used immediately for all
specimen or chemical contamination of the eyes.

6.3 Contaminated glassware (and other consumables) and sharp waste must be
placed in special bags (clearly indicated), and sterilized (or incinerated) before
disposal.

9
 6.4 Work areas where spillage risk is great may be protected by plastic-backed
absorbent covers, which should be changed regularly.

6.5 Corrosive spills are to be contained and absorbed with special acid spill
granulate.

6.6 Centrifuges, water-baths, and specimen mixers are to be cleaned regularly.

7. Disinfection and sterilization:

Following are the most commonly used disinfectants and sterilization techniques,
detailed information about each disinfectant and sterilization techniques are offered in
the Biosafety Manual for UNRWA Laboratory Personnel, First edition, 2000.

7.1 Chemical disinfectants:

a) Chlorine (sodium hypochlorite).

b) Formaldehyde.
c) Glutaral (glutaraldehyde).
d) Phenolic compound.
e) Alcohol and alcohol mixtures.
f) Iodine and iodophors.
g) Hydrogen peroxide.

7.2 Sterilization:

The term “sterilization” means the total inactivation of all forms microbial life in
terms of their ability to reproduce. About 95 percent of all sterilization
operations are done by steam under pressure in the autoclave; sterilization by
chemicals is less reliable.

Methods of sterilization:

a) Sterilization by steam:

Steam sterilization by autoclave is the method of choice for reusable medical

instrument including needles, syringes and other instruments commonly
used in health care settings.

An autoclave is a chamber in which steam sterilization is carried out. The

whole of the material to be sterilized shall be in contact with saturated steam
at the required temperature for the necessary length of time. For reliable
sterilization, an exposure for 20 minutes at 121°C and 15 Ib/sq. in is used.

Precautions in the use of autoclaves: There are hazards inherent in the

operation of all pressurized vessels. The following rules should be observed:

i. Qualified technicians should regularly inspect the chamber and door seals.
A preventive maintenance programme, including a check on gauges and
controls, should be carried out at regular intervals.

10
 ii. All materials should be in small shallow containers, to aid the removal of
air and permit good heat penetration.

iii. The chamber should not be tightly packed, or heat penetration will be
inadequate, and some of the load will not be sterilized.

iv. If the autoclave is not fitted with an interlocking safety device that
prevents the door being opened when the chamber is pressurized, it is
essential that the main steam valve is closed, and the chamber
temperature allowed to fall to below 80 C before the door is opened. The
door should then be opened a few millimeters (“cracked”), to allow steam
to escape safely, and left in that position for 5 minutes before the
autoclave is unloaded.

v. Operators should wear gloves and visors, to protect the arms, hands, face
and neck when they open the autoclave, even when the temperature of
the contents has been reduced to 80 C.

vi. Biological sterility indicators or thermocouples should be placed at the

centre of each load. Regular monitoring with thermocouples and
recording devices in a “worst case” load is highly desirable. Operating
cycles can be determined in the light of the findings.

vii. Responsibility for operation and routine care should be assigned to trained
individuals.

viii. The drain screen filter at the bottom of the chamber should be removed
and cleaned daily

ix. Care should be taken to ensure that the relief valves of pressure cooker
autoclaves do not become blocked by paper, etc. in the load.

b) Sterilization by dry heat:

Sterilization by dry heat requires higher temperature and a longer period of

heating. The most widely used type of dry heat is the hot air oven.
Sterilization for 2 hours at 180°C is adequate. This method is appropriate for
instruments that can withstand a temperature at 180°C.

c) Sterilization by boiling:

This is the simplest and most reliable method for inactivating most
pathogenic microbes when sterilization equipment is not available. A high
level of sterilization is achieved when instruments, needles, and syringes are
boiled for 20 minutes.

7.3 Incineration:

Incineration is a useful method of disposing of laboratory waste either with or

without prior autoclaving.

11
8. Fire in the Laboratory:

8.1 Common causes of fires in laboratories are:

* Electrical overloading.
* Poor electrical maintenance.
* Overlong gas tubing and electricity leads.
* Equipment left switched on unnecessarily.
* Naked flames.
* Deteriorated gas tubing.
* Misuse of matches.
* Carelessness with flammable materials.
* Flammable and explosive chemicals stored in ordinary refrigerators.

8.2 All staff should know instructions for evacuation in case of fire.

8.3 Suitable fire extinguishers should be available, and fire drills should be conducted
regularly.

Fire fighting equipment should be placed near to the doors of rooms and at
strategic points in corridors and hallways (as advised by local fire prevention
officers).

This equipment should include hoses, buckets (of water and sand), and the
following fire extinguishers: water, carbon, dioxide, “dry powder”, foam, and
bromochlordifluoromethane (BCF).

The shelf life of these extinguishers should be ascertained, and arrangement

made for them to be inspected and maintained.

Types and uses of fire extinguishers: (Table I.2)

Type Use for Do not use for
Water Paper, wood fabric Electrical fires, flammable liquids,
burning metals
CO2 powder Flammable liquids and gases, Alkali metals, paper
electrical fires
Foam Flammable liquids Electrical fires

BCF Flammable liquids, electrical fires

8.4 Emergency exits should be clearly marked.

8.5 Smoke detectors and sprinklers should be installed, if possible.

8.6 Equipment and reagents, which are prone to initiate or propagate fire, should be
identified and removed whenever possible.

12
9. Electrical hazards:

Electric shock is life threatening; electrical faults may cause fires. It is therefore
essential that all electrical installations and equipment are inspected and tested
regularly, including grounding (earthing), and are maintained by qualified electricians.
Laboratory staff should not attempt to service any kind of electrical equipment.

Voltages vary from country to country, but even low voltages can be hazardous. Care
should always be taken to ensure that fuses of the correct rating are interposed
between the equipment and the supply. Circuit breakers and ground (earth) fault
interrupters should be fitted into laboratory electrical circuits.

Laboratory staff should be made aware of the following hazards:

- Wet or moist surfaces near to electrical equipment.

- Long flexible electrical connecting cables.
- Poor and perished insulation on cables.
- Overloading of circuits by use of adapters.
- Sparking equipment near to flammable substances and vapors.
- Electrical equipment left switched on but unattended.
- Use of the wrong extinguisher (water or foam instead of CO2 or BCF) on electrical
fires.

10. Chemical Hazards:

Chemical hazards could be attributed to the following effects. (Detailed information

regarding each effect are offered in the Biosafety Manual for UNRWA Laboratory
Personnel, First edition, 2000).

a) Storage of chemicals.

b) Incompatible chemicals.

c) Toxic effects of chemicals.

d) Explosive chemicals.

e) Charts describing methods for dealing with spillage’s of various chemicals should
be displayed in a prominent position.

11. Waste Disposal:

Waste disposal includes the following procedures. (Detailed information about each
procedure is offered in the Biosafety Manual for UNRWA Laboratory Personnel, First
edition, 2000).

a) Detailed orders for handling waste should be developed.

b) Handling and disposal of contaminated material and waste.

c) Final disposal.

13
12. Safety Checklist:

This checklist is intended to assist in assessments of the safety status of biomedical

laboratories which includes the following areas. (Detailed information about each area
are offered in the Biosafety Manual for UNRWA Laboratory Personnel, First edition,
2000).

a) Laboratory Premises.

b) Storage facilities.

c) Sanitation and staff facilities.

d) Heating and ventilation.

e) Lighting.

f) Services.

g) Security.

h) Fire Prevention.

i) Electrical hazards.

j) Personal protection.

k) Health and safety of staff.

l) Laboratory equipment.

m) Infectious materials.

n) Chemical substances.

13. References:

a) Biosafety Manual for UNRWA Laboratory Personnel, First Edition, 2000.

b) Laboratory Biosafety Manual, Second Edition, World Health Organization-Geneva,

1993.

c) Quality Systems for Medical Laboratories, Guidelines for Implementation and

Monitoring, World Health Organization-Regional Office for the Eastern
Mediterranean, 1995.

d) Guidelines for the safe Transport of Infectious Substances and Diagnostic

Specimens, World Health Organization-Geneva, 1997.

14
V. Quality Control

Important decision regarding the diagnosis and treatment of patients is sometimes based on
the results of laboratory tests. The laboratory should therefore have a system for assessing the
quality of work.

1. Source of Variance in Laboratory Work

True analytical errors are those which occur during the actual performance of the analysis
which can be classified into systematic and random errors.

1.1 Systematic Errors:

On repeated analysis of the sample we may also find values at wrong level (too
high or too low). These systematic errors can be due to the Analytical Methods,
Technical Performance, Reagents, Measuring Equipment and the Technicians.

Repeated analysis of the sample should be also done by the Field Laboratory
Superintendent during his supervisory visits to the laboratories.

1.2 Random Errors:

Manifest themselves by variations in the results of the repeated analysis of the

same sample. These variations may be caused by several variable factors which
include variations in apparatus, temperature, weighing, etc., it is recognized that
random errors cannot be avoided, therefore, for the use of analytical results, we
have to employ the concept of the "mean value" and "standard deviation".

On repeated analysis of the same substance, either positive or negative errors are
usually equally distributed. These Random Errors can be reduced by more precise
methods and more accurate equipment.

2. Provision of Quality Laboratory Results Requires the Following:-

2.1 Control of the Quality and Quantity of Laboratory Supplies.

Besides the specification of the quality to order, there are important points which
must be considered:

a) When to order a particular item.

b) How much to order (quantity).

c) Estimation of expected usage.

d) Purchasing.

15
2.2 Control of the Quality of the Patient Specimen.

Laboratory technicians must be conscious of factors other than disease that may
affect test results such as:

a) Patient's identity, age and sex

b) Patient's diet (time after last meal)
c) Time of the day (diurnal variations)
d) Exercise and patient position (supine versus upright)
e) The effect of drugs on laboratory result
f) Specimen nature, quantity, quality, labelling and specimen container and
preservation.

2.3 Selection and Continuing Education of Laboratory Personnel:

A good deal of time and effort should be devoted for selection of suitable
candidates and continuing education of recruited staff. Turnover of personnel is a
real problem facing many laboratories.

The pre-employment interview for selection of laboratory personnel should serve

two basic functions:

a) Provides the opportunity for the employer to get to know the applicant, his
qualifications, background and previous employment record. Subjective
information concerning his maturity, personal appearance and attitude.

b) Gives the applicant essential information about the nature of the job, benefits
and opportunities for advancement.

In continuing education of the laboratory staff, they will be able to join a

rapidly growing movement of today's society and the rapid introduction of new
technologies.

Continuing education is important to support learning activities throughout the

life time.

2.4 Selection and Maintenance of Equipment:

Any type of equipment malfunction could potentially affect patient care. So, when
a decision has been made to purchase equipment, specific points should be outlined
in the purchase order. This includes:

a) Clear specification of the required equipment

b) Installation of the equipment
c) Training of personnel
d) Warranty and evaluation period
e) The manufacturer's operation manual.

The purpose of preventive maintenance programme is to ensure that equipment

operates properly and safely. This can be accomplished by checking critical
operating characteristics of an instrument, and performing the recommended
maintenance on a scheduled basis.

16
 Preventive maintenance can be divided to:

a) Function verification: includes checks and tests to ensure that the equipment
is working properly and is correctly calibrated.

b) Maintenance: includes replacement, adjustment or repair, in order to prolong

the life of equipment.

2.5 Communications:

Continued communication with the clinicians using the laboratory services is an

important factor for quality control in the laboratory.

2.6 Evaluation of New Laboratory Procedures:

Ensuring the quality of laboratory services involves many different factors, but
particular emphasis has been placed on the evaluation of technical quality of
laboratory tests performed. For analytical results to be reliable, the method used
should be both Precise and Accurate.

Precision : is a measure of reproducibility. If the individual results of the repeated

analysis of the same substance show little dispersion, we consider that
the method yields reproducible values, or works with high precision.

Accuracy: refers to how close the assay value for a particular substance to its true
value.

In order that a laboratory be in a good standard, it must have some method of

evaluating new procedures. The most important considerations are shown in the
list below. These guidelines can be followed with slight modifications for every
method, kit or instrument to be introduced into the laboratory.

a) Select a method to evaluate.

b) Become familiar with the method and instrumentation: (specificity, sensitivity,

linearity).

c) Keep accurate and complete records.

d) Examine all the data before making a decision.

e) Don't change the procedure during the evaluation.

f) Change the instrumentation used.

2.7 Implementation of an Internal Control Programme:

a) Quality Assessment:

Is referred to as Internal Quality Control when the checking of results is made

by a laboratory's own staff and as External Q.C. when the checking is carried
out by an outside laboratory.

17
 b) Analytical Process Variance:

Is the variance between sampling and measurements. The effective measure

to present variance is to routinely perform Q.C. within both internal and
external Q.C. programmes.

To establish an internal quality control system, a suitable control material for

the analyses to be measured must be identified. This material should mimic
the physical nature of the patient specimens analyzed as closely as possible.

Usual control materials are obtainable commercially as either frozen or

lyophilized sera or urine.

There are various methods for establishing and maintaining an internal quality
control system including the Shewhart-Charts (Figure I.1). The analysis of
control should be included in each run of analytical determinations for a specific
analyte.

The first step in forming a Shewhart Control Chart is the establishment of a

mean ( Χ ) and standard deviation (s) for each level of analyte in the selected
quality control materials.

ΣΧi
Χ=
n

Xi = observed values
n = number of values

Σ ( Χi − Χ ) 2
S=
n -1

∑ [Χi − ( Χ) 2
] = Sum of each difference from the mean squared.

105 mg/d1 .............................................................................……………………..+2s

100 mg/dl ...........................................................................……………………….+1s

x x x x x
( Χ ) 95 mg/dl x x _______
x x x x x
90 mg/dl .................................................................................……………………-1s

85 mg/dl ................................................................................…………………...-2s

. _ . _ . _ . _ . _ . _ . _
0 5 10 15 20 25 30 days

(Figure I.1)

18
3. How to Put Quality Control into Action

Before introducing an assay method into routine practice, one should determine the
reproducibility of the method under the best possible (optimal) conditions, optimal
coefficient of variation (OCV), and under routine conditions of work, routine coefficient of
variation (RCV).

3.1 Method of Establishing the Optimal Coefficient of Variation:

To determine the OCV perform the following:

a) Analyze 20 samples of the same control serum under optimal conditions.

b) Record the result and calculate:

ΣΧi
- The mean value: Χ=
n

Σ ( Χi − Χ ) 2
- The standard deviation: S =
n -1

Where:

(Xi - Χ ) = difference of the observed value (Xi) from the mean ( Χ )

n = number of observed values

S
- The optimal coefficient of variation: OCV = x100
Χ

c) Plot the results of the tests in a chart prepared as follows:

- Draw 5 horizontal lines on a graph paper to record the mean value, ± 1s

from the mean and ± 2s from the mean.

- Enter on the vertical axis the mean value and the value for each of the ± 1s
& ± 2s from the mean.

- Number the horizontal axis form 1-20 corresponding to the 20 tests. An

example of plotting of OCV glucose values are shown in (Figure I.2)

19
 ( Χ ) = 6.7 mmol/1

s = 0.15 mmol/l

7.0 mmol/1 ...........................................................................……………………..+2s

6.9 mmol/l ........................................................................………………..……….+1s

x x x x x
( Χ ) 6.7 mmol/l x x _______
x x x x x
6.5 mmol/l .............................................................................………………………-1s

6.4 mmol/l ...............................................................................……….…………...-2s

. _ . _ . _ . _ . _ . _ . _
0 5 10 15 20 25 30 days

(Figure I.2) - OCV Result for Glucose Assay

d) Examine the results and the chart:

- Check whether the OCV is within the acceptable limits for the particular
method. If the OCV is acceptable, the reproducibility of the method under
optimal conditions is satisfactory.

- Check whether the results obtained for the control serum are within the
acceptable limits. If the results are acceptable, the accuracy of the
method is satisfactory, indicating that the reagents and reference
solutions have been accurately prepared and that the instrument is
working satisfactorily.

- Check the chart for any Upward or Downward Trend in the distribution
around the mean, which may indicate the presence of a systematic
error.

If any of the above checks are unsatisfactory, the method must be

investigated and the fault corrected.

3.2 Method of Establishing the Routine Coefficient of Variation:

To determine the RCV perform the following:

a) Analyze the same control serum along with the routine tests. Include a control
as an extra test whenever a batch of tests is analyzed, until a total of 20
readings for the control serum have been recorded.

b) When the control serum has been analyzed 20 times under routine conditions
of work, calculate:

- The mean value ( Χ )

- The standard deviation (s)
- The routine coefficient of variation (RCV)

20
 c) Check that the RCV does not exceed the value stated in the test method.

d) Chart the results and check that the results of the control serum are within the
acceptable limits and that there is no upward or downward drift of the result.

e) If the RCV and chart are satisfactory, set up a daily quality control chart for the
method as follows:

- Draw 5 horizontal lines on a graph paper to record the mean value and ± s
from the mean.
- Mark on the vertical axis the acceptable values for the mean ±1s and ±2s.
- Mark on the horizontal axis the days of the month (Figure I.3).

If any of the above checks are unsatisfactory, the patients' tests must not
be reported. The error must be investigated and the analysis repeated.

................................................................................………………… +2s

................................................................................………………… +1s

Mean ( Χ ) __________________________________________________

................................................................................………………… -1s

................................................................................………………… -2s

. _______ . _______ . _______ . _______ . _______ . _______ . _______

0 5 10 15 20 25 30 days

(Figure I.3) - Layout of a quality control chart for daily use.

4. Interpretation of Quality Control Charts:

A variety of statistical control techniques have been used in clinical laboratories. Tabular
records with appropriate calculations can be used to implement the techniques, but
graphical display is often easier to interpret. Therefore, control charts have been
accepted as a more effective way to implement most control techniques. The levey-
Jennings control chart has been the most widely used technique.

4.1 Levey-Jennings Control Chart:

The control results are plotted on the Y-axis versus time on the X-axis. This chart
shows the expected mean value by the solid line in the centre and indicates the
control limits or range of acceptable values by the dashed lines. The usual way of
interpreting this control chart is to consider the run to be in control when the
control values fall within the control limits, and to be out of control when a result
exceeds the control limits.

21
4.2 Types of changes commonly observed in quality control data:

a) Dispersion : Dispersion is observed due to random error or slight imprecision.

Random error shows a wider range of scatter of the points on the
control chart within the control limits (Figure I.4-A).

b) Shift : An abrupt change, or systematic shift, may be observed when

there is a sudden development of certain analytical problems that
cause a sudden consistent change in values in either side of the
mean (Figure I.4-B) & (Figure I.4-C).

c) Trend : A trend, or systematic drift of the values, occurs when the

analytical method suffers a progressively developing problem
(Figure I.4-D).

When changes in control data indicate that the performance of an analytical

method has deteriorated, the analyst must determine the cause of the problem. It
is generally useful first to try to classify the error as random or systematic because
the different kinds of errors suggest different sources. Random errors show a wider
range of scatter of the points on the control chart, while systematic error can be
seen when the points drift or shift to one side of the central line. Further
information on the nature of the systematic and random errors were previously
mentioned.

22
 Interpretation of quality control charts
(Figure I.4-A to D)

+2 SD20

+1 SD15

Χ10

-1 SD 5

0
-2 SD
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A. Shwehart/Levey-Jennings chart showing the analysis is in control.

+2 SD20

+1 SD15

Χ 10
-1 SD 5

-2 SD 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14

B. Levey-Jennings chart showing a sudden shift in values

+2 SD20

+1 SD15

Χ10
-1 SD5

-2 SD 0
1 2 3 4 5 6 7 8 9 10 11 12 13

C. Levey-Jennings chart showing a shift of more than 5 values on the

same side of the mean and therefore out of control.

+2 SD20

+1 SD15

Χ 10

-1 SD 5

-2 SD 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

D. Levey-Jennings chart showing a gradual trend toward lower values.

23
4.3 The Allowable Limits of Laboratory Errors (ALE)

The evaluation of the Accuracy and Precision of a laboratory is meaningful only

with respect to establishment of limits of acceptable error. The maximum limits of
total analytical error can be specified on both theoretical and practical grounds.

The normal (reference) range for many parameters is defined as ± 2s of the main
values observed in an apparently healthy population if it is assumed that the
variability of a healthy population is ± 1s of the normal population range, the
Laboratory error, defined as 2s of the range of analytical values of a reference
specimens having the normal mean concentration, should be less than these
limits.

That is to say laboratory error (± 2s, Analytical Range) - individual variation (±1s,
Normal Population Range).

From this equation it follows that the laboratory error should be less than ± ¼ the
normal population range and expressed as the percentage of the normal mean
values.

1 4 normal range
Allowable Limits of Errors (%) = x100
normal mean

Example: Normal range of serum chloride = 98 - 108 meq/L

1 4 (108 - 98)
A.L.E.= x100 = 2.4%
103

The normal population range (± 2s of the mean):

To illustrate the relationship of the standard deviation and the mean to the normal
curve, consider data which are normally distributed as in (Figure I.5) 68.3% of the
area under the normal curve lies between the mean and ±1 standard deviation,
that is, from 1 standard deviation below the mean to 1 standard deviation above
the mean. Also, 95.5% of the area lies between the mean and ±2 standard
deviations, and 99.7% of the area lies between the mean and ±3 standard
deviations. Further, 95% of the area lies between the mean and ±1.96 standard
deviations.

(Table I.3)
Group Concent. (mg/dl) 60 - 79 80 – 99 100 – 119 120 – 139 140 – 159 160 – 179

Frequency 2 7 25 86 252 559

180 – 199 200 – 219 220 – 239 240 – 259 260 – 279 280 – 299

810 867 764 521 318 146

300 – 319 320 – 339 340 - 359 360 - 379 380 – 399 400 – 419

66 22 7 4 2 1

420 – 439 440 – 479 480 – 499 500 – 619 620 – 639

1 0 1 0 1

24
 (Figure I.5)

Frequency

Group Concentration (mg/dl)

5. Types of Quality Control:

5.1 Internal Laboratory Quality Control

Procedure for utilizing the results of only one laboratory for quality control
purposes.

5.2 External Laboratory Quality Control

External quality control is a procedure for utilizing, for quality control purposes, the
results of several laboratories which analyze the same specimens. Many different
types of scheme are in use throughout the world. Extensive national and
international schemes have been organized by government agencies or professional
bodies. In some countries, laboratories are required by law to participate in
external QC scheme, while in others participation is on a voluntary basis.

6. Terminology:

The term Quality Control in clinical chemistry refers mainly to the monitoring of precision
and accuracy of the performance of analytical methods.

6.1 Accuracy

Agreement between the mean estimate of a quantity and its true values.

6.2 Inaccuracy

Numerical difference between the mean of a set of replicate measurements and the
true value. This difference (positive or negative) may be expressed in the units in
which the quantity is measured, or as a percentage of the true value.

6.3 Precision

Agreement between replicate measurements. It has no numerical value.

25
6.4 Imprecision

Standard deviation or coefficient of variation of the results in a set of replicate

measurements. The mean value and the number of replicates must be stated and
the design used must be described for particular type of imprecision, such as
between laboratory, within-day, or between-day.

6.5 Trend

A trend is a gradual decrease or increase in the values of a test system.

6.6 Shift

A shift is a sudden consistent change in values in either side of the mean.

6.7 Mean

The arithmetic mean is the average.

6.8 Standard Deviation

The standard deviation (s) is the distribution range around the mean value.

Σ ( Χi − Χ ) 2
S=
n -1

Where:
Χ = Mean value
Xi = Data values
n = Number of points

6.9 Specificity

Total number of negative results

Specificity =
Total number of uninfected patients

The greater the specificity of a test, the fewer the number of false - positive results.

6.10 Sensitivity

Total number of positive results

Sensitivity =
Total number of infected patients

The greater the sensitivity of a test, the fewer the number of false - negative
results.

26
 6.11 Coefficient of Variation (C.V.)

It is the standard deviation(s) expressed in percentage and is calculated as follows:

S
CV = x100
Χ

6.12 Control

Substance which is chemically and physically similar to the unknown sample and
the constituents of which are of known concentration.

6.13 Standard

Substance of constant composition and of sufficient purity to be used for

comparison purposes.

7. Literature

a) Whitehead, T.P.: Quality control in clinical chemistry J. Wiley and Sons, New York,
1977.

b) International Federation of Clinical Chemistry: Approved recommendation (1978) on

quality control in clinical chemistry. J. Clin. Chem. Cli. Biochem. 18: 69-77, 1980.

c) Lipovac, V.: Basic laboratory control for diabetes. World Book of Diabetes in Practice.
Krall, L.P. ed. Alberti, K.G.M.M. assoc. ed., Excerpta Medica, Oxford-Amsterdam-
Princeton, 1982.

d) Albert, K.G.M.M. and Skrabalo, Z.: Standardization of biochemical methods in

diagnosis and management of diabetes with particular reference to developing
countries. IDF Bll., 27: 17-25, 1982.

e) Whitby, L.G., Mitchell, F.L. and Moss, D.W. Quality control in routine clinical
chemistry Ad. Clin. Chem. 10: 102, 1967.

f) Selected Methods of Clinical Chemistry, Vol. 9, Willard R., Faulkurs and Samuel
Meiles, American Association for Clinical Chemistry, Washington, D.C. 1982, PP: 17-
37.

g) Clinical Diagnosis and Management by Laboratory Methods, Henry, 20th edition,

2001.

27
VI. Preparation of Solutions

1. Percent solutions:

Percent solutions are either weight per volume (W/V) or volume per volume (V/V). As an
example of a W/V solution, let us consider a 5% solution of Sodium Chloride.

To prepare this solution weigh 5 gr (W) of the salt, add it to a 100 volumetric flask and
bring the volume to 100 ml mark with distilled water (V). The volume per volume (V/V)
solutions require no weighing, only volume measurements. For example, to prepare a
70% solution of ethyl alcohol, measure 70 ml of absolute (100%) ethyl alcohol and add
30 ml of distilled water. There is a mathematical relationship between the varying
percentage solutions which enables us to readily calculate quantities of distilled water
required in order to change a high percentage of the solution to a lower percentage.

The formula used is: % x V = %1 x V1

Where:
% = Percent of solution available
V = Volume of solution available
%1 = Percent of solution required
V1 = Volume of solution required

Example 1:

If we want to prepare 200 ml of 70% of ethanol solution from 95% ethanol solution,
using the formula, we get:

% x V = %1 x V1

95% x V = 70% x 200 ml

70 x 200
V = = 147.37ml
95

This tells us that we take 147.37 ml of 95% solution and add distilled water to make the
volume to 200 ml.

Example 2:

If we want to prepare 500 ml of 0.3% from a 10% solution, using the formula, we get:

% x V = %1 x V1

10 x V = 500 x 0.3

500 x 0.3
V = = 15ml
10

This tells us that we take 15 ml of 10% solution and add distilled water to make the
volume to 500 ml.

28
2. Molar (M) Solutions:

Are defined as those solutions containing one gram-molecular weight of substance per
litre of solution.

Calculations of different dilutions of Molar solutions are aided by the use of the formula:

V x M = V1 x M1

Where:
V = Volume of solution available
M = Molarity of solution available
V1 = Volume of solution to be prepared
M1 = Molarity of solution to be prepared.

3. Normal (N) Solutions:

Are defined as those solutions containing one gram-equivalent weight of the substance
per litre of solution.

3.1 A gram-equivalent weight of an acid equals the molecular weight of the acid in
grams divided by the number of replaceable hydrogen ions.

Example 1:

Molecular weight of HCl = 36.5 (H=1, Cl=35.5)

There is one replaceable H ion

36.5
∴ equivalent weight of HCL = = 36.5
1

Example 2

Molecular weight of H2SO4 = 98 (H=1, S=32, O=16)

There are two replaceable H ions

98
∴ equivalent weight of H 2 SO 4 = = 49
2

(Table I.4)
Table of Commonly used Concentrated Acids

Acid Mol. Weight Spec. Gravity Molarity Normality

Con. HCL 36.46 1.19 12 12

Con. H2SO4 98.08 1.84 18 36
Conc. HNO3 63.02 1.42 16 16

29
3.2 A gram-equivalent weight of a base equals the molecular weight of the base in
grams divided by the number of replaceable hydroxyl groups.

Example 1:

Molecular weight of NaOH = 40 (Na=23, O=16, H=1)

There is one replaceable OH group,

40
∴ equivalent weight of NaOH = = 40
1

Example 2:

Molecular weight of Ba(OH)2 = 171 (Ba=137, O=16, H=1)

There are two replaceable OH groups,

171
∴ equivalent weight of Ba(OH) 2 = = 85.5
2

3.3 A gram equivalent weight of a salt equals the molecular weight of the salt in grams
divided by the number of electrons which are given up or taken on during the
reaction under consideration.

Example 1:

Molecular weight of Ag NO3 = 170 (Ag=108, N=14, O=16)

AgNO3 + NaCl AgCL + NaNO3

on molecule of AgNO3 forms one molecular of AgCL

170
∴ equivalent weight of AgNO 3 = = 170
1

Example 2:

Molecular weight of K2Cr2O7 = 294 (K=39, Cr=52, O=16)

K2Cr2O7 + 6KI + HCl 2CrCl2 + 9KCl + 6I + 7H2O

Six molecules of KI are taken on during the reaction in presence of HCl.

294
∴ equivalent weight of K 2 CR 2 O 7 = = 49
6

30
 Calculations of different dilutions of normal solutions are aided by the use of the
formula:

V x N = V1 x N1

Where:
V = Volume of solution available
N = Normality of solution available
V1 = Volume of solution to be prepared
M1 = Normality of solution to be prepared.

Another formula that will be useful in the preparation of normal solutions from
concentrated acids is:

MW
V=
Valence x Sp. grav. x %Conc.

V = ml concentrated acid required per litre to prepare a normal solution

Example:

If we have concentrated sulphuric acid on hand, according to the label on the

bottle, the specific gravity is 1.83, the concentration is 92%, the valency is 2 and
the molecular weight (MW) is 98.076.

98.076
V= = 29.1 ml
2x1.83x0.92

V = ml conc. H2SO4 required per litre to prepare a Normal H2SO4 solution

In order to make the normal solution of H2SO4, take one litre volumetric flask and
add approximately 900ml of distilled water. Then slowly add exactly 29.1ml of
H2SO4. Then mix carefully since heat is generated in this solution and bring the
volume up to a litre with distilled water.

It must be remembered that any acid prepared in this way is only approximate.

VII. International System of Units (SI-Units)

Over the years numerous different measurement systems have been proposed, but all of them
for one reason or another proved unsatisfactory. The exception is a version of the metric
system which was given the name International System of Units "SI Units".

Following the introduction of SI units, medical scientists prepared systematic list of names,
some of these names are the same as the traditional ones. In other cases, the traditional
names were inaccurate, misleading or ambiguous. New names were therefore introduced to
replace them.

All SI units are based on seven SI base units. Only four of them are commonly used.

31
(Table I.5)
Quantity Name Symbol
Length Meter M
* Mass Kilogram Kg
Time Second s
** Amount of Substance Mole mol
Electric Current Ampere A
Thermodynamic temperature Kelvin K
Luminous intensity Candela Cd

* Mass is the correct term for what is commonly called "Weight".

** Amount of Substance and its unit mole are very important in medicine. When two or more
chemical substances react together, they do not do so in relation to their mass.

For example, in the reaction: NaOH + HCl NaCl + H2O

1 Kg of Sodium Hydroxide does not react with 1 Kg of Hydrochloric Acid. On the contrary,
1 mole of Sodium Hydroxide reacts with 1 mole of Hydrochloric Acid. Whenever chemical
substances interact, they do so in relation to their relative molecular mass.

Measurements would be difficult because these units are too large or too small. For many
purposes to overcome this difficulty, the SI incorporates a series of prefixes called SI
prefixes, which when added to the name of a unit multiply or divide that unit by a certain
factor giving multiples or submultiples of the unit.

(Table I.6)
Name of Prefix Symbol Factor
Mega M Mult. by 1 million (X106)
Kilo K Mult. by 1 thousand (X103)
Centi C div. by 1 hundred (X10-2)
Milli m div. by 1 thousand (X10-3)
Micro u div. by 1 million (X10-6)
Nano n div. by 1000 million (X10-9)

The blood contains many different kinds of cells, these cells are suspended in the blood. The
quantity of blood cells is defined as "the number of specified particle (cells) in a mixture divided
by the volume of the mixture (number per litre).

In making the conversion to recommended SI units, the following guidelines are followed:

a) All reference ranges have been converted to SI units except in cases where the
measurements are not quantitative.

b) The order of magnitude of the factors are calculated to make the values in SI units
convenient numbers i.e. with prefixes, a number not greater than 1000 or smaller than
0.001.

32
 c) The number in "SI Units" is equal to the number in conventional "units" times the "Factor".

SI unit = Conventional unit X Factor.

d) For compounds where relative molecular masses are not definitely known e.g. proteins,
reference intervals are converted to mass amounts per litre.

e) Enzyme units are given as the international unit per litre *(U/L). Although the SI unit for
catalytic activity (the Katal) has been defined as the number of moles of substrate
converted per second under defined conditions.

f) The pH scale is retained for measurement of hydrogen ion concentration

* U/L: The quantity of enzyme that will catalyze the reaction of one micromole (umole)
of substrate per minute.

One of the benefits of the SI system application can be recognized in the following table which
gives the concentration of the cation and anion for normal plasma in SI units (mmol/L). Note
that there is an exact quality of concentration of total anion charge and of total cation charge.

(Table I.7)
Cation Charges Mmol/L Conc. mmol/L Anion Charges Concentration
Na+ 142 Cl- 103
K+ 4 HCO3- 27
Ca++ 5 HPO4- 2
Mg++ 2 SO4- 1
Organic acids 5
Trace elements 1
Protein 16
154 154

VIII. Care of Equipment

* Concept of Preventive Maintenance For Laboratory and Medical Equipment

Any type of equipment malfunction could potentially affect patient care. The purpose of
preventive maintenance programme is to ensure that equipment operates properly and
safely.

Preventive maintenance can be divided into two categories.

a) Function verification:
This includes checks and tests to ensure that an instrument is working properly and
is correctly calibrated.

b) Maintenance:
This includes adjustment, repair or replacement to prolong the life of an instrument.

33
** Instrument Selection and Implementation

Preventive maintenance programmes begin by selecting instruments that will last

effectively for a reasonable period of time.

The routine operation of the instrument should be considered.

Before purchasing an instrument, inquiries should be made at laboratories using similar

equipment regarding the performance record of their particular instrument and the quality
of service provided by the manufacturer.

Specific points should be outlined in the purchase order. These should include:

a) Specifications of the instrument

b) Installation of the instrument
c) Training of personnel
d) Evaluation period
e) Warranty period
f) The manufacturer's operation manual
g) A list of recommended spare parts.

As soon as an instrument is delivered and installed, it should be evaluated.

*** Documentation

The key to a good preventive maintenance programme is organization. This includes

function checks and routine maintenance for each instrument.

Performing these checks at scheduled intervals, and carefully documenting the

information and any repair work of service done to the instrument. It is important that
these records be kept up to date.

Organization of a programme begins with a careful inventory of all equipment and

instrumentation. This inventory can be in the form of:

- Card file
- Note book
- Computer listing

It should include the following information for each item of equipment.

- Name of instrument
- Manufacturer
- Model number
- Serial number
- Purchase date
- Service representative
- Service phone number
- A list of spare parts

34
 Equipment Card File

Name of Equipment
Location
Manufacturer
Model Number
Serial Number
Locator Card Number
Purchase Date
Service Representative
Ser. Rep. Phone Numb.

Date Problem Action Taken & Comments Signature

1. Care of the Microscope

1.1 Keep the microscope covered with a clean plastic or cloth cover when it is not in
use.

1.2 Take special care to protect the microscope from dust.

1.3 Take special care to protect the microscope lenses and prisms from fungal growth
in hot humid periods. This can be done by keeping the microscope in an air-
conditioned room.

1.4 Clean the immersion oil from the immersion objective every day; use a soft cloth
dampened with Xylene and polish with a clean lens tissue.

1.5 Clean the oculars with a soft, lint-free cloth; as an alternative, use lens tissue or
facial tissue, if available.

1.6 Do not use the tissue or cloth for the oil immersion objective to clean the oculars.

1.7 Do not use alcohol to clean painted surfaces of the microscope.

1.8 Do not try to clean parts of the microscope that are difficult to reach unless you
have been trained to do so.

Recommended spare parts:

Halogen lamp, Fuses, Dust cover and Eye piece dust caps.

35
2. Care of the Spectrophotometer

Spectrophotometer is the widely used instrument in the laboratory, which directly affects
the precision and accuracy of analytical tests. This instrument must receive regular
preventive maintenance.

2.1 Keep your instrument always covered with a plastic dust cover when not in use.

2.2 Do not turn your instrument on, before removing its plastic dust cover.

2.3 Don't wipe the outside of the instrument with alcohol or any solvent, use a partly
damp cloth.

2.4 Clean and dry immediately any spills on the instrument.

2.5 Warm your instrument for at least 10 minutes before use.

2.6 Turn your instrument off while not in use.

2.7 Plug the instrument into a grounded outlet.

2.8 Put the instrument in an appropriate location away from any centrifuges or shakers.

2.9 Clean the interior of the instrument with an air gun or vacuum at least once every
three months to eliminate dust.

2.10 Clean the light source once every three months, using a lens paper.

2.11 Check wavelength calibration monthly by a didymium filter and calibrate as needed.

2.12 Absorbency calibration should be carried out every six months, and checked
monthly. This can be achieved by checking the linearity of the dichromat standard
calibration curve.

(Dilutions of 1% potassium dichromat in 0.05 M H2SO4 solution can be used to

check linearity). Monthly checks can be achieved by using a 0.05g dichromat
solution which should be given absorbency of 0.536 ± 0.005 at 350 nm.

The following checks should be made:

Checks Period

a) Linearity checking Monthly

b) Zero Transmittance/100% Absorbance Daily
c) Internal cleaning of dust Quarterly
d) Cleaning of sample holder Monthly
e) Cleaning of photocell/Detector Quarterly
f) Cleaning/replacement of Tungsten lamp Quart./100 working hrs.
g) Matching A/T Quarterly

36
 a. Linearity Checking:

Linearity checking of spectrophotometer is necessary to insure that the

equipment is valid to Obey Beer’s Law. This is done for example, by using a
stock glucose standard lg./dl that will be used to obtain standards of
different concentrations 100 mg/dl. 200 mg/dl. 300 mg/dl, 400 mg/dl and
500 mg/dl.

The five prepared standards will be used as serum samples to measure the
absorbance of glucose against a blank reagent. Let the obtained readings
be A1,A2 ,A3 ,A4 and A5 respectively.

Use a graph paper to construct the X-axis and Y-axis where the test and
principle of test should appear in the upper middle of the paper and the date
of the test should appear on the upper right side. Divide the Y-axis into five
equidistant points; 100. 200. 300. 400 and 500 mg/dl.

If the kit pamphlet tells that the test is linear up to 500 mg/dl then you
should obtain a straight line of function y = ax (affiant function – straight
line passes through the origin).

However, if at least any point of the five points was deviated away from the
line, this mostly will be found at higher concentrations, this means there is
deviation of Beer’s Law and the spectrophotometer should be checked by a
Biomedical Engineer and rechecking should be made.

All linearity charts obtained on monthly basis should be kept in a special file
for spectrophotometer linearity checking.

b. Zero Transmittance/100% Absorbance:

Pull the sample holder out and insert the occluder, a black cylindrical bar
supplied with the spectrophotometer – and if not available reinsert the sample
holder after rotating it 90, now transmitted light should be zero 0.00 or ± 0.1
as a maximum accepted; whereas, the absorbed light should be over-range
1999 with flashing. If the above was not obtained, then the
spectrophotometer should be checked by an authorised maintenance
technician.

c. Internal cleaning of dust:

If you are using Milton Roy spectrophotometer. Use a screwdriver to loosen the
screw of the lamp compartment. Also, remove the main hard cover of the
spectrophotometer. Using a smooth cloth clean the internal in general. Then
using an air gun, if available, blow the whole area to remove all dust that you
have made free. After that, use a paper lens to clean the lamp, mirrors.
Lenses, i.e. whatever optical.

d. Cleaning of sample holder:

Pull out the sample holder and clean thoroughly using soap and water. Keep
to dry then insert back in place.

37
 e. Cleaning of Photocell/Detector:

Carefully, loosen the screw from the bottom of the spectrophotometer just
under the sample compartment. Remove the detector house cover. Very
carefully pull out the circuit holding the photocell. Clean the photocell using a
swap of Methanol. Then clean using a paper lens. Put back in place, return the
detector house cover in place.

f. Cleaning/Replacement of Tungsten Lamp:

Tungsten lamp should work for a period of time after which it should be
replaced even if it is working.

If a grey-black filter was formed this means:

i) The Tungsten wire inside the lamp has lost its full capacity i.e. the quantity
of energy supposed to be produced has diminished.

ii) The internal lining of the lamp caused by burned Tungsten will act as a filter
that will not permit all produced energy that is already diminished to come
out.

Therefore, if Tungsten lamp formed a grey-black filter from inside, it should be

replaced and Tungsten lamp alignment procedure should be followed as
instructed in the spectronic-manual.

If grey colour was not noticed, cleaning using a paper lens is enough.

g. Matching A/T:

Set you spectrophotometer on for 5-10 minute. Choose wavelength 399 nm

using air as blank obtain 39.9 transmittance then change the mode to
absorbance you should obtain 0.399. If not the spectrophotometer should be
checked by a maintenance technician.

Recommended spare parts:

Tungsten lamp, Visible lamp, Deuterium lamp, Detector assembly, Sample
compartment cover, Sensitised paper for spectronic standards, Plastic dust
cover and Fuses.

3. Care of the Centrifuge (Test tube/Haematocrit)

The centrifuge is used extensively in the clinical laboratory to:

- Separate cells from blood in preparing serum or plasma.

- Clarify fluids.
- Separate suspended solid particles from solution.
- Concentrate and purify various biological and chemical agents.
- Perform certain analysis i.e. quantitative separation of solids from liquids.

38
 Centrifugal force is expressed both in terms of Revolution Per Minute (rpm) or Gravity (g).

g = 1.118 x 10 -5 x r x (rpm) 2

g = relative centrifugal force

1.118 = gravitational force

1.118x10-5 = Constant

r = radius in centimetres between axis of rotation and the centre of the

centrifuge tube.

rpm = revolutions per minute.

For the centrifuge to function and reproduce speed accurately, the following preventive
maintenance steps should be carried out at regular intervals:

a) Clean up major spills and broken tubes immediately.

b) Wipe the interior and exterior with a damp cloth once a month.

c) Lubricate according to the manufacturer's recommendation.

d) Check if the unit is balanced and free of vibration every three months.

e) Check the brushes and replace them if they are worn out.

f) Match the centrifuge tube carriers of the same weight in the opposite position.

Recommended spare parts:

Carbon brushes of proper size and Fuses of proper strength.

4. Care of the Distiller

The distiller (still) is used in the clinical laboratory for the preparation of distilled water.
Distilled water is used for:

a) Rinsing glassware after washing.

b) Preparing different reagents.

The still should be fastened to a wall at a convenient height and the water supply should
be taken from a controlled level tank to get a steady flow of water.

Operation

To ensure a good out-put, you should check the outlet of cooling water coming out from
the cooling system that will make condensation of vapor. If this water is coming out hot,
this is not good, and you should open the water tape a little bit more to ensure that this
water is coming out cold, i.e. condensation process is good and efficiency should be
accepted.

39
 When you want to stop distillation. All what you want to do is to turn power OFF, BUT
KEEPING THE RUNNING WATER ON, to avoid forming calcium carbonate on heating
elements.

a) Open the water supply to the still and wait until water flows from the overflow outlet.

b) Switch on the electrical current.

c) Regulate the raw water tap until the still runs at a constant temperature.

d) Distilled water should have a pH value of 6.5 - 7.5 and a maximum conductivity of
5 µmhos.

e) The double heater still has an output 3.5-4.5 L/hr.

Cleaning

a) Disconnect the electrical and water supplies.

b) All elements should be checked on a regular basis.

c) The elements are de-scaled as necessary.

d) A nylon scrubber is used for removing deposits of hard scale.

e) A chemical scale remover can be used, but this should be followed by lengthy run
rinsing with tap water.

Note: a solution of 10% HCl can be used for cleaning.

Recommended spare parts:

Coiled beating elements for “Manesty” water distiller 220V – 1200W.

5. Care of the Analytical Balance

5.1 The analytical balance is used in the clinical laboratory to determine accurately the
weight of chemicals or other materials. All balances need some care for proper
functioning.

5.2 The analyst should avoid all extraneous forces such as:

- Air currents
- Heating effects - direct sunlight
- Changes in relative humidity
- Magnetic influences
- Vibrations
- Fingerprints

40
 5.3 The analyst should keep weighing pans and other parts of the balance away from
the dust.

Follow the following steps regularly:

a) Keep the Analytical Balance covered.

b) Return the scale of the balance to zero after each use.
c) Clean up spillage after each use.
d) Keep the area near the balance dry, clean and dust-free.
e) Check the accuracy of the balance every three months with calibrated weights.

6. Care of the Water Bath

The water bath is used in the clinical laboratory to carry out certain analytical reactions.
The most commonly used temperature for a water bath is 37°C, but other temperatures
are also used. Inconsistent or changeable reactions cause falsely increased or decreased
test results.

This equipment has a heating element, a thermostat, a thermometer and a safety system
which includes constant level device to stop heating if the level of water went down below
the heating element and a temperature control knob that will stop heating if a certain
temperature was exceeded.

The following steps should be followed at regular intervals to ensure proper functioning of
the unit:

a) Check and record the temperature of the water bath daily.

b) Check the water level daily. If it is too low, fill with distilled water.
c) Clean the bath thoroughly and fill it with distilled water.
d) Cover the water bath when not in use.

Recommended spare parts:

Thermometer with temperature range from 25 to 100 °C.

7. Care of the Refrigerator

The refrigerator is an essential piece of equipment in any laboratory. It stores reagents

and patients' samples at low temperature 2-8°C.

Refrigeration prevents deterioration of reagents and samples in three ways:

a) It prevents or retards microbial growth.

b) It retards the decomposition of reagents, e.g. enzymes and co-enzymes decompose
rapidly at room temperature.
c) It retards the reaction between the various ingredients within a single reagent.

Refrigeration failure or improper functioning may result in expensive losses as well as

inconveniences in the smooth operation of the laboratory.

41
 For good service from a refrigerator, carry out the following preventive maintenance
steps:

a) Check and record the temperature of the refrigerator daily.

b) Adjust the temperature once a week if necessary. The temperature of the

refrigerator should be between 2 and 8°C.

c) Defrost the refrigerator at least once every three months.

d) Remove any dust from the condensing coil at the back of the refrigerator at least
once a year.

e) Clean up any spillage in the refrigerator immediately.

f) Always keep the back of the refrigerator at least 15 cm away from the wall to provide
adequate ventilation.

Use an alcohol-in-glass thermometer for freezers.

8. Care of the Thermometer

The mercury thermometer is used in the clinical laboratory to measure temperatures of:

- Water baths
- Incubators
- Refrigerators
- Heating blocks
- Freezers

The thermometer consists of:

- A graduated capillary tube.

- A mercury-containing bulb.

Accuracy of the thermometer depends on the integrity of the mercury column, therefore:

a) Check the mercury column monthly.

b) Avoid rapid heating and cooling.
c) Do not use thermometers with broken mercury columns. Regenerate the continuity
of the column by cooling the bulb to a very low temperature so as to withdraw all the
mercury back into the bulb.

9. Urine Refractometer:

Washing with water after a batch of measurements is necessary. Calibration should be

made monthly using distilled water that should read 1.000. If 1.000 was not obtained for
distilled water, calibrate using the screw on the bottom of the prism.

No spare parts are needed.

42
43
44
PART II - EXAMINATIONS FOR PARASITES

I. Stool Specimens:

1. Collection of Stool Specimen

2. Macroscopic Examination

3. Microscopic Examination
3.1 Direct Method
3.2 Concentration Method

4. Bench Aids for the Diagnosis of Intestinal Parasites

5. Various Structures Seen in Stool Preparations

6. Occult Blood

II. Urine Specimens

III. Vaginal and Urithral materials

IV. Blood Specimens

V. Skin Specimens

VI. References

45
46
I. Stool Examination

Parasitic diseases are often present with non-specific symptoms and signs. Most parasitic
diseases cannot be diagnosed by physical examination alone, and laboratory investigation is
necessary to decide whether or not the patient is infected with a parasite and what species of
parasite is present.

1. Collection of Stool Specimen

The reliability of the results obtained will depend largely on the care taken in collecting
the specimen. The following precautions should be taken in collecting specimens for
parasitological examination.

1.1 Collection of a sufficient quantity. This is necessary in order to:

a) Permit detection of parasites, if present, in low concentration.

b) Prevent rapid drying of stools. The specimen should contain at least 4cucm.

1.2 Provision of a container for the patient's use. The patient should be given a waxed
cardboard/light plastic box (container) for collection of the specimen.

1.3 Examination of stools while fresh:

a) Stools must be examined within one hour of collection.

b) If a number of specimens are received at the same time, pick out:

- Liquid stools and those containing mucous or blood. Examine them first,
because they may contain motile amoebae (Trophozoite) that die quickly.

1.4 Things not to do:

a) Never leave stool specimens exposed to the air in containers without lids.
b) Never set aside stool specimens for examination after 2 to 3 hours.
c) Never accept stools mixed with urine or water.
d) Never place the container of the stool specimen on the examination request
form.

1.5 Quality of Specimens:

Specimen should be obtained before any type therapy initiated, since antibiotics,
antihelmintics, antidiarrhoeal drugs, antacids, laxatives, soap, and hypertonic salt
enemas suppress parasites. At least 1 week should be allowed to elapse after
treatment before reexamination of the stool for parasites.

1.6 Preservatives used to preserve stool specimens:

a) Formalin
b) Polyvinyl alcohol (PVA) Fixative.

47
2. Macroscopic (Gross) Examination

Note the following characteristics of the specimen:

2.1 Consistency of stool

a) Formed : may contain cysts, eggs, and larvae.

b) Soft : may contain trophozoite, cysts, eggs and larvae.

2.2 Character of stool: may be bloody, mucoid, watery or pussy.

2.3 Presence or absence of whole worms or parts of worms in strained specimen, e.g.,
proglottides, scolices, or adult organisms such as pinworms, roundworms,
tapeworms, hookworms.

3. Microscopic Examination

3.1 Direct Method:

Can be performed by the following preparations:

a) Saline wet mounts : Trophozoite (motility), cysts, eggs and larvae.

b) Eosin in saline : Trophozoite (motility), cysts, eggs and larvae – an

optional technique.

c) Iodine wet mounts : Cysts, eggs and larvae.

Procedure:

a) Mark the number of the specimen on the slide.

b) Put two drops of normal saline in the middle of the slide.

c) Using an applicator, take a small portion of the stool from inside and from the
surface of the specimen.

d) Mix the sample with the drops of saline solution on the slide.

e) Place a coverslip over the mixture.

f) Examine the preparation under the microscope use the 10x and 40x objectives.
Reduce the amount of light by lowering the condenser to increase the contrast.

g) Repeat the steps (a) to (f) by using a drop of working iodine instead of normal
saline if you suspect the presence of ova, cyst or trophozoite.

48
 Reagents:

a) Normal Saline:
Dissolve 8.5g sodium chloride (NaCl) in one litre of distilled water.

b) Lugol Iodine (stock):

Iodine 1g
Potassium Iodide (KI) 2g
Distilled water (DW) 100 ml

Dissolve the KI in about 30ml D.W., add the iodine and mix until dissolved,
complete to 100ml with D.W. and store in a brown bottle.

c) Working Iodine Solution:

Dilute 5 times the stock iodine with D.W.

3.2 Concentration Method:

This procedure should be used whenever possible especially if the number of

organisms in the stool is low and direct wet mount may not detect parasites.

The direct wet mount examination is necessary because the protozoan trophozoite
will not be seen in the concentration method.

Can be performed by two preparations:

a) Saline wet mounts : Cysts, eggs and larvae.

b) Iodine wet mounts : Cysts, eggs and larvae.

Procedure:

a) Add 5ml of 10% formalin to about 1g of faeces.

b) Mix until you get a suspension.

c) Filter the suspension through a gauze filter into a clean centrifuge tube.
Discard the gauze filter with residue.

d) Add 2ml of ether or ethyl acetate and mix well for at least one minute.

e) Centrifuge for one minute.

f) Loosen the fatty plug at the top with a stick applicator, pour away the
supernatant by inverting the tube.

g) Mix the sediment at the bottom of the tube.

h) Transfer one drop to a slide for examination.

i) Use the X10 and X40 objectives to examine the whole area under the coverslip
for ova, cysts or larvae.

49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
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68
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5. Various Structures Seen in Stool Preparations:
(Figure II.2)

Various structures that may be seen in stool preparations:

I-8. Various yeast cells. 9. Macrophage with nucleus. 10-11. Deteriorated

macrophage without nucleus. 12-13. Polymorphonuclear leucocytes. 14-15.
Pollen grains. 16. Charcot-Leyden crystals.
Diagnostic Micrology (page 654).

71
 6. Occult Blood

Blood in the stool may be grossly visible or it may be in small amounts and revealed only
by specific test - occult blood. The presence of blood in the faeces is always abnormal
and denotes haemorrhage into the alimentary tract.

Principle:

Haem compounds catalyze the oxidation of organic substances such as benzidine,

Orthotolidine or 4-aminophenazone by hydrogen peroxide.

Procedure:

a) Place a small portion of faeces on a piece of filter paper and spread it on a small
area.

b) Add one drop of Glacial acetic acid on the stool area.

c) Add on drop of 4-Aminophenazone on the same area.

d) Add one drop of hydrogen peroxide reagent on the same area.

e) A positive reaction is shown by the appearance of a blue ring at the junction of the
two fluids.

Reagents

a) Glacial Acetic Acid 30%

Glacial Acetic Acid 30 ml
Distilled water 70 ml

b) 4-Aminophenazone 5%
4-Aminophenazone 0.5 g
Ethyl Alcohol (95%) 10 ml

c) Hydrogen peroxide 3%
Hydrogen peroxide concentrated 3 ml
Distilled water 97 ml

II. Urine Specimens

Urine specimens are usually examined for Schistosoma haematobium eggs. Trichomonas
vaginalis trophozoites may also be seen

In areas where schistosomiasis is endemic, the first indirect evidence of infection is haematuria
and/or proteinuria, detectable using a reagent strip. Gross haematuria indicates heavy
infection.

72
1. Collection of Urine for Diagnosis of Schistosoma Infection

The number of ova in the urine varies throughout the day, being highest in urine obtained
between 10h 00 and 14h 00. The specimen should be collected between these times and
consist of a single terminal urine of at least 10 ml. Ask the patient to pass the urine into
a clean container and examine the urine at once.

If the urine must stand for an hour or longer, add 1 ml of undiluted formalin (37%
formaldehyde solution) to each 100 ml of urine. This will preserve any eggs that might
be present.

Note : If formalin is not available, 2 ml of ordinary household bleach can be added to each
100 ml of urine.
Warning : Formalin and bleach are corrosive, and dangerous if swallowed.

2. Examination of Urine

The two methods used for detection of Schistosoma haematobium ova are
sedimentation and filtration. The sedimentation method is less sensitive but cheaper
and simpler to perform. The filtration technique is used in public health care mainly
when quantitative information is required.

Sedimentation Method:

Materials:

- Centrifuge, with head and cups to hold 15ml. tube.

- Centrifuge tube, conical, 15ml.
- Coverslips.
- Flask, conical for urine collection.
- Microscope slides.
- Pen or marker for labelling.
- Pipettes, Pasteur, with rubber bulbs

Technique:

a. Shake the urine specimen well.

b. Allow the urine to sediment for 1 hour. Withdraw the supernatant, transfer the
sediment into the centrifuge tube, and centrifuge at 2000g for 2 minutes.

c. Examine the deposit of the centrifuged sample for the presence of ova, using the
(x 10) objective to screen the whole of the deposit.

Do not increase the centrifugation time and do not exceed 2000g as this may rupture
the ova and release miracidia.

* Process as soon as possible.

* Shake container before pouring.
* Label slides / tubes / papers carefully.

73
 3. Identification

Schistosomia haematobium eggs are large, about 120-150µm long, and have a
terminal spine at one end. An embryo (the miracidium) can be seen inside the egg.
Sometimes, it is necessary to determine whether the eggs are viable. This can be
done if the specimen is fresh and no preservatives have been added. Look carefully at
the eggs to see if the embryos are moving. This is the best indication of viability. If
no movement is seen, look for the “flame cells”. There are 4 flame cells, one at each
corner of the embryo. Use high-power, dry magnification, reduce the illumination
slightly and look for the rapid movement of cilia (short hairs) in the cell.

III. Vaginal and Urethral Material

Vaginal and urethral materials are examined for the presence of Trichomonas vaginalis, a
flagellate parasite of the urogenital system. It parasitizes both men and women, but men are
usually asymptomatic. Trichomonas vaginalis is usually identified in wet mounts of vaginal and
urethral material. (In stained preparations these organisms are badly distorted and may not be
recognizable).

1. Collection of Specimens

Materials:

- Centrifuge, with head and cups to hold 15 ml conical tube.

- Coverslips.
- Cotton swabs, sterile.
- Microscope slides.
- Pipettes, Pasteur, with rubber bulbs.
- Pen or market for labelling.

Technique:

a. With a sterile cotton swab, collect the vaginal or urethral discharge.

b. Put the swab immediately into a sterile tube containing about 3 ml of sterile saline.
The top of the stick can be broken off if it is too long for the tube.

c. Smears for staining can be made if desired. For these, collect more material with
a second sterile swab and smear on the slide. Allow to dry.

d. Label tubes and slides with patient’s name or number, and the date of collection.

Note : if the patient can come to the laboratory, wet mounts can be examined directly;
tubes are not needed.

2. Direct Examination of Vaginal and Urethral Smears

a) Obtain some of the vaginal or urethral discharge with a sterile swab and put into a
drop of saline on a microscope slide.
b) Cover with a coverslip and examine with the (x10) and (x40) objectives for motile
flagellates.

74
 3. Centrifuged or Sedimented Material

a) If a swab in saline is received, remove the excess fluid from the swab by
squeezing it against the side of the tube. Discard the swab.

b) Centrifuge the tube for 2 minutes. If a centrifuge is not available, let the tube
stand for 10 minutes to allow any sediment to settle on the bottom.

c) With a pipetting remove the supernatant fluid. Do not disturb the sediment.

d) Take a drop of the sediment and put on a microscope slide.

e) Cover with a coverslip and examine with (x10) and (x40) objectives for motile
flagellates.

In wet mounts, flagellates can be identified by their pattern of movement.

Trichomonas trophozoites move with a nervous, jerky or jumpy movement. Since T.
vaginalis is the only species of Trichomonas that inhabits the urogenital system, there
is no need to study the morphological features or to differentiate it from T. hominis,
which lives in the intestine. On rare occasions, ciliated bodies from epithelial cells of
the genital tract may be mistakenly identified as some sort of parasitic organism.

IV. Blood Specimens

Blood is examined for the following parasites:

* Plasmodium
* Trypanosoma
* Microfilariae
* Leishmania

The most commonly used technique for blood examination is stained blood films. Giemsa stain
is usually used to stain the films; either thick films or thin films may be used, depending on the
circumstances.

The most economical use of slides is achieved by making a combination thick and thin slide,
i.e., a thick film and a thin film on the same slide. However, combination films must dry
thoroughly (8-10 hours or overnight) before they can be satisfactorily stained. Slides for
malaria should be stained the same day.

Sometimes, the physician may need a diagnosis quickly. In these cases, make thin films and
thick films on separate slides.

Direct wet mounts of fresh whole blood (or centrifuged blood) are usually used for detection of
microfilariae and trypanosomes. This only gives evidence of infection and stained films are
necessary for confirmation of the species present.

In areas where malaria, trypanosomes, and/or microfilariae may all be present, both wet and
stained films should be prepared and examined. If neither trypanosomes nor microfilaria occur
in the region, only stained films need to be made for detection of plasmodia.

75
1. Stained Blood Films:

1.1 Collection of Specimens

Carefully attention to technique that is necessary in the collection of blood and

the preparation of blood films. One should always be aware that a number of
viral, bacterial, and parasitological diseases may be transmitted in blood.

Preparation of a thick and thin blood film on the same slide

For routine malaria microscopy, thin and thick films are made on the same slide.
The thin film is used as a label but, if well prepared, is also available for species
confirmation. The thick film should be used for examination.

Technique

After patient information has been recorded in the appropriate form or register,
the blood films are made as follows:

a) With the patient’s left hand, palm upwards, select the third finger. (The big
toe can be used with infants. The thumb should never be used for adults or
children). Use cotton wool lightly soaked in alcohol to clean the finger –
using firm strokes to remove dirt and grease from the ball of the finger.
With a clean cotton towel dry the finger, using firm strokes to stimulate
blood circulation.

b) With a sterile lancet puncture the ball of the finger using a quick rolling
action. By applying gentle pressure to the finger, express the first drop of
blood and wipe it away with dry cotton wool. Make sure no strands of
cotton remain on the finger.

76
c) Working quickly and handling clean slides only by the edges, collect the
blood as follows:

- Apply gentle pressure to the finger and collect a single small drop of
blood, on to the middle of the slide. This is for the thin film.

- Apply further pressure to express more blood and collect two or three
larger drops, on to the slide about 1 cm from the drop intended for the
thin film as illustrated.

- Wipe the remaining blood away from the finger with cotton wool.

d) Thin film. Using another clean slide as a “spreader”, and with the slide with
the blood drops resting on a flat, firm surface, touch the small drop with the
spreader and allow the blood to run along its edge. Firmly push the
spreader along the slide, away from the largest drops, keeping the spreader
at an angle of 45°. Make sure the spreader is in even contact with the
surface of the slide all the time the blood is being spread. The blood film
should not extend to the edges of the slide, in order to prevent infection of
the investigator.

77
 e) Thick film. Always handle slides by the edges, or by a corner, to make the
thick film as follows: using the corner of the spreader, quickly join the larger
drops of blood and spread them to make an even, thick film. The blood
should not be excessively stirred but can be spread in a circular or
rectangular form with 3-6 movements.

f) Allow the thick film to dry in a flat, level position protected from flies, dust,
and extreme heat. Label the dry film with a pen or marker pencil by writing
across the thicker portion of the thin film the patient’s name or number and
date (as shown below). Do not use a ball pen to label the slide.

g) Wrap the dry slide in clean paper, and dispatch with the patient’s record
form to the laboratory as soon as possible.

h) The slide used for spreading the blood films must be disinfected and could
then be used for the next patient, another clean slide from the pack being
used as a spreader.

1.2 Staining Blood Films with Giemsa Stain

Regular Method for Staining Thick and Thin Blood Films on the Same Slide

For optimum staining, the thick and thin films should be made on separate slides
and different concentrations and times used for staining. This is often not
possible and the thick and thin films are generally made on the same slide.
When this is done, good-quality staining of the thick film is of primary
importance. Best results are obtained if the blood films have dried overnight.

a) Fix the thin film by adding 3 drops of methanol, or by dipping it in a

container of methanol for a few seconds. With prolonged fixation it may be
difficult to demonstrate Shüffner’s dots and Maurer’s dots. To permit
dehaemoglobinization, the thick film should not be fixed; therefore avoid
exposure of the film to methanol or methanol vapour.

78
 b) Place the slides back to back in a staining dish.

c) Prepare a 3% Giemsa solution in buffered, distilled or deionized water,

pH7.2, in sufficient quantity to fill the number of dishes being used. Mix the
stain well.

d) Pour the stain gently into the dish, until the slides are totally covered.

e) Allow to stain for 30-45 minutes out of sunlight.

f) Pour clean water gently into the dish to float off the iridescent scum on the
surface of the stain. Alternatively, gently immerse the whole dish in a vessel
filled with clean water.

g) Gently pour off the remaining stain, and rinse again in clean water for a few
seconds. Pour the water off.

h) Remove the slides one by one and place them in a slide rack to drain and
dry, film side downwards, making sure that the film does not touch the slide
rack.

(Table II.1) - Morphological features of malaria parasites in thin blood films

Stage P. Vivax P. Ovale P. Malariae P.Falciparum

Infected red cell Enlarged; Enlarged; may be Size normal or Size normal;
Shüffner’s dots oval with fimbriae; smaller than normal Maurer’s clefts may
present Shüffner’s dots be seen
present

Ring stage (early Quite large; one or Compact; two rings Compact; two rings Small and delicate;
trophozoite) two chromatin per rbc; rare per rbc; rare often two
dots; may be two chromatin dots;
rings per rbc. often two or more
rings per rbc;
accolé forms
common

Late Trophozoite Large; amoeboid; Small; not Smaller; compact; Moderate size;
pigment seen as amoeboid; pigment often band-shaped; usually compact;
fine rods coarse pigment coarse pigment granular

Mature schizont Large; merozoites Smaller than Small but Rare in peripheral
large (12-24 in P.Vivax; (6-12) merozoites (6-12) blood; merozoites
number); merozoites; large; “daisy head” (8-26)
coalescent pigment pigment darker appearance Small; single
than in P.Vivax characteristic;
pigment mass
pigment coarse

Gametocytes Spherical; Similar to, but Resemble P.Vivax Crescent shaped;

compact; single smaller than but smaller, less single nucleus
nucleus; pigment P.Vivax numerous and
diffuse and coarse Shüffner’s dots
absent

79
Figure II.3 – Malaria Parasites

80
 The Plus System for counting malaria in thick film:

A simpler method of enumerating parasites in thick blood films is to use the plus
system. This indicates the relative parasite count and entails using a code of 1-4
pluses, as follows:

+ = 1-10 parasites per 100 thick-film fields

+ + = 11-100 parasites per 100 thick-film fields
+ + + = 1-10 parasites per thick film field
+ + + + = more than 10 parasites per thick-film field

This system should be used only when it is not possible to undertake the more
acceptable parasite count per µl of blood.

2. Trypanosoma

Trypanosomes may be distorted in thick films. If organisms cannot be recognized in

thick film, look for them in the thicker area of the thin film. They are between the red
blood cells. Look at the length, the shape, and the size of the kinetoplast of the
parasites, Figure II.4.

3. Microfilariae:

The following characteristics are used for the identification of microfilariae:

- Presence or absence of a sheath,
- Presence or absence of nuclei in the tip of the tail,
- Innerbody – can or cannot be demonstrated,
- Size of the microfilaria, Figure II.5.

81
 Remember

a. Use a (x10) objective to locate microfilariae.

b. Search the blood film systematically.
c. Use high-power, dry (x 40) or oil-immersion objectives to examine microfilariae for
specific identification.

V. Skin Specimens

The parasites are most easily obtained in slit-skin smears from the nodular edge of the sore,
which is held between finger and thumb to cause blanching. Using a small scalpel blade, an
incision a few millimetres long is made through the intact epidermis into the dermis and the
exposed surface is scraped to obtain tissue juice and cells. Smears are stained with
Giemsaor another equivalent stain and examined directly . Smears that contain blood, pus,
or epithelial debris are unusable.

Smears are stained with Giemsa stain and examined under oil-immersion (see illustration
below), The pH of the buffered saline used in the Giemsa stain should be 6.8 for Leishmania
(not 7.2 as used for malaria).

Figure II.6 (a & b)

A. Leishmania promastigotes B. Leishmania amastigotes

The parasite density is graded according to the following table:

(Table II.2)
Grade Average parasite denisty
6+ > 100 parasites/field
5+ 10-100 Parasites/field
4+ 1-10 parasites/field
3+ 1-10 parasites/10 field
2+ 1-10 parasites/100 fields
1+ 1-10 parasites/1000 fields
0 0 Parasites/1000 fields

82
VI. References:

a) Manual of Basic Techniques for a Health Laboratory, WHO, Geneva, 2nd Edition,
2003.

b) Bench Aids for the Diagnosis of Intestinal Parasites, WHO, Geneva, 1994

c) Medical Parasitology, Markell E. K. and Voge. M., 1981

83
84
 PART III - EXAMINATION OF URINE

I. Collection of Urine Specimen

II. Physical Characteristics

III. Strips for Urine Testing

IV. Microscopic Examination of Deposits

V. Illustrations of Urine Deposits

VI. Pregnancy Test

VII. References.

85
86
I. Collection of Urine Specimen

Urine specimens must be collected in the correct way in suitable containers. If the specimen is
not collected properly, the laboratory findings will be unreliable.

1. Time of Collection

Have the patient collect the midstream specimen in the health centre (fresh), if possible.

2. Specimen Containers

2.1 Use clean, dry container

2.2 If the specimen is intended for bacteriological examination, a suitable sterile

container must be used.

3. Quantity of Urine to Collect

Collect at least 25 ml in a suitable container.

II. Physical Characteristics:

Variations in odour, colour, and turbidity may be caused by the handling of the specimen
(standing, refrigeration) and may not reflect pathogenic changes. If the physical appearance
is important, a fresh specimen should be requested and examined immediately after voiding.

1. Odour:

The odour of urine may be modified by the presence of acetone (as in diabetes mellitus,
starvation, and dehydration, which imparts a fruity odour), or by bacterial decomposition,
which produce an ammoniacal odour. An offensive odour may be the results of bacterial
action in the presence of pus. Some food (garlic and asparagus) and in some medication
(menthol) also affect the odour. In phenylketonuria the odour has been described as
"mousy" but in general, the odour is not of diagnostic significance.

2. Colour:

Normally, urine is some shade of yellow, because of a mixture of pigments, such as:
uroerythrin, urochrome, and porphyrin. The colour varies with specific gravity; if the
urine is diluted, it is a straw coloured, and if concentrated, almost deep orange. It is
influenced by a variety of metabolic products, food, drugs and pigments. On standing,
urine darkens, because of the oxidation of the colourless urobilinogen to coloured urobilin.
The colour of urine varies according to the following conditions:

2.1 Very pale yellow or greenish yellow, almost colourless:

Chronic kidney disease, diabetes insipidus, diabetes mellitus, high dilution, iron
deficiency.

2.2 Yellow:

Cascara, food colour, normal urine, phenacetin.

87
 2.3 Orange:

Carotene, concentrated urine (fever, inadequate water intake, excessive water

loss), food colour, rhubarb, riboflavin.

2.4 Green or blue-green:

Bile pigment, blue diaper syndrome, indican, methylene blue, robaxin, vitamin B
complex.

2.5 Pink, red or reddish orange:

Beets, blood (smoky if red cells are intact), chromogenic bacteria, food colour,
haemoglobin, myoglobin, phenolsulfonephthalein, porphyrin.

2.6 Black, grey, or brown:

Alkapton bodies (ochronosis, homogentisic acid, alkaptonuria), Bilirubin, Iron

compounds (injectable), Melanin, Methaemoglobin, Phenol poisoning and Porphyrin.

3. Transparency and Turbidity:

Freshly voided urine is clear.

Causes of turbidity:

3.1 Temperature and pH:

Diffuse clouding may occur or a sediment may form on standing because of

changes in the pH and in the temperature, which may be responsible for
precipitation of solutes from a supersaturated solution.

3.2 Amorphous phosphate and carbonates:

They are soluble in acid urine but may be precipitate in alkaline urine. They
dissolve in addition of 5-10% acetic acid, amorphous phosphates without and
carbonates with gas formation.

3.3 Urate:

They are soluble in neutral or alkaline urine but may precipitate in acid urine. They
are often pink and dissolve on heating. If protein is present, the cloudiness may
increase on heating.

3.4 Oxalate:

Clearing is produced by 12% hydrochloric acid.

3.5 Pus (pyuria), blood and epithelial cells.

In alkaline urine pus is usually mucoid; it is crumbly in acid urine. About 200 white
cells/cu mm or about 500 red cells/cu mm produce turbidity.

88
 3.6 Bacteria:

They are not removed by filtration through filter paper unless some inert substance
such as kaolin is added first; even then the results are not satisfactory.

3.7 Fat (lipuria):

Fat globules impart milky appearance to the specimen but may be removed and
cleared by extraction with ether.

3.8 Chyle:

Chyluria may be parasitic (filarial) or non parasitic (as thoracic duct obstruction,
trauma, or tumour) in origin and imparts a cream colour to the urine. Obstructed
lymph vessels may force chylous fluid of cholesterol into the excretory urinary
apparatus and into the urine. Shaking the specimen with ether will clear the urine
sample.

III. Strips for Urine Testing

The following precautions are necessary when using test strips in order to obtain reliable
results:

a) Follow exactly the manufacturers' instructions regarding the use and storage of reagent
strips.

b) Protect the reagent strips from moisture, excessive heat and light. The strips must not be
refrigerated.

c) Do not use strips that show any discoloration of the test areas.

d) Avoid any contamination of strips.

e) Use fresh urine and mix it before testing. Avoid prolonged contact with the urine, and
shake off the excess urine.

f) Read the reaction in a good light at the times stated by manufacturer. Compare the
reaction by holding the strip close to the chart on the bottle label.

g) Control the performance of the reagent strips by checking regularly the strip reactions with
reference solutions.

1. Strips for Urine Glucose

Glucosuria is present with the following conditions:

a. Renal Glycosuria
b. Rapid Intestinal Absorption
c. Endocrine Disorders
d. Sepsis
e. Head injury
f. Massive oral glucose intake, especially after poor diet.
g. Lead poisoning.

89
 The principle of the strip tests for detecting glucose in urine is based on enzymatic
reaction summarized as follows:-

Glu cos e Oxidase

Glu cos e + Oxygen ⎯⎯ ⎯ ⎯ ⎯ ⎯ ⎯⎯→ Gluconolactone + Hydrogen peroxide

Peroxidase
Hydrogen peroxide + Chromogen ⎯⎯ ⎯ ⎯ ⎯ ⎯→Water + Oxidized Chromogen

(coloured)

Since the reactions are enzyme based, the pH, Temperature and Timing of the tests
are important. The optimum pH is between 5 and 6, and the optimum temperature is
between 20-25°C.

The glucose reaction can be affected by a number of substances which may give a false
reaction. The most commonly interfering substances are:

- Ascorbic Acid, certain Antibiotics and Drug metabolites can be oxidized by the
hydrogen peroxide in preference to the chromogen, which can lead to a reduction of
the sensitivity of the strip.

- Increased amount of acetoacetate in poorly controlled diabetes may interfere in a

similar way.

- High concentration of Catalase (in severe E. coli infections) can destroy hydrogen
peroxide and cause false negative results.

- Oxidizing disinfectants, by oxidizing the chromogen, may result in false positive

reactions.

2. Strips for Urine Ketones

The principle of the strip tests for detecting ketonuria is based on the nitroprusside
reaction. Acetoacetate and acetone in an alkaline medium react with sodium
nitroprusside to produce a purple-coloured compound.

Weak false positive reactions may occur if the urine contains L-DOPA, large amounts of
phenyl-pyruvic acid (in phenyl-ketonuria), and the phthalein compounds (used in the
intravenous liver function test).

3. Strips for Urine Proteins

The strips for detecting proteinuria are impregnated with the indicator tetrabromphenol
blue or tetrabromphenol-phthalein ethyl ester, and buffered to an acidic pH. In the
presence of protein there is a change in the colour of the indicator from light yellow to
green blue depending on the amount of protein present.

The strips are very sensitive, detecting as little as o.3 g/L of protein. The reaction is
unaffected by turbidity in the urine. The strips detect mainly albumin, and other proteins
give only weak positive reactions.

90
False positive results may occur:

- If the urine is strongly alkaline (over pH 9) as in bacterial infections, caused for

example, by Proteus organism.

- If the specimen is collected during or after an infusion of plyvinylpyrolidone (plasma

expander), or during therapy with drugs such as quinine, trimethoprim or
phenazopyridine.

- If the urine contains traces of Cetavlon, or other disinfectant based on quaternary

ammonium compounds or chlorohexidine.

Alternative and confirmatory method: Sulfosalicylic acid turbidity method

Principle:

Protein denatured by acid, precipitates and renders the urine specimen more turbid as its
concentration increased.

Reagents:

Sulfosalicylic acid 3% aqueous (3 g/dl distilled water).

Procedure:

To 1 ml centrifuged urine add 3 ml reagent. Mix and allow to stand for 5 min.

Method of Reporting:

(Table III.1)
Protein
Turbidity
mg/dl
None (no turbidity) <7.5
Trace (perceptible turbidity) 20
1+ (print can easily be seen and read when viewed through test tube) 30-100
2+ (print can be seen but not easily read when viewed through test tube) 100-250
3+ (print cannot be seen when viewed through test tube) 250-450
4+ (precipitate formed) >450

Combined use of strip and sulfosalicylic acid test:

If both methods are positive, proteinuria is present; if both tests are negative, proteinuria
is absent. If the strip is +1 and the sulfosalicylic acid is negative, there is probably no
pathogenic concentration of protein in urine. If the reverse is found, then there may be
Bence Jonse protein or one of the heavy-chain proteins and should be confirmed by
immunologic methods.

91
4. Strips for Urinary Infection (Nitrite):

Urinary infection caused by E.coli and other pathogenic bacteria is characterized by the
reduction of nitrate to nitrite in the urine. The principle of the detection of nitrite in urine
is based on the Griess reaction. Aromatic amine sulphanilamide reacts with nitrite in
acidic media producing a diazonium compound which with 3 hydroxy 1,2,3,4
tetrahydrobenzo-h-quinoline leads to formation of an azo-dye(which is red in colour).
Intensity of the red colour is proportional to the concentration of nitrite.

The strips detect the concentration of nitrite as low as 7 umo 1/1.

False results may occur:

- If the urine contains high concentration of vitamin-C (false negative results) or drug
phenazopyridine (false positive results).

- If the patients are on parenteral feeding or on a diet without vegetables (false

negative results).

5. Strips for Urine Bilirubin

Test for bilirubin is not necessarily part of the routine urinalysis. It is usually ordered if
liver disease is suspected. This test is useful in the differentiation of haemolytic jaundice
and hepatic jaundice.

The test is based on the coupling of bilirubin with diazonium salt in an acid medium. The
colour ranges through tan or tannish-purple. The test area will detect bilirubin in
concentration as low as 0.2 - 0.4 mg/dl.

False-negative results can occur in the presence of large amount of ascorbic acid and
nitrite. False-positive results occur in urine from patients receiving chlorpromazine, or
metabolites of drugs.

Alternative method: Fouchet test (Harrison's modification of Gmelin test)

Principle:

Barium Chloride precipitates phosphates that entrain and concentrate bile pigments,
which are tested for the oxidation reaction.

Reagent:

To make Fouchet reagent mix 25g trichloroacetic acid, 1 dl distilled water, and 10 ml 10%
aqueous ferric chloride.

Procedure:

To about 10 ml urine, add about 1g barium chloride, mix and filter. Spread the filter
paper out and, when partly dry, drop a little Fouchet reagent on the precipitate.

92
 Result:

A green colour of biliverdin is positive and indicates the presence of bile (bilirubin) in the
urine.

Interfering substances:

Salicylate and phenazopyridine give an interfering purple colour.

Differential diagnosis of jaundice based on urine bilirubin and urobilinogen test.

(Table III.2)
Liver Pathology Urine Urine
And Type of Jaundice Bilirubin Urobilinogen

Normal 0 Trace

Hepatitis (viral, toxic, drug): Hepatocellular

↑ ↑
jaundice
Biliary obstruction (extrahepatic and
↑ 0
intrahepatic); obstructive jaundice

Haemolytic jaundice 0 ↑

Liver cell dysfunction (cirrhosis, infections,

0 ↑
metastases, heart failure, hyperthyroidism)

6. Strips for Urine Urobilinogen

This is a useful screening test in the diagnosis of liver function. Urobilinogen is normally
found in the urine in concentration less than one Ehrlich unit per 100 ml of urine. The
test should be done on a fresh urine sample, because urobilinogen is unstable.

This test is based on the Ehrlich aldehyde reaction, which contains p-

dimethylaminobenzaldehyde which reacts with urobilinogen in a strongly acid medium to
produce a pink-red colour.

False-negative results can occur in the presence of formalin and nitrite.

False-positive results can occur in urine from patients receiving phenazopyridine.

7. Strips for Urine Specific Gravity

Specific gravity measurement is actually measuring the ionic concentration of the urine.
The test is based on the pKa change of polyelectrolytes in the reagent area which
disassociate releasing hydrogen ions and causing the pH to change. The reagent area
which contains a pH indicator (bromothymol blue) measures the change in pH. The
colour ranges from deep blue-green in urine of low specific gravity through green and
yellow-green in urine of increasing specific gravity. Lower specific gravity results may
occur in urine contain glucose or urea (greater than 1%).

93
Higher specific gravity results may occur in urine with moderate or high amounts of
proteins (100-750 mg/dl or greater).

The specific gravity of distilled water is 1.000 at 20 °C. Specific gravity of urine should
be corrected according to the urine temperature. Add 0.001 for every 3 °C above 20 °C
and subtract 0.001 for every 3 °C below 20 °C.

Alternative Method: Refractometer

The refractive index of a solution, defined as a ratio of the velocity of light in the solution
to that in vacuum, is a property of a solution that increases at a fairly liner rate with
increases in the amount of dissolved solute. Thus the measurement of the refractive
index of the urine servers the same purpose as a measurement of specific gravity, an
index of the amount of solids excreted by the kidneys. As with specific gravity the
increase with the concentration is not the same for all substances, but an average value
serves as it does for the specific gravity. Refractometers are calibrated in terms of
specific gravity. The commercial hand refractometer for urine analysis are convenient,
accurate and rapid. They require few drops, and the measurement takes only few
seconds.

Normal values:

Random specimen, 1.005 - 1.030 (highest in the morning)

24 hr specimen, 1.015 - 1.018
Middle age and over: progressive decrease of specific gravity.

Interpretation of specific gravity measurement:

Normal specific gravity is primarily influenced by the electrolytes and nitrogenous waste
products. The first morning specimen should have a specific gravity between 1.015 and
1.025.

Increase of specific gravity:

Values over 1.020 are seen in decreased fluid intake, fever, sweating, vomiting and
diarrhoea. The increased values are also encountered in diabetes melitis (glycosuria),
congestive heart failure, adrenal insufficiency, and proteinuria and when preservatives or
x-ray contrast media are added to urine specimen.

Decrease of specific gravity:

Low specific gravity (less than 1.009) is seen in exaggerated oral or intravenous fluid
intake), following the administration of diuretics, and in hypothermia. The renal
concentrating power is impaired or lost in glomerulonephritis in pyelonephritis, and in the
absence of antidiuretic hormone (diabetes insipidus).

Fixed specific gravity:

In sever renal damage the specific gravity is fixed at 1.010, the value of the glomerular
filtrate.

94
 8. Strips for Red Cells and Haemoglobin

This test is based on the peroxidase-like activity of haemoglobin which catalyzes the
reaction of common hydroperoxide and 3,3 , 5,5 tetramethylbenzidine. The resulting
colour ranges from orange through green or dark blue. The significance of the "trace"
reaction may vary among patients; and the clinical judgment is required for assessment in
an individual case. Development of green spot (intact erythrocytes) or green colour free
haemoglobin/myoglobin) on the reagent area indicates the need for further investigation.
This test is highly sensitive to haemoglobin (it is slightly less to intact erythrocytes) and
thus complements the microscopic examination.

Interpretation:

A positive test may be caused by the presence of red cells, haemoglobin, or myoglobin.
Elevated specific gravity or elevated protein may reduce the reactivity of the blood test.

False Positive Test: May be caused by pus (peroxidase of white cells), bacteria, iodides,
bromides, or sodium hypochlorite if used to disinfect the containers. Ascorbic acid which
is added to some antibiotics in large quantities inhibits the colour development.

False Negative Test: The screening test also detects myoglobin which occurs in urine,
but less often than haemoglobin.

Blood in Urine: Haematuria and haemoglobinuria. The term haematuria implies the
presence of more or less intact red cells in the urine while haemoglobinuria denotes the
presence of dissolved haemoglobin.

Haematuria is usually accompanied by some degree of haemoglobinuria because of the

disintegration of red cells in the urine on standing.

IV Microscopic Examination of Urine (Urinary Deposits)

Urinary sediment provides useful information both for prognosis and diagnosis. Urine usually
contains microscopic elements such as cells, crystals and casts in suspension form. These
elements can be collected by centrifugation and a drop of the deposit is examined
microscopically.

Procedure:

a) A fresh, mid-stream urine sample should be collected in the laboratory in a clean labelled
container.

b) Pour 10, 12 or 15 ml of well-mixed urine into a labelled conical centrifuge tube.

c) Centrifuge at 1500 rpm for 5 minutes.

d) Pour off the supernatant urine without shaking.

e) Suspend the sediment by shaking the tube.

f) Place one drop on a clean slide and cover with a coverslip.

95
g) Lower the condenser and examine under the microscope, using the X10 objective.

h) Look for crystals, casts, parasitic ova or larvae.

i) Use the X40 objective and look for cells, mucus, crystals, casts, parasitic ova or larvae.

k) Record the result and mention the quantity of each element seen under the microscope.

(Table III.3) - Elements found in urine, their expected range, and clinical significance.
Clinical Significance of
Microscope Element Expected Range
Increased No.
Red Blood Cells 0-2/HPF More than 2/HPF indicates:
Bleeding, Renal Stones, Pyelonephritis, Cystitis,
Prostitis, Tuberculosis.

Red Blood Cell Casts None Acute inflammatory disorder in the glomeruli.

White Blood Cells 0-8/HPF More that 8/HPF indicates:

Bacterial infection in the urinary tract.

White Blood Cell Casts None Indicates Renal infection.

Yeast Cells None Occasionally present in urine containing glucose.

Hyaline Casts None or Fever, Exercise, Nephrotic Syndrome.

occasional

Epithelial Cells (Renal) Few Nephrosis, poisoning from heavy metals or toxins.

Granular Casts None or Glomerulonephritis, Pyelonephritis, lead poisoning.

occasional

Bacteria (fresh urine) None Urinary tract infection (confirm by culture).

Crystals
a. Acid urine: Uric Acid and Calcium oxalates may appear in normal
- Amorphous urates None or urine as it cools.
- Uric Acid occasional
- Calcium oxalate
- Cystine None Cystine crystals: Diagnostic of cystinuria

b. Alkaline urine: None or

- Amorphous occasional
phosph.
- Triple phosph.
- Calcium phosph.

All crystals should be reported, because they may lead to kidney stones.

96
(Table III.4) - Urinalysis Abnormalities Found in Various Urinary System Diseases
Diseases Macroscopic Findings Microscopic Findings
Acute glomerulonephritis Gross haematuria Erythrocyte and blood casts
“Smoky” turbidity Epithelial casts, Waxy casts
Proteinuria Hyaline and granular casts
Erythrocytes, Neutrophils
Chronic Haematuria Granular and waxy casts
glomerulonephritis Proteinuria Occasional blood casts, Epithelial casts
Erythrocytes, Leukocytes
Lipid droplets
Acute pyelonephritis Turbid Numerous neutrophils ( many in clumps)
Occasional odor Few lymphocytes and histocytes
Occasional proteinuria Leukocyte casts, Epithelial casts
Renal epithelial casts
Granular and waxy casts
Erythrocytes, Bacteria
Chronic pyelonephritis Occasional proteinuria Leukocytes, Erythrocytes
Granular and epithelial casts
Occasional leukocyte casts
Broad waxy casts, Bacteria
Nephrotic syndrome Proteinuria Fatty and waxy casts
Fat dropets Cellular and granular casts
Oval fat bodies and/or vacuolated renal
epithelial cells.
Acute tubular necrosis Haematuria Necrotic or degen. renal epithelial cells
Occasional proteinuria Neutrophils and erythrocytes
Granular and epithelial casts
Waxy casts, Broad casts
Epithelial tissue fragments
Cystitis Haematuria Numerous leukocytes, Erythrocytes
Transitional epithelial cells
Histiocytes and giant cells
Bacteria, Absence of casts
Dysuria-pyuria syndrome Slightly turbid Numerous leukocytes, bacteria
Erythrocytes, No casts
Acute renal allograft Haematuria Renal epithelial cells
rejection Occasional proteinuria Lymphocytes and plasma cells
(lower nephrosis) Neutrophils, Renal epithelial fragments
Renal epithelial casts
Granular, bloody, and waxy casts
Urinary tract neoplasia Haematuria Atypical mononuclear cells with enlarged,
irregular hyperchromatic nuclei.
Neutrophils. Erythrocytes
Transitional epithelial cells
Viral infection Haematuria Enlarged mononuclear / multinucleated cells.
Occasional proteinuria Lymphocytes and plasma cells
Neutrophils, Erythrocytes

97
V. Illustrations of Urinary Deposits

Figure III.1

Calcium oxalate (acid urine) Amorphous phosphates (alkaline urine)

Uric acid (acid urine) Amorphous urates (acid urine)

Triple phosphates (neutral or alkaline urine) Cystine (acid urine)

Urates (alkaline urine)

98
Figure III.2

Red blood cells

Few red cells (normal):

0-2 red cells per field

Moderate number of red cells:

10-30 red cells per field

Many red cells:

over 30 red cells per field

Leucocytes (white cells)

Few leucocytes (normal):

0-10 leucocytes per field

Moderate number of leucocytes:

10-20 leucocytes per field

Many leucocytes:
20-30 leucocytes per field

Many leucocytes seen in

clumps: clumps of more than
20 degenerate leucocytes.

Full field:
Clumps and may degenerated
Leuccytes

Pus casts Eggs of Schistosoma haematobium Renal cells

Blood casts Yeasts Granular casts

99
Figure III.3

1. Renal tubular epithelial cell containing brown pigment; iron, unstained (X260).
2. Renal tubular epithelial cell positive with Prussian blue stain (hemosiderinuria) (X260)
3. Dysmorphic erythrocytes (X160).
4. Neutrophils with dilute acetic acid (X200).
5. Eosinophils (X500).
6. Squamous epithelial cell, Pyridium stained (X200)
7. Transitional epithelial cells, Papanicolaou.
8. Renal tubular epithelial cells in renal fragment (X200).
9. Renal tubular epithelial cells and neutrophil. Papanicolaou stain (X 430).
10. Oval fat body (X160).
11. Oval fat body with attached fat droplets. Brightfield (X160).
12. Oval fat body with attached fat droplets. Polarized (X160).

100
Figure III.4

13. Hyaline casts: A, brightfield, and B, phase-contrast microscopy (x 100).

14. Waxy cast (x 200).
15. Finely granular cast becoming waxy (x 200).
16. Erythrocyte cast (x 200).
17. Leucocyte cast. Papaniclaou stain (x 200).
18. Cellular cast (x 200).
19. Renal tubular epithelial cell cast. Papanicolaou stain (x 430).
20. Mixed (leukocyte and renal tubular epithelial cell) cast (x 200).
21. Cellular cast (x 200).
22. Granular cast (x 200).
23. Fatty cast. Brightfield, non-polarizing (x 160).
24. Fatty cast. Positive Oil Red O (x 200).
25. Haemoglobin cast (x 200).

101
Figure III.5

26. Acid urates (x 160).Candida: budding spores (X200).

27. Uric acid crystals (x 160).
28. Large uric acid plate, laminated (x 160).
29. Hexagonal uric acid crystals. Brightfield (x 50)
30. Hexagonal uric acid crystals. Polarized (x 50).
31. Calcium oxalate crystal (x 200).
32. Calcium oxalate. Unusual oval form (x 200).
33. Calcium phosphate (large clear plate). Almost amorphous phosphates (x 64).
34. Triple phosphate (x 50).
35. Calcium phosphate (fine sheaves) (x 160).
36. Ammonium biurate (X160).
37. Cystine (hexagonal, laminated) (x 200).
38. Tyrosine crystals (x 160).

102
Figure III.6

39. Leucine crystals (x 160).

40. Sulfadiazine (X160).
41. Ampicillin (X40).
42. Renografin (meglumine diatrizoate). Brightfield (x 160).
43. Renografin (meglumine diatrizoate). Polarized (x 160).
44. Bacteria (x 200).
45. Candida. Budding Yeast (x 200).
46. Candida. Pseudohyphae (x 160).
47. Muscle Fiber (x 200).
48. Pollen grain (x 160).

103
VI. Pregnancy Test

Introduction/Principle:

Human Chorionic Gonadotropin (hCG) is a hormone that is elevated during pregnancy, it

could be detected in urine or blood. Its detection aid in early prediction of pregnancy in
general. The test is a rapid chromatographic immunoassay for the qualitative detection of
(hCG). The test utilizes a combination of antibodies, including a monoclonal (hCG) antibody,
to selectively detect elevated levels of (hCG).

Sensitivity:

Based on the above, low concentrations of (hCG) cold be detected by this method. In most
cases, if the pregnancy test strip could detect (hCG) with a concentration of 25 IU/L, then
the pregnancy could be detected at the first day after missed period.

Specimen:

Urine specimen is collected in a dry and transparent plastic container, even though the first
morning urine sample is recommended. If the urine is turbid, then allow the sample to stand
for a time, or centrifuge the urine and use the supernatant.

Procedure:

Timing is critical.

For procedural steps, please refer to the manufacturer’s instruction as per the kit insert.

VII. References:

1. Cheesbarough, M: Medical Laboratory Manual for Developing Countries, Vol.1, Stephen

Austin and Sons Lts. Hertford, England, 1981, pp 380-382 and 437-440.

2. Sister Laurine Graff. A Handbook of Routine Urinalysis, 1983, pp 21-48 and 64-65.

3. Clinical Diagnosis and Management by Laboratory Methods, Henry, 20th edition, 2001.

104
105
106
PART IV – HAEMATOILOGY AND BLOOD BANK

I. Blood Collection
1. Capillary Blood
2. Venous Blood
3. Use of Anticoagulants

II. Blood Cell Counting

1. Red Blood Cells (RBCs) Count
2. White Blood Cells (WBCs) Count
3. Platelets Count
4. Reticulocytes Count

III. Blood Film Examination

1. Preparation of a thin blood film
2. Preparation of a thick blood film
3. Staining of thin blood film with Wright’s stain

IV. Haemoglobin Determination

1. Cyanmethaemoglobin Method
2. Haemoglobinometer

V. Haematocrit

VI. Erythrocyte Sedimentation Rate (ESR)

VII. Clotting Time

VIII. Bleeding Time

IX. Sickle Cell Test

X. ABO and Rh Grouping

XI. Direct Antiglobulin Test (Direct Coomb’s Test)

XII. Indirect Antiglobulin Test (Indirect Coomb’s Test)

107
108
I. Blood Collection

1. Capillary Blood

Capillary blood is obtained from the tip of a finger in adults and from the great toe or the
heel in infants. Wash area with 70% alcohol, dry with sterile gauze, and puncture the
skin with a sterile disposable blood lancet that is designed to penetrate no deeper than
2mm.

Use a sterile gauze to wipe away the first drop of blood and collect the subsequent drops.
Avoid squeezing to obtain blood, since it will alter the composition of blood specimen. If
the blood is difficult to obtain, warm or allow it to remain in hanging position for some
time.

Advantages:

a) Capillary blood can be obtained with ease.

b) Capillary blood is the preferred material for making peripheral blood smear.

Disadvantages:

a) Only a small specimen can be obtained, and repeated examinations require new
specimens.
b) Blood in microtubes frequently haemolyses, and haemolysis interferes with most
laboratory tests.
c) Test results on capillary blood cannot be compared with test results in venous blood.
d) The finger is not only sensitive but difficult to adequately sterilize in the time usually
available. In patients with lowered resistance to infection, a specimen taken from
the finger is much likely to lead to infection than one taken from the arm.
e) Red and white cell counts and enumeration of Platelets and Reticulocytes should not
be performed on capillary blood, because of the difficulty in standardizing capillary
blood flow.

2. Venous Blood:

Venous blood is necessary for most tests that require anticoagulation or larger quantities
of blood, plasma or serum that can be provided by capillary blood.

Venous blood is usually obtained from one of the capital fossa veins, although other veins
may be chosen. The vein is congested by placing a tourniquet on the upper arm and
tighten it sufficiently to prevent venous blood return. Clean the vein with 70% Ethyl
Alcohol, after the vein entered, loosen the tourniquet and obtain blood by gentle suction.

At the end, use a sterile gauze to apply pressure over the puncture site. Remove the
needle from the syringe, and quickly transfer blood into a test tube which may or may not
contain anticoagulant. If an anticoagulant has been added, mix blood gently by inverting
the stoppered tube several times.

Advantages:

a) Multiple and repeated examinations can be performed on the same specimen.

b) Aliquots of the specimen may be frozen for future reference.
c) There is no variation in blood values if specimens are obtained from different veins.

109
 Disadvantages:

a) The venous method is somewhat lengthy procedure that requires more preparation
than the capillary method.
b) Prolonged stasis produced by the tourniquet must be avoided, because it produces
haemoconcentration.

3. Use of Anticoagulants:

3.1 Ethylenediamine Tetra-Acetic Acid (EDTA)

It is the sodium or potassium salt of Ethylenediaminetetraacetic Acid. EDTA

prevents the blood from clotting by removing and binding calcium. EDTA can be
used for both haematology and some chemistry tests. It is considered an excellent
anticoagulant and it is widely used. The amount of EDTA required for 10cc or less
of blood is 10mg of the dry powder or 0.1cc of a 10% solution. The EDTA in the
test tube does not have to be evaporated, it may be used in liquid form.

3.2 Trisodium Citrate:

Trisodium citrate prevents the blood from clotting by removing and binding calcium.
Preparation of trisodium citrate: Weigh 3.8g of trisodium citrate and dissolve in
100ml distilled water. The ratio of blood to sodium citrate solution is 9:1, i.e. for
9ml blood add 1ml sodium citrate solution for coagulation tests and 4:1 for ESR
estimation.

3.3 Mixture of Ammonium Oxalate & Potassium Oxalate:

This mixture of oxalate salts prevents the blood from clotting by removing and
precipitating calcium. This anticoagulant should not be used for chemistry tests as
Urea and Potassium.

Preparation:

Weigh 12g of ammonium oxalate and 8g of potassium oxalate, dissolve in 1L

distilled water. For 5cc blood or less, place 0.5cc of the above solution in a test
tube and evaporate to dryness at room temperature or in an oven which is not over
80oc.

3.4 Heparin:

Heparin prevents the blood from clotting by neutralizing thrombin. It is used at a

concentration of 10-20 iu per ml of blood. Heparin is the best anticoagulant to use
for osmotic fragility tests. However, heparinized blood should not be used for
making blood films.

3.5 Oxalate with Sodium Fluoride:

Sodium Fluoride acts as glycolytic inhibitor. Oxalated blood collecting tubes with
Sodium Fluoride are used for plasma glucose testing.

110
 Approximate Keeping Time of EDTA Blood

(Table IV.1)
Test Keeping Time
White Cell Count 24 hr
Red Cell Count 24 hr
Haemoglobin Determination 1 hr
Stained Red Cell Examination 1 hr
Sedimentation Rate 2 hr
Haematocrit Reading 24 hr
Reticulocyte Count 1 hr
Red Cell Indices 3 hr
Platelet Count 1 hr
Blood Grouping 48 hr
Differential White Cell Count 1 hr

II. Blood Cell Counting:

1. Red Blood Cell (RBC) Count

Measurement of the total number of circulatory RBCs is important because the number of
RBCs and the amount of haemoglobin,they contain, must remain within certain limits to
maintain good health. The main function of the red cell is to carry oxygen from the lungs
to the body tissue and to transfer carbon dioxide from the tissue to the lungs. The
process is achieved by means of the haemoglobin in the red cells that combines easily
with oxygen and carbon dioxide.

Equipment:

a. 10 ul Automatic pipet or Red cell pipet (Fig.IV-3)

The red cell diluting pipet contains a red bead in the bulb and has ten divisions on
the capillary end, with points 0.5 and 1.0 numbered. If the blood is drawn to the 0.5
mark and the pipet filled with diluting fluid, the resultant dilution is 0.5:100 or 1:200,
the dilution used in routine counting. One volume of diluting fluid remains in the
stem, does not enter the bulb, is blown out first before the counting chamber filled
and therefore does not dilute the blood.

b. Counting Chamber (Haemocytometer)

The most commonly used method employs the Improved Neubauer counting
chamber (Fig.IV-1). There are two Chambers per Haemocytometer, each consists of
nine large squares, each measuring 1mm2 in Fig.IV-2.

The Central square (No. 5) of the chamber is subdivided into 25 smaller squares,
each 1/25mm2. Five of these squares in Fig.IV-4, are marked A, B, C, D and E, each
of these 25 squares is further divided into 16 squares of 1/400mm2 each. The five
squares marked A, B, C, D and E are used for RBCs counting.

111
Procedure:

a) Add 10ul of the patient whole blood sample to 2ml of red cell solution. If you use
red cell pipet, draw blood to 0.5 mark and complete with red cell solution to 101
mark.
b) Mix well the diluted blood. (The dilution of blood is 1:200).
c) If you use red cell pipet, discard the first 5 drops before charging the counting
chamber.
d) Fill the counting chamber, taking care not to overfill beyond the ruled area, and
check if air bubbles present.
e) Count the cells is 5 squares of red cell count area R as shown in the figure.

Figure IV.1- Improved Neubauer haemocytometer counting chamber ruling.

Figure IV.2- Counting chamber rulings and dimensions. Haemocytometer of counting

chamber has two ruled areas etched on its surface each consisting of a 3mm square
divided into nine large squares (W) each measuring 1mm2. Centre square, which is used
for red cell counting, is subdivided into 25 smaller squares (R), each occupying an area of
0.04mm2. Red cells in five R squares are enumerated. Depth of the chamber is 0.1mm.
The four large corner squares, each 1mm2 in area are subdivided into 16 smaller squares
and are used for leucocyte counting.

112
 Figure IV.3 Filling the Counting Chamber

Figure IV.4 - Improved Neubauer ruling for one counting chamber. White cell count is
done on the four large corner squares (1,2,3 and 4) of each of two counting chambers.
Red cell count is done on square 5 (A, B, C, D, and E) of each of two counting chambers.
Platelet count is done on two large corner squares (1 and 3) of each of two counting
chambers.

113
Calculation:

Each "R" section (Figure IV-1) has an area of 0.04mm2 and depth of 0.1mm. The
volume of 1 "R" is found as follows:

Area of 1 " R" X Depth of 1 " R" = Vol. of 1 " R"

0.04 mm 2 X 0.1 mm = 0.004 mm 3

Number of “R” sections X Vol. of 1 “R” = Total Vol.

5 X 00.004 = 0.02 mm3

Vol. Desired
Vol. Correction Factor =
Vol. Used

1.0mm3
Vol. Correction Factor = = 50
0.02mm3

Red Cell Count = No. of cells in 5 "R" X dil. factor X Vol. Correction
(Per cu.mm) = Number of cells X 200 X 50
= Number of cells X 10000

Expected Range:

Males = 4.5 - 5.5 x 106/cu.mm

Females = 4.0 - 5.0 x 106/cu.mm
Children = 4.2 - 5.2 x 106/cu.mm

Reagent:

Diluting Fluid
Trisodium citrate 3g
Concentrated formaldehyde (37%) 1ml
Distilled water 100ml

Red Cell Indices:

i. Mean Cell Volume (MCV)

The mean cell volume is the volume of average erythrocyte.

Normal range: 76-96 fl

PCV x 10
MCV =
RBC million

ii. Mean Cell Haemoglobin (MCH)

The mean cell haemoglobin is the weight of haemoglobin in average erythrocyte.

Normal range: 27 - 32 pg

Hb x 10
MCH =
RBC million

114
 iii. Mean Cell Haemoglobin Concentration (MCHC)

The mean cell haemoglobin concentration is the haemoglobin concentration of the

average erythrocyte.

Normal range: 30% - 36%

Hbx100
MCHC =
PCV

2. White Blood Cell (WBC) Count

Measurement of the total number of circulating white blood cells (WBCs) is an important
procedure in the diagnosis and prognosis of the disease process. The main function of
leucocytes is to fight infection by phagocytosis and production of antibodies.

Since leucocytes are affected by so many diseases, the leucocyte count serves as a useful
guide to the severity of the disease process. The increase of white blood cells above
10x109/L is called leucocytosis and the decrease of white blood cells below 4X109/L is
called leucopenia.

Procedure:

a) Add 50ul of whole blood sample to 0.95ml of diluting fluid into a labelled test tube.
If you use white cell pipet, draw blood to 0.5 mark and continue with the diluting
fluid to 11 mark.

b) Shake well (The dilution of the blood is 1 in 20).

c) Prepare the counting chamber and attach the cover glass by pressing it carefully into
place.

d) Discard first 4 drops if you are using white cell pipet.

e) Fill the counting chamber (take care not to overfill beyond the ruled area).

g) Allow the cells to settle for 3 minutes.

h) Place the chamber on the stage of the microscope, use the 10X objective, and
reduce the light by lowering the condenser.

i) Count WBC in 4 big (W) squares at the corners of the counting chamber see (Figure
IV.2).

115
Making the Calculations

Each "W" section (Fig.IV-2) has an area of 1mm2 and depth of 0.1mm.

The volume of "W" = 1mm2 X 0.1 = 0.1cu.mm

The volume = number of "W" X volume of "W"

1cu.mm
Vol. Correction Factor = = 2.5
0.4cu.mm

Number of WBCs = Number of cells in 4 "W" (0.4cu.mm) x dilution

Fac. X correction factor.

Number of WBCs = Number of cells in 4 "W" (0.4cu.mm) x 20 x 2.5

Reagents

Diluting Fluid:
Acetic Acid, glacial 4 ml
Distilled Water 200 ml
Aqueous methylene blue (0.3gm%) 20 drops

Expected Range:

4 - 10 X 103/cu.mm

Common Sources of Error:

a) Failure to have required blood volume.

b) Failure to mix well.

c) Failure to discard the first 4 drops.

d) Failure to properly charge the counting chamber.

The least Frequent Sources of Error are:

a) Inaccurate pipet or counting chamber.

b) Moist or unclean pipet.

c) Excessive pressure in finger when obtaining the blood.

d) Too little or too much diluting fluid.

e) Slowness in manipulation, thus allowing the blood to clot.

f) Air bubbles in the pipet.

g) Air bubbles in the counting chamber.

116
 h) Presence of yeast or other contaminants in the diluting fluid.

i) Presence of many nucleated red cells causing a high white cell count.

j) Mistakes in counting or calculations.

Correction for Nucleated Red Cells:

In some anaemia, such as thalassaemia and erythroblastosis fetalis, many nucleated red
may be found in the blood. Since these nucleated red cells are not dissolved by the white
cell diluting fluid, they are counted as white cells. This would give us an erroneously high
white cell count. Consequently, the count must be corrected.

If you find large numbers of nucleated cells in the stained red cell examination, correct
the white cell count with the formula given below:

100
Corrected WBC = Uncorrected WBC x
100 + A

Where:
100 = white cells counted in the differential white cell count.

A = Number of nucleated red cells counted while counting the 100 white cells of the
differential count.

Absolute Eosinphils Count:

Absolute Eosinphils Count can be roughly calculated from total and differential WBCs counts as
follows: If a patient with 9000 WBCs/cu.mm has 3 eosinophils per 100 WBCs from
differential count, the absolute eosinophils count will be

3 x 9000
Absolute Eosinphils Count = = 270 cu.mm
100

Reference range for Absolute Eosinophils Count is 40-350/cu.mm, it increases with

allergic and parasitic infections.

3. Platelet Counts:

Platelets are the smallest elements in the blood. These cells are nonnucleated, round or
oval shaped. Platelets activity is necessary for blood clotting. A deficiency of platelets
leads to prolonged bleeding time. The life span of a platelet is approximately 5-7 days.

Abnormally increased number of platelets occurs in cancer, splenectomy, iron deficiency

anaemia, and cirrhosis, while abnormally decreased number of platelets occur in
idiopathic thrombocytopenic purpura (ITP), pneumonia, allergic conditions, infection and
toxic effects of many drugs.

117
 Procedure

a) Pipette 0.95 ml of diluting fluid into a labelled test tube.

b) Add 50ul (0.05 ml) of the patient whole blood sample.

c) Shake well. The dilution of the blood is (1) in (20).

d) Fill one side of a Neubauer counting chamber and allow the platelets to settle for 10-
20 minutes in a moist Petri-dish.

e) Count the same area as for red cell count.

The platelets will appear as small refractile bodies under the x 40 objective.

Calculation

Platelet Count = No. of cells in 5 "R" X dil. Fac X Vol. Corr. (per cu.mm)
= No. of cells X 20 X 50

Expected Range

(150 – 400) x 103/cu.mm

Reagents

Diluting fluid (1% Ammonium Oxalate)

Ammonium oxalate 1g
Distilled water 100 ml

4. Reticulocytes Count

Principle

Reticulocytes are immature red cells that pass into the blood stream from the bone
marrow. The number of reticulocytes in the blood indicates the degree of activity of the
bone marrow. The number increases when the marrow is very active.

Method

a) Filter a little of the cresyl blue solution into a test tube.

b) Add equal quantity of venous blood collected in EDTA dipotassium salt.

c) Mix gently and leave for 10 minutes at 37oC or 15 minutes at room temperature.

d) Shake the tube and make a thin smear of the mixture.

e) Examine the smear using the oil-immersion objective.

f) Examine at least 1000 red cells

118
 g) Count the total number of red cells and the number of reticulocytes.

h) Calculate the percent of reticulocytes as follows:

Number of reticulocytes
x 100
1000

Note: In order to decrease the microscopic field and thus make it easier to count
the cells, place a piece of paper containing a "Window" in the eyepiece of
the microscope (Figure IV.5).

Figure IV.5

Reticulocytes are juvenile red cells that contain fine, deep violet granules
(remnants of the ribosomes and the ribonucleic acid present in the precursor cell)
arranged in a network.

Reference values

Adults & children = 0.2 - 2.0%

Infants = 2-6%

Reagent

(Saturated Solution of Brilliant Cresyl Blue)

Brilliant cresyl blue 1.0 g
Trisodium citrate 0.4 g
Sodium chloride solution (0.85%) 100 ml

Conditions accompanied with abnormal reticulocytes count:

Reticulocytosis: Reticulocytopenia:
Hereditary spherocytic anaemia Aplastic anaemia
Sickle cell anaemia Pernicious anaemia
Thalassaemia
Paroxysmal noctoral Hb-Uria
Acquired autoimmune Hem-anaemia
Acute posthemorrhagic anaemia

III. Blood Film Examination

1. Preparation of a thin blood film

a) Collect a drop of blood about 3-4 mm in diameter at one end of the slide.
b) Hold the slide with one hand. Using the other hand, place the edge of the spreader
just in front of the drop of the blood.
c) Draw the spreader back until it touches the drop of blood.
d) Let the blood run along the edge of the spreader.

119
 e) Push the spreader to the end of the slide with a smooth movement (all the blood
should be used up before you reach the end).
f) Check that the film is satisfactory:

- There should be no lines extending across or down through the film.

- The film must be smooth at the end, not ragged and lined.
- The film must not be too long.
- The film must not be too thick.
- The film must not contain holes because a greasy slide has been used.

(Table IV.2)
Common faults in preparing thin blood films
Fault Cause
The end of the film is lost The drop of the blood is too big
The film ends in a thick line The spreader has been lifted up too early
The end of the film is ragged The edge of the spreader is uneven
Lines along the film Blood is clotting when the film is made
Lines across the film The spreader was pushed forward jerkily
Holes in the film Greasy slide

2. Preparation of a thick blood film

a) Collect 3 drops of blood about 3-4 mm in diameter at the centre of the slide.
b) Using the corner of the spreader, quickly join the three drops of blood and spread
them to make an even, thick film. The blood should not be excessively stirred but can
be spread in a circular or rectangular form with 3-6 movements.

3. Staining of thin blood film with Wright’s stain

The stained thin blood films can be used to study the morphology of RBCs, WBCs and
platelets, besides the WBCs differential count.

Principle:

Wright’s stain is a methyl alcohol solution of an acid dye and a basic dye. The acid is
known as eosin, it is red in color. The basic is known as methylene blue, it is blue in
color.In the staining process, a buffer solution is used to control the acid-base balance of
the stain.

Procedure:

a) Prepare a thin blood film.

b) Fix the film by absolute methyl alcohol for 2-3 minutes. The smear will change from
red to light brown.
c) Completely cover the slide with stain. After 3 minutes, add an equal volume of buffer.
Blow gently to ensure uniform mixing. Agreen metalic sheen will appear.
d) After an additional 5 minutes rinse thoroughly with tap water. Wipe the back of the
slide to remove all traces of stain. Air dry the slide and add a drop of immersion oil.

120
e) Use the X40 & X100 objectives to examine the blood film for cells morphology and
for WBCs differential, see (Figures IV.6and 7).

Reagents

a) Wright’s stain solution Eosine 0.3% (W/V)

Methylene blue

b) Phosphate Buffer (pH 6.8) Na2 HPO4

KH2 PO4

Schematic drawing of a blood film made on a slide.

The film has been spread from left to right. An indication is given of the way the leucocytes are
distributed (see text).

Blood films made on slides.

Left: a well-made film. Left centre: a film which is too long. Too wide, grossly irregular in
thickness and which has been made on a greasy slide. Right centre: a film which is too thick.
Right: a film which has been spread with an irregularly-edged spreader and which sows long tails.

Figure IV.6

Method of examining the blood smear for the differential white cell count. When a blood
smear is made, the large cells tend to accumulate on the edge of the smear, whereas the
small cells tend to stay in the middle of the smear. If the cells are counted only on the
edges or only in the middle of the smear, it would not be a true representative sample of
the patient's cells. Therefore, the smear is examined by following the path of the arrow
shown above. In following the path of the arrow, note that you are moving toward the
thicker end of the smear. When you look in the microscope, however, it will appear that
you are moving in the opposite direction. As you move the smear, of course, the oil is
dragged along on the smear.

121
(Table IV.3)

Neutrophilic Segmented Cell Neutrophilic Band Cell

Size : Medium Size : Medium

Nucleus : Broken up into segments Nucleus : Shaped like a band

Cytoplasm : Contains small pink or brownish granules Cytoplasm : Contains small pink or brownish granules

Comments : May be confused with a neutrophilic band Comments : May be confused with neutrophilic
cell. When in doubt, call cell a neutrophilic segmented cell. When in doubt, call cell a
segmented cell. neutrophilic segmented cell.

When found : 55 to 75% in normal blood; increased in When found : 2 to 6% in normal blood; increased in
appendicitis, pneumonia. appendicitis, pneumonia.

Eosinophilic Segmented Cell Basophilic Segmented Cell

Size : Medium Size : Small or medium

Nucleus : Usually has 2 lobes or segments Nucleus : Usually indistinct; appears buried under
large purple or purplish-black granules.

Cytoplasm : Contains large red granules Cytoplasm : Contains large purple or purplish-black
granules

Comments : Eosinophilic segmented cell has large red Comments : Easily identified by the large purple or
granules whereas neutrophilic segmented purplish-black granules scattered through-
cell has small pink or brownish granules. out the cell.

When found : 1 to 3% in normal blood; increased in When found : 0 to 1% in normal blood.

asthma, hay fever, etc.

Lymphocyte Monocyte

Size : Small, medium, or large Size : Large

Nucleus : Closely knit and usually round Nucleus : Spongy and sprawling

Cytoplasm : Light blue; may contain a few reddish Cytoplasm : Light grey; may contain very tiny reddish
granules, cytoplasm may be sparse and granules
even absent in some small lymphocytes

Comments : Large lymphocyte may be confused with Comments : Monocyte may be confused with large
monocyte. Nucleus of large lymphocyte is lymphocyte. Nucleus of monocyte is
closely knit and usually round. Nucleus of spongy and sprawling. Nucleus of
monocyte is spongy and sprawling. lymphocyte is closely knit and usually
round
When found : 20 to 35% in normal blood; increased in When found : 2 to 6% in normal blood; increased in
infectious mononucleosis, lymphocytic tuberculosis and monocytic leukaemia.
leukaemia, and many other diseases.

122
Figure IV.7 - White Cells Found in a Normal Differential White Cell Count

123
IV. Haemoglobin Determination

1. Cyanmethaemoglobin Method

Haemoglobin is the main component of red cells. It is composed of two pairs of globin
chains and a haem compound which contains iron. The main function of haemoglobin is
to carry oxygen (O2) from the lungs to the body tissue cells and to transfer carbon
dioxide (CO2) from the tissue cells to the lungs. The oxygen combining capacity of the
blood is directly proportional to the haemoglobin concentration rather than to the RBCs
count. The haemoglobin determination is useful to screen and measure for the severity
of anaemia and to follow its response to treatment.

Principle:

The basis of the method is dilution of blood in a solution containing potassium cyanide
and potassium ferricyanide. Haemoglobin, Methaemoglobin and Carboxyhaemoglobin are
all converted to Cyanmethemoglobin. The absorbance of the solution is then measured in
a photoelectric colorimeter or spectrophotometer at a wavelength of 540nm.

Sample:

Blood may be taken directly from a finger (or heel) puncture without use of anticoagulant
or may be collected in a test tube containing Ethylene Diamine Tetra Acetate Tri-
Potassium (EDTA, K3) as an anticoagulant.

Method:

One reagent blank per series is required.

(Table IV.4)

Blank Standard Test (sample

Drabkin's solution 5ml 5ml 5ml

Haemoglobin standard - 20ul -
Sample (blood) - - 20ul
Mix thoroughly all blood specimens to be tested by repeated inversion immediately
before testing.

Mix and let stand for approximately 5 minutes to ensure complete reaction.

Read at 540nm (nano-meter) against blank.

Calculation:

Ab
Cb g/dl = x Cs
As

Cb = The concentration of Haemoglobin in a given sample.

Ab = The absorbance of the sample
As = The absorbance of the standard
Cs = The concentration of the standard.
Haemoglobin g/dl X 0.62 = Haemoglobin (Fe) mmol/L

124
Example:

Adjust the spectrophotometer to read 0.0 absorbance against blank in all the following
examples:

a) Absorption Mode:

For determination of Haemoglobin in an unknown sample, the following results were

obtained.

Concentration (Cs) of Haemoglobin standard = 15 g/dl

Absorbance of Haemoglobin standard (As) = 0.45
Absorbance of unknown (sample or control) (Ab) = 0.34

Ab
Concentration of unknown (g/dl) = x Cs
As
0.34
Concentration of unknown (g/dl) = x 15 = 11.3
0.45

b) Concentration Mode:

i. Press mode selector until the concentration lid is lit. Place the standard solution
in sample compartment.

ii. Using the Concentration/Factor Adjust Control, set the concentration value of the
standard (15g/dl) on the digital display.

iii. Insert the samples in the sample compartment and read results in the
concentration unit.

c) Factor Mode:

If the factor is already known for your test, you can enter this value by:

i. Pressing the mode until the factor lid is lit.

ii. Using the Concentration/Factor Adjust Control to set the display to the desired
factor value (33).

iii. Insert the samples in the sample compartment and read results directly in
concentration unit.

How to Calculate your Factor:

Ab
Cb = x Cs
As
Cs 15
Factor (F) = = = 33
As 0.45
Cb = F X Ab = 33 X 0.34 = 11.3 mg/dl

125
 Reference values:

Adult Male 13.5 – 18 g/dl

Adult Female 12 – 16 g/dl
Newborn 16 – 20 g/dl

Reagents:

a) Drabkin's Reagent
- Potassium Ferricyanide K3Fe (CN)6 = 0.2 g
- Potassium Cyanide KCN = 0.05 g
- Sodium Bicarbonate NaHCO3 = 1.0 g
Dissolve in succession in distilled water and dilute to 1000 ml.

b) Standard:

Cyanmethemoglobin standard: The concentration is usually given as g per dl.

Note: Cyanide is a well-known lethal chemical, therefore reasonable care must be

exercised in handling this solution.

2. Haemoglobinometer (HemoCue)

General:

The HEMOCUE B-Hemoglobin system consists of disposable microcuvettes with reagent

in dry form and a single purpose designed photometer. The microcuvette is used for
measuring the sample, as reaction vessel and a measuring cuvette. No dilution is
required.

Photometer, transformer (battery eliminator) and control cuvette are provided.

HEMOCUE could be operated using five batteries, type AA, inserted in the battery
compartment.

Capillary, venous or arterial whole blood may be used.

Procedure:

a) Put the switch at the back of the photometer to the position ”Power On”.

b) Pull the cuvette holder to insertion position. The display shows “Hb” and after 15
seconds “Ready” with three blinking dashes.

c) Insert the Control cuvette to verify that the calibration is stable.The obtained value
should not exceed the value printed on the cuvette +/- 0.3 g/dl.

d) Take the cuvette out of the container. Recap the container immediately.

e) Use only the middle or the ring finger for finger prick. Clean using a disinfectant
and allow to dry.

126
 f) Apply finger prick, wipe off the first 2-3 drops of blood.

g) Fill the cuvette completely in one continuous process. Wipe off the excess blood
from the outside of the cuvette. Inspect the filled cuvette for air bubbles inside the
internal circle of the cuvette that should be avoided.

h) Place the filled cuvette into the cuvette holder immediately and push it into the
measuring position.

Result will be displayed after 15-45 seconds.

Quality Control:

A QC chart should be established on monthly basis, for every HEMOCUE, and the user
should be trained on the issue. The control cuvette should be checked once every day,
before performing the first Hb test of that day. Obtained control result should be
plotted on the QC chart.

If the HEMOCUE will be operated using batteries, then the alkaline batteries are
recommended.

Cleaning / Care:

Cuvette holder : could be pulled out and cleaned using water and soap, dry well
before insert in place.
Control Cuvette : should be kept in the box and protected from dust and dirt. The
control cuvette may be cleaned using a 70-90 % ethanol without
any additives.

HEMOCUE could be gently wiped using a damp smooth cloth.

For more details, refer to the operating manual.

V. Haematocrit (Packed Cell Volume, PCV)

This is one of the simplest, most accurate and most valuable of all haematological
investigations.

By means of haematocrit, haemoglobin and red cell count the absolute indices can be
calculated.

High speed micro haematocrit centrifuge has become commercially available, it provides a
centrifugal force of about 12,000 g which gives a constant packed cell volume after a 3-minute
centrifugation.

Method:

a) Capillary or venous blood is drawn into 75mm long, 1.5mm bore heparinized capillary tubes
from finger, lobe of the ear or the heel (infants).

b) Seal the capillary tube at the end with a commercially available sealing compound or by
heating the end of the tube over a spirit lamp.

127
 c) Place the capillary tubes in the numbered slots in the centrifuge head, making sure that the
number of the slot corresponds with the specimen number.

The end of the sealed tube should point outward away from the centre.

d) Centrifuge for 3 minutes.

e) After centrifugation the tubes will show 3 layers:

i. Top : a column of plasma

ii. Middle : a thin layer of white cells.
iii. Bottom : a column of red cells.

f) Hold the tube against the scale of the reading device so that the bottom of the column of
red cells is aligned with the horizontal zero line.

g) Move the tube across the scale until the line marked 1.0 passes through the top of the
plasma column. Make sure that the tube is vertical.

h) The line that passes through the top of the column of red cells gives the Haematocrit value.

Results:

Before the introduction of SI units, the haematocrit (erythrocyte volume fraction = packed cell
volume, PCV) was reported as a percentage rather than a decimal fraction e.g. PCV = 45%.

In using SI units, the results become PCV = 0.45 (Vol. Fraction).

Expected Range:

Men 0.4 - 0.54 Volume fraction

Women 0.37 - 0.47 " "
Children (5) years 0.38 - 0.44 " "
Infants (3 months) 0.35 - 0.40 " "
Newborn 0.44 - 0.64 " "

VI. Erythrocyte Sedimentation Rate (ESR)

This test is based on the fact that inflammatory processes cause an alteration in blood proteins,
resulting in aggregation of red cells, which makes RBCs heavier and more likely to fall rapidly
when placed in a special vertical tube. The faster the sedimentation rate, the higher the ESR.
ESR is a non-specific test, because abnormal results indicate a pathological state rather than a
functional disturbance.

Principle:

The red cells settle to the bottom of a long graduated tube held in a vertical position leaving a
layer of plasma above. The height of the column of plasma after 1 hour indicates the
sedimentation rate of the erythrocytes (red cells).

128
Procedure:

a) Add 1.6ml of whole blood or collected with K2 EDTA to 0.4ml of the trisodium citrate
solution.

b) Mix and draw the citrated blood into the westergren tube up to the 0 mark.

c) Place the tube in the Westergren stand in an upright position.

d) Wait for one hour.

e) Read the height of the column of plasma in mm starting from the 0-mark.

The ESR increases with temperature above 23oC. Use the following chart for correction of ESR
reading:

Figure IV.8

Reference values:

Men 1 - 10mm/hr
Women 3 - 15mm/hr

129
 Reagent:

Trisodium Citrate 3.8%

Trisodium Citrate anhydrous 3.8 g
Distilled water 100 ml
Mix and keep in the refrigerator

Conditions Accompanied by Increased ESR:

Rheumatic fever Multiple myeloma Anaemia

Rheumatic arthritis Nephrosis Leukaemia
Coronary thrombosis Metallic poisoning Pregnancy
Pneumonia Syphilis Agranulocytosis
Nephritis Tuberculosis Menstruation
Cancer

Sources of Error in the Sedimentation Rate

a) Unclean sedimentation rate tubes: Dirt, water, alcohol, ether, etc., will cause haemolysis
and decrease the sedimentation rate.

b) Excessive anticoagulant: Excessive anticoagulant will decrease the sedimentation rate.

c) Partially clotted blood will decrease the sedimentation rate.

d) Old blood: The blood must be fresh; therefore it must be used within 2 hours after
withdrawal. As the blood stands, the red cells become more spherical and thus less
inclined to assume rouleau formation. This decrease in rouleau formation decreases the
sedimentation rate.

e) Failure to mix blood: The test tube containing the blood should be completely inverted 10
to 12 times before filling the sedimentation rate tube.

f) Use of cold unmixed blood.

g) Air bubbles in the column of blood.

h) Inclined sedimentation rate tube. The sedimentation rate tube must be placed in an exact
vertical position

VII. Clotting Time:

Clotting time is the time required for the solid clot to form. The basis for this test is that whole
blood will form a solid clot when exposed to a foreign surface such as a glass test tube.

As this test is the least effective test in the diagnosis of actual haemostatic failure, it has been
replaced by the partial thromboplastin time (PTT) test.

Principle:

A venous blood sample is collected in a glass tube. The time it takes for the blood to coagulate
(clot) is measured.

130
Method:

a) Collect 2ml venous blood using a plastic syringe.

b) Start the stop watch as soon as the blood enters the syringe.

c) Remove the needle from the syringe and fill each of two glass tubes with 1ml blood.

d) Plug the tubes and place them in a water bath at 37oC.

e) After about 3 minutes remove the 1st tube from the water bath. Tilt the tube to see
whether the blood has clotted.

f) If the blood has not clotted, return it to the water bath and examine it at 30 second
intervals.

g) After the blood in the first tube has clotted, examine the second tube immediately.

h) The coagulation time is reported as the clotting time of the second tube.

Results:

Report the clotting time in minutes to the nearest half minute.

Reference values:

5 - 12 minutes.

Conditions Accompanied by Increased Clotting Time:

Factor V deficiency Vitamin K deficiency

Factor VII deficiency Heparin therapy
Haemophilia (factor VIII deficiency) Dicumarol therapy
Factor IX deficiency(christmas disease) Presence of circulating antibodies
Factor XI deficiency Anaemia and Leukaemia
Factor XII deficiency Afibrinogenemia
Haemorrhagic disease of newborn Pneunomia

Sources of Error in the Clotting Time:

a) Dirty test tubes

b) Tissue juices mixed with blood.

c) Air bubbles in the blood. This may be caused by a faulty venipuncture. (Either failure to
have the needle completely in the vein or having needle loosely attached to the syringe).

d) Excessive agitation of blood. This may occur during the transfer of the blood from the
syringe to the test tube. The blood should be allowed to flow gently down the inside of the
test tube and not forcefully squirted into the test tube.

131
 Coagulation will be Retarded by the Following:

a) Temperature below 35oC.

b) Temperature above 45oC.
c) Other factors affecting coagulation are: Diameter of the test tube. The smaller the
diameter, the more rapid the clot formation is, all test tubes should be the same diameter.

VIII. Bleeding Time:

Bleeding time measures the primary phase of haemostasis, the interaction of the platelets with
the blood vessel wall and the formation of the haemostatic plug.

This test is of significant value in detecting vascular abnormalities and of moderate value in
detecting platelet abnormalities or deficiencies.

Principle:

A small cut is made with a lancet in the lobe of the ear. The time takes the blood flow from the
puncture to stop is measured and called "Bleeding Time".

Method:

a) Gently clean the lobe of the ear with alcohol swab.

b) Puncture the lobe of the ear deeply with a sterile lancet.
c) Start the stop-watch.
d) Leave the blood to flow freely without need to squeeze the ear lobe.
e) Blot the blood drop with a filter paper.
f) Continue blotting the blood until no more blood appears.
g) Stop the stopwatch and note the time.

Result:

Report the bleeding time to the nearest half minute.

Reference values:

1 - 5 minutes.

IX. Sickle Cell Test:

The purpose of this test is to detect sickle cell disorder (Anaemia or Trait). Sickle cell anaemia
is caused by an abnormal form of haemoglobin known as haemoglobin-S, which tends to
precipitate in such a way that the red cell takes the sickling shape.

Principle:

One drop of blood is mixed with one drop of a sodium metabisulfite reagent on a slide. If the
red cells contain an abnormal haemoglobin, they will become sickle-shaped (or half-moon
shape). The reagent sodium metabisulfite removes oxygen from the cells, allowing sickling to
take place.

132
 Method:

a) Place a small drop of capillary blood in the centre of a slide.

b) Add an equal drop of the fresh sodium metabisulfite solution.
c) Mix carefully and cover with a cover slide, make sure that no air bubbles form. Seal with
Vaseline or DPX.
d) Place the slide in a wet chamber.
e) Examine under the microscope after 15 minutes using the X40 objective. If the result is
negative, re-examine after one hour, then after a further hour and after 24 hours.
f) The test is negative if the red cells remain round.
g) The test is positive if the cells become sickle-shaped, or banana-shaped, often with spikes.

Reagents:

Sodium metabisulfite 2% Aqueous Solution:

Sodium metabisulfite (Na2S2O5) 0.2g
Distilled water 10ml
Always should be freshly prepared before use.

X. ABO and Rh-Grouping

1. Slide Method

The slide method is only a little less satisfactory than the tube method. Agglutination is
rapid on flat slide; this method is useful when only one or two samples of blood are to be
tested.

Grouping Sera should be obtained ready for use from a known source and should be
stored according to the recommendations of the manufacturer. They must be kept free
from bacterial growth to avoid non-specific false-positive results.

Procedure:

a) Put one drop of the patient's whole blood to each labelled square on a special slide
(A, B, AB and D).

b) Add one drop of each grouping anti-serum (anti-A, anti-B, anti-AB and anti-D)
respectively.

c) Mix the blood and the anti-serum in each square with a wooden-stick.

d) The results may be read within 1-5 minutes.

e) Presence of agglutination means a positive reaction.

f) Doubtful presence or absence of agglutination may be checked by viewing under the

microscope.

133
2. Tube Method:

Principle

ABO grouping by the test tube gives more reliable results than the slide method. All
doubtful cases or results that are difficult to read by slide method must be checked by the
test tube method.

Procedure

a) Wash a small quantity of blood 3 times by using normal saline.

b) Suspend 3 drops of red cells deposit in 5ml of normal saline (the result is 3%
suspension of red cells).

c) Label 3 tubes A, B and D and add the reagents according to (Table IV.5)

(Table IV.5)

Tube # A Tube # B Tube # D

Anti-serum One drop One drop One drop

(Anti-A) (Anti-B) (Anti-D)
3% red cells suspension One drop One drop One drop

Shake and let the tubes to stand for 10 minutes at room temp.
Centrifuge for 1 minute at low speed (1000 rmp).
Shake the bottom of the tube, examine the deposit of red cells.

Results:

Positive Agglutination (+):

The red cells form one or more clumps with a clear supernatant fluid.

Negative Agglutination (-):

The red cells resuspend easily without any visible clumps.

3. Technical Errors:

The first step in resolving a discrepancy is a careful repeat of the entire test procedure,
paying careful attention to details possible pitfalls are list below:

a) Partial drying of the slide in cell groupings may be misinterpreted as agglutination.

b) Overcentrifugation or undercentrifugation may result in false positive or false

negative readings respectively.

c) Rough dislodgement of the centrifuged cell button may disrupt small agglutination
and result in false negative reading.

134
 d) The cell-serum mixture may be inadvertently heated, resulting in false negative
reading.

e) The use of improper concentration of cells or old cells may lead to a false negative
reading.

f) Failure to observe haemolysis will result in false negative readings.

g) Dirty glassware will simulate clumping and result a false positive reading.

h) The specimen may be identified incorrectly.

XI. Direct Antiglobulin Test (Direct Coombs Test)

Procedure:

a) Prepare a 2-4% suspension in normal saline of the cells to be tested.

b) Add 2 drops of the prepared suspension of cells to a tube marked “test” and 2 drops to
a tube marked “auto control”.

c) Wash cells 3 times by filling both tubes with fresh normal saline and centrifuging
according to calibration of centrifuge for cells washing. Decant supernatant completely
after each washing (Cord blood specimens must be washed a minimum of six times).

d) After the third washing add two drops of coombs anti-human serum to the dry cell
button in the tube marked “test”. Do not resuspend the cells in the “test” tube with
saline before adding the Coombs serum.

e) Add two drops of saline to the tube marked auto control.

f) Resuspend the cell buttons in both tubes with gentle shaking and centrifuge according
to calibration of centrifuge for the antiglobulin technique.

g) Read macroscopically by holding the tubes to a light source and gently a gitating them.
Examine for agglutination. Read microscopically only if results are macroscopically
doubtful.

XII. Indirect Antiglobulin Test (Indirect Coombs)

Procedure:

a) Label three test tubes as 1, 2 and 3.

b) Add 2 drops of patient’s serum to each tube.

c) To tube No. 1 add two drops of screening cells No. 1.

To tube No. 2 add two drops of screening cells No. 2.
To tube No. 3 add two drops of screening cells No. 3.

d) Add to each tube 2 drops of 22% bovine serum albumin.

135
 e) Spin, read macroscopically.

f) Incubate at 36oC for 15 - 40 minutes.

g) Spin, read macroscopically, if no agglutination go to next step.

h) Wash 3 times.

i) Add to each tube 2 drops of Anti Human Globulin.

j) Spin, read macroscopically & microscopically.

Any agglutination or haemolysis detected at any step, means Indirect Doomb’s is
positive.

XIII. References:

1. M.J. Lynch, S.S. Raphael, L.D. Mellor, P.D. Spare & M.J.H. Inwood.: Medical Laboratory
Technology & Clinical Laboratory, 2nd edition the W.B. Saunders Co. 1969.

2. Dacie, J.V. and Lewis, S.M. Practical Haematology, 7th edition, .A. Churchil, Ltd., London,
1991.

3. Manual of Basic Techniques for a Health Laboraoty, WHO, Geneva, 2nd edition, 2003.

136
137
138
 PART V – BIOCHEMISTRY

I. Glucose
1. Glucose by Blood Glucose Meter
2. Glucose by GOD – PAP Method
3. Oral Glucose Tolerance Test (OGTT)

II. Urea

III. Creatinine
(Jaffe Method with Deproteinization)

IV. Creatinine
(Without Deproteinization)

V. Uric Acid

VI. Cholesterol

VII. HDL-Cholesterol
(Precipitation Method)

VIII. HDL-Cholesterol
(Direct Enzymatic Colorimetric Method)

IX. Triglycerides

X. Bilirubin Direct & Total

XI. Total Protein

XII. Albumin

XIII. AST (SGOT)

XII. ALT (SGPT)

XIII. Akaline phosphatase

139
140
I. Glucose

1. Glucose by Blood Glucose Meters

It is a quick and simple technique for determination of blood glucose by blood glucose
meters, please follow the manufacturer's instructions for measurement procedure.

2. Glucose by GOD - PAP Method

Principle:

The glucose is determined after enzymetic oxidation in the presence of glucose oxidase
enzyme (GOD). The formed hydrogen peroxide reacts under catalysis of peroxidase with
phenol and 4-aminophenazone to a red-violet quinoneimine dye as indicator. The
increase of absorbance at 500 nm is directly proportional to the glucose cocentration in
the sample.

Glucose + O2 + 2H2O ⎯ ⎯
⎯→ Gluconate + 2H2O2
GOD

2H2O2 + 4 - aminophenazone + phenol ⎯ ⎯

⎯→ quinoneimine + 4H2O
POD

(Coloured complex)

Clinical Significance:

The detection of glucose in body fluids is important in the diagnosis of diabetes and in the
investigation of hypoglycaemia.

Sample:

Serum, plasma
The glucose is stable for 24 hours at 2-8 °C, if serum or plasma is separated within 30
min.

Procedure: (HUMAN kit Cat. No. 10260)

Method without deproteinization

One reagent blank per series is required.

(Table V.1)
Reagent Blank Sample or Standard

Sample --- 10ul

Reagent 1 1ml 1ml

Mix and incubate for 10 min. at 20-25 °C or 5 min. at 37 °C. Measure the absorbance of
the standard and the sample against the reagent blank at 500 nm within 60 minutes.

141
Calculation:

Ab
Cb (mg / dl) = x Cs
As
Cb = the concentration of glucose in a given sample.
Cs = the concentration of glucose in a given standard.
(mg/dl x 0.0555 = mmol/L)

Adjust the spectrophotometer to read 0.0 absorbance against blank in all calculation
modes.

a) Absorption Mode:

For determination of glucose in an unknown sample, the following results were

obtained:

Concentration (Cs) of glucose standard = 100 mg/dl

Reading (As) of glucose standard = 0.360

Reading (Ab) of unknown (sample or control) = 0.270

AbxCs
Cb (mg / dl) =
As

0.270x100
Concentration of unknown (mg / dl) = = 75
0.360

b) Concentration Mode:

i. Press mode selector until the concentration lid is lit. Place the standard solution in
sample compartment.

ii. Using the Concentration/Factor Adjust Control, set the concentration value of
standard (100 mg/dl) on the digital display.

iii. Insert samples in the sample compartment and read results directly in
concentration unit.

c) Factor Mode:

If the factor is already known for your test, you can enter this value by:

i. Pressing the mode until the factor lid is lit.

ii. Using the Concentration/Factor mode adjust control to set the display to the
desired factor value (278).

iii. Insert samples in the sample compartment and read results directly in
concentration unit.

142
 How to calculate your Factor:

Ab
Cb = x Cs
As
Cs 100
Factor (F) = = = 278
As 0.360

Cb = Ab x F = 0.270 x 278 = 75 mg/dl

Reference values:

(70 - 110 mg/dl) or (3.9 - 6.1 mmol/L)

Reagents:

a) Reagent 1:

Phosphate buffer (pH 7.5) 0.1 mol/l

4-aminophenazone 0.25 mmol/l
Phenol 0.75 mmol/l
Glucose oxidase >15 KU/l
Peroxidase >1.5 KU/l
Mutarotase >2.0 KU/l
Stabilizers

b) Glucose standard 100 mg/dl or (5.55 mmol/l)

Quality Control:

Each batch of specimens should include at least two serum control specimens having
stated values in the range 45-450 mg/dl one of which is unknown to the operator. An
Optimal Coefficient Variance (OCV) of around 3% should be obtainable. Routine
coefficient of variance should not exceed 6%.

3. Oral Glucose Tolerance Test (OGTT):

Procedure for confirmation of diabetes:

a) The patient should adhere to his usual diet in the 3 days prior to the test.

b) Water intake is not restricted during the overnight fast, but smoking is not permitted.

c) A fasting blood sample is taken after an overnight fast of 10-12 hours.

d) 75 g of glucose in 250 ml of water should be administered orally after an overnight

fasting. If OGTT is performed in children, 1.75g of glucose per kg body weight is
given, up to a total of 75g.

e) A blood sample should be collected 2 hours after the oral glucose load. OGTT is
recommended for diagnostic purposes as well as for epidemiological studies. If
nausea, fainting, sweating, or other autonomic nervous system over activity occurs,
a specimen for glucose should be drawn immediately and the procedure
discontinued and repeated at a later date if indicated.

143
II. UREA (Enzymatic colorimetric method)

Principle:

Urea is hydrolysed in the presence of water and urease to produce ammonia and carbon
dioxide. In a modified Berthelot reaction the ammonium ions react with hypochlorite and
salicylate to give a green dye. The increase of absorbance at 578 nm is proportional to the urea
concentration in the sample.

Clinical Significance:

The determination of serum urea is presently the most widely used screening test for the
evaluation of kidney function. The test is frequently requested along with the serum creatinine
test since simultaneous determination of these two compounds appears to aid in the differential
diagnosis of prerenal, renal and postrenal hyperuremia. Hyperuremia may also indicate liver
disease or dehydration.

Sample:

Serum, plasma (all anticoagulants except ammonium heparin can be used) or diluted urine
1+99 with distilled water.
Do not use lipemic sera. Serum or plasma can be stored for up to 3 days at 4 °C.

Procedure: (HUMAN kit Cat. No. 10505)

One reagent blank per series is required.

(Table V.2)
Reagent Blank Sample or Standard
Sample or standard --- 10 ul
Reagent 1 1 ml 1 ml
Mix and incubate for 5 min. at 20 - 25 °C or 3 min. at 37 °C
Reagent 2 1 ml 1 ml
Mix, incubate for 10 min. at 20 - 25 °C or for 5 min. at 37 °C. Measure the absorbance of the
sample (Ab) and the standard (As) against the reagent blank within 60 min. at 578 nm.

Calculation:

Ab
Cb (mg / dl) = x Cs
As

Cb = the concentration of urea in a given sample.

Cs = the concentration of urea in the standard solution.
(mg/dl x 0.166 = mmol/L)

a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.

144
 b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

15 - 50 mg/dl
(2.5 - 8.3 mmol/L)

Reagents:

a) Reagent 1:
Phosphate buffer 120 mmol/l
Sodium salicylate 60 mmol/l
Sodium nitroprusside 5 mmol/l
EDTA 1 mol/l
Urease > 5 KU/l

b) Reagent 2:
Phosphate buffer 120 mmol/l
Sodium hydroxide 400 mmol/l
Sodium hypochlorite 10 mmol/l
Irritates eyes and skin. Upon contact with the eyes, rinse thoroughly with water and
consult a doctor.

c) Reagent 3:
Urea 80 mg/dl or 13.30 mmol/l
BUN 37.28 mg/dl
Sodium azide 0.1 %

Quality Control:

The following variances should not be exceeded:

- OCV within 4%
- RCV not exceeding 8%.

III. Creatinine (Jaffe method with deproteinization)

Principle:

Creatinine forms a coloured complex with picric acid in alkaline medium. The rate of formation
of the creatinine-alkaline picrate complex is proportional to the concentration of creatinine in
the specimen. The amount of complex formed is determined spectrophotometrically at 520nm.

Alkali
Creatinine + picric acid ⎯ ⎯⎯→ coloured complex

145
Clinical Significance:

Determination of blood creatinine particularly when made in connection with measurement of

blood urea is of value in assessment of kidney function.

Sample:

Serum, Heparinized plasma, Diluted urine (1+49)

Samples are stable for 24 hours at 2-8 °C

Procedure: (HUMAN kit Cat. No. 10051)

Deproteinization:

Pipette into centrifuge tubes

a) Trichloroacetic acid 0.5 ml

b) Sample or standard 0.5 ml
c) Mix well, centrifuge for 10 minutes.

One reagent blank per series is required.

(Table V.3)
Reagent Blank Sample or Standard
Distilled water 0.5 ml --
Supernatant -- 0.5 ml
Working reagent 0.5 ml 0.5 ml
Mix, incubate for exacty 20 min. at 20-25 °C and read the absorbance of the standard (As)
and sample (Ab) against reagent blank at 520 nm.

Calculation:
Ab
Cb (mg / dl) = x Cs
As

Cb = the concentration of creatinine in a given sample.

Cs = the concentration of creatinine in a given standard
Conversion factor: mg/dl x 88.4 = umol/l or mg/dl x 0.0884 = mmol/l

a) Bsorption Mode
Same procedure as for glucose by GOD-PAP Method.

b) Oncentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

Adult 0.6 - 1.3 mg/dl 53 - 115 umol/L

Children 0.3 - 0.6 mg/dl 26 - 53 umol/L

146
 Reagents:

a) Reagent (1) Creatinine Standard 2mg/dl (177 umol/L)

b) Reagent (2) Picric Acid 35 mmol/L
c) Reagent (3) Sodium Hydroxide 1.6 mol/L (6.4%)
d) Trichloro Acetic Acid 1.2 mol/L (19.6%)

Working Reagent:

Prepare a 1+1 mixture of Reagents (2) and (3), stable for five hours at room temperature in a
dark place or a bottle.

Creatinine clearance:

mg creatinine / dl urine x ml urine / 24hrs

Creatinine clearance (ml / min) =
mg creatinine / dl blood x 1440

Reference values:

Men 98-156 ml/min.

Women 95-160 ml/min.

IV. Creatinine (Jaffe method without deproteinization).

Method:

Creatinine forms, in alkaline solution, an orange-red coloured complex with picric acid. The
creatinine concentration is determined by a fixed time kinetic measurement.

Principle:

Creatinine + picric acid → Creatinine-picrate-complex

Contents, reagent composition

1. 1 x 100 ml Picric acid 35 mmol/l

2. 1 x 100 ml Sodium Hydroxide 1.6 mol/l
3. 1 x 25 ml Creatinine standard 2 mg/dl or 176.8 µmol/l

Reagent Preparation:

- Dilute sodium hydroxide (bottle 2) with dist. water in the ratio 1 + 4. Store the solution in a
plastic bottle.
- Mix picric acid (bottle 1) and diluted sodium hydroxide in the ratio 1 + 1.
- The standard is ready for use.

Reagent stability:

The reagents/diluted sodium hydroxide are stable up to the state expiry date when stored at
15-25°C. The combined working reagent is stable for 32 hours at (15-25°C).

147
Specimen:

Serum, heparinized plasma and urine.

Avoid hemolysis!
Stability: 24 hours at 2-8°C.
Dilute urine 1 + 49 with dist. water.

Assay:

Wavelength : Hg 492 nm (490-510 nm)

Optical path : 1 cm
Temperature : 25°C
Measurement : Against air (increasing absorbance)
Warm the reagents and cuvettes up to 25°C. Temperature must be kept constant (± 0.5°C) for
the duration of the test.

Pipetting scheme:

(Table V.4)
Pipetting into Cuvettes Macro Semi Micro
Serum / Plasma / Dil. Urine
200 µl - 100 µl -
(1 + 49)
Standard - 200 µl - 100 µl
Working Reagents 2 ml 2 ml 1 ml 1 ml
Mix and start the stop-watch. After 30 seconds read the absorbance A1. Read the
absorbance A2 after exactly 2 minutes.
A2 − A1 = ∆A (sample) or ∆A (standard)

Calculation:

1. Serum/Plasma

∆A (sample) ∆A (sample)
C = 2.0 (mg/dl) or C = 176.8 × (µmol/l)
∆A (standard) ∆A (standard)

2. Urine:

∆A (sample)
C = 100 (mg/dl) or
∆A (standard)

Creatinine concentration in 24h urine:

C= mg/dl x ml urine/24h x 0.01 (mg/24h)
C= mg/24h x 0.00884 (mmol/24h)

mg creatinine/dl urine x ml urine/24

Creatinine Clearance = (ml/min)
mg creatinine/dl serum x 1440

Conversion of (mg/dl) into (µmol/l) and vice versa:

(mg/dl) x 88.402 = (µmol/l)
(µmol/l) x 0.0113 = (mg/dl)

148
Linearity:

The test is linear up to a creatinine concentration of (13 mg/dl or 1150 µmol/l), in urine (500
mg/dl or 44200 µmol/l. Dilute samples with a higher concentration in serum, plasma or diluted
urine 1 + 5 with physiological saline (0.9%) and repeat the assay. Multiply the result by 6.

Reference values 3, 4:

(Table V.5)
Serum (mg/dl) (µmol/l)
Men 0.6 – 1.1 53 – 97
Women 0.5 – 0.9 44 – 80
Urine 1000 – 1500 mg/24 hours
Creatinine clearance:
Men 98 – 156 ml/min.
Women 95 – 160 ml/min.

Quality Control:

All control sera with creatinine values determined by this method can be employed.

Automation:

Special adaptations for automatic analyzers should be made.

Notes:

1. The reaction is highly sensitive to temperature. The reaction temperature must be kept
constant.

2. Picric acid is poisonous when inhaled, swallowed or in contact with the skin. If the picric
acid comes into contact with the skin or mucous membranes wash with polyethylenglycol
400, in emergency with plenty of water.

3. Sodium hydroxides causes strong corrosion. If the sodium hydroxide comes into contact
with the skin or mucous membranes wash copiously with water. Wash splashes in the
eyes with plenty of water and consult the ophthalmologist.

4. The assay can be affected by the presence of reducing compounds. The interference can
be partially eliminated by boiling the urine for a short time.

5. A slight precipitant in the sodium hydroxide is insignificant.

149
V. Uric Acid (Enzymatic colorimetric method)

Principle:

Uric acid is oxidized in the presence of uricase which is used to improve specificity. The
hydrogen peroxide produced reacts with 3.5-dichloro-2-hydroxybenzene sulfonic acid (DCHBS)
and 4-aminophenazone in the presence of catalase to produce a red-violet quinoneimine
complex. The increase in absorbance at 520 nm produced by quinoneimine production is
proportional to the amount of uric acid in the sample.

Uricase
Uric acid + O2 + 2H2O ⎯ ⎯⎯
⎯→ Allantoin + CO2 + H2O2

H2O2 + DCHBS + 4-aminophenazone

¯ Peroxidase

N (4-antipyryl)-3Chloro-5-Sulfonate-p-benzo-quinoneimine+HCL+4H2O

Clinical Significance:

Determination of uric acid is of great value in the diagnosis of gout and in the assessment of
renal function.

Sample:

Serum, heparinized or EDTA plasma, diluted urine (1:10). Uric acid is stable in refrigerated
specimens for three days and frozen for six months.

Procedure: (HUMAN kit Cat. No. 10037)

One reagent blank per series is required.

(Table V.6)
Reagent Blank Sample or Standard

Sample -- 20 ul

Working reagent 1 ml 1 ml
Mix, incubate for 15 min. at 20 - 25 °C or 7 min. at 37 °C. Measure the absorbance of the
sample (Ab) and the standard (As) against the reagent blank within 30 min. at 520 nm.

Calculation:
Ab
Cb (mg / dl) = x Cs
As

Cb = the concentration of uric acid in a given sample.

Cs = the concentration of uric acid in a given standard.
(mg/dl x 59.5 = mmol/L)

a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.

150
 b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

Men 3.7 - 7.8 mg/dl Women 2.7 - 7.3 mg/dl

226 - 468 umol/L 160 - 430 umol/L

Reagents:

a) Buffer solution (1)

Phosphate buffer pH 7.0 50 mmol/L
DCHBS 0.3 mmol/L

b. Enzyme reagent (2)

4-aminophenazone 0.3 mmol/L
peroxidase 1000 u/L
uricase 200 u/L

Preparation of working reagent:

Reconstitute the contents of vial-2 with 15ml buffer from bottle-1 (stable for 21 days at 2-
8°C).

c. Uric acid standard (3) 8 mg/dl (476 umol/L).

VI. Cholesterol (Enzymatic colorimetric method)

Principle:

Cholesterol is determined after enzymatic hydrolysis by cholesterol esterase and oxidation by

cholesterol oxidase. The indicator quinoneimine is formed from hydrogen peroxide and 4-
aminoatipyrine in the presence of phenol and peroxidase.

Cholesterolester + H2O ⎯ ⎯
⎯→ Cholesterol + fatty acids
CHE

Cholesterol + O2 ⎯ ⎯
⎯→ Cholestene-3-one + H2O2
CHO

2 H2O2 + 4-aminoantipyrine + phenol ⎯ ⎯

⎯→ quinonemine + 4H2O
POD

Clinical Significance:

The level of cholesterol in the blood plasma or serum reflects the concentrations of the
lipoproteins. The HDL and LDL are the cholesterol-rich lipoprotein fractions. There is an inverse
relationship exists between the plasma levels of thyroxin and cholesterol. Diabetes Mellitus is
associated with hypercholesterolemia and hypertiriglyceridemia, the more uncontrolled the
diabetes, the greater the elevation of lipids. There is a statistically significant correlation
between high serum cholesterol level and the incidence of coronary artery disease,
atherosclerosis and heart disease.

151
Sample:

Serum, heparinized or EDTA plasma.

Procedure: (HUMAN kit Cat. No. 10017)

One reagent blank per series is required.

(Table V.7)
Reagent Blank Sample or Standard

Sample / standard -- 10 µl

Reagent (1) 1 ml 1 ml
Mix, incubate for 20 min. at 20 - 25 °C or 10 min. at 37 °C. Measure the absorbance of the
sample (Ab) and the standard (As) against the reagent blank within 60 min. at 500 nm.

Calculation
Ab
Cb (mg / dl) = x Cs
As
Cb = the concentration of cholesterol in a given sample.
Cs = the concentration of cholesterol in a given standard.
(mg/dl X 0.0258 = mmol/L)

a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.

b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

Child 120 - 200 mg/dl (3.1 - 5.2 mmol/L)

Adolescent 120 - 210 mg/dl (3.1 - 5.4 mmol/L)
Adult 150 - 250 mg/dl (3.9 - 6.5 mmol/L)

Reagents:

a) Buffer solution (1)

Phosphate buffer (pH 6.5) 30 mmol/l
4-Aminoantipyrine 0.25 mmol/l
Phenol 25 mmol/l
Peroxidase > 5 KU/l
Cholesterolesterase >150 U/l
Cholesteroloxidase >100 U/l
Sodium azide 0.05 %

b) Cholesterol standard 200 mg/dl

152
 Quality Control:

The following variance should not be exceeded.

- OCV within 7%
- RCV within 14%

VII. HDL – Cholesterol (Precipitation Method)

Principle:

Chylomicron, VLDL (very low density lipoproteins), and LDL (low density lipoproteins) are
precipitated by addition of phosphotungstic acid and magnesium chloride to the sample. After
centrifugation the supernatant fluid contains only the HDL (high density lipoproteins) -fraction,
which is assayed for HDL Cholesterol with HUMAN kit Cat. No. 10017.

Sample:

Serum, heparinized or EDTA plasma.

Procedure: (HUMAN kit Cat. No. 10018)

a) Precipitation

i. Pipette into centrifuge tube

500 ul of patient's sample
1000 ul of undiluted precipitant

ii. Mix and let stand for 10 minutes at room temperature.

iii. Centrifuge for 10 minutes at 4000 rpm.

b) Determination of HDL-Cholesterol

i. Separate the clear supernatant from the precipitate within 1 hour.

ii. Determine the cholesterol content with HUMAN kit Cat. No. 10017.

Calculation of HDL – Cholesterol:

Ab
Concentration of HDL - Cholesterol (mg / dl) = x Cs x F
As

Ab = Absorbance of supernatant solution

As = Absorbance of standard solution
Cs = Concentration of a given standard
F = Dilution Factor (=3)
(mg/dl x 0.0258 = mmol/L)

153
 a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.

b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Calculation of LDL Cholesterol:

Triglycerides
(mg / dl) LDL - Cholesterol = Total - Cholesterol - - HDL - Cholesterol
5

Triglycerides
(mmol / l) LDL - Cholesterol = Total Cholesterol - - HDL - Cholesterol
2.2

The values obtained are reliable, provided that:

- No chylomicron are present in the sample.
- The triglycerides concentration does not exceed 400mg/dl or 4.5 mmol/L.

Reference values:

a) HDL - Cholesterol:

Men 35-60 mg/dl (0.97-1.55 mmol/L)

Women 45-70 mg/dl (1.18 - 1.92 mmol/L)

b) LDL - Cholesterol:

< 150 mg/dl (< 3.88 mmol/L)

Reagents:

Precipitant:
Phosphotungstic acid 0.55 mmol/L
Magnesium chloride 25 mmol/L
Store at 15 to 25°C.

VIII. HDL – Cholesterol (Direct Enzymatic Colorimetric Method)

Intended Use:

HUMAN’s HDL Cholesterol direct is a homogeneous enzymatic assay for the quantitative
determination of HDL cholesterol (HDL). HDL is regarded as a protecting lipid component
against coronary heart disease (CHD). Together with LDL cholesterol (calculated by Friedewald
formula) it is of diagnostic importance to estimate the individual risk for CHD.

154
Method:

The assay combines two specific steps: in the 1st step chylomicrons, VLDL and LDL cholesterol
are specifically eliminated and destroyed by enzymatic reactions. In the 2nd step remaining
cholesterol from the HDL fraction is determined by well established specific enzymatic reactions
in the presence of specific surfactants for HDL.

Reactions Principle:

1st Step:

+CHO
LDL, VLDL,
⎯CHE
⎯⎯ ⎯→
Specific conditions Cholestenone + H2O2
and Chylomicrons

2 H2O2 ⎯Catalase
⎯⎯ ⎯→ 2 H2O + O2

2nd Step:

+CHO
⎯CHE
⎯⎯ ⎯→
HDL Specific conditions Cholestenone + H2O2

H2O2 + chromogen ⎯Catalase

⎯⎯ ⎯→ quinine pigment

Contents, Reagent Composition in the Test:

(Table V.8)
Enzymes 1 X 60 ML Enzymes (white cap)
Good’s buffer, pH 7.0 (20°C) 100 mmol/l
Colesterol esterase 600 U/l
Cholesterol oxidase 380 U/l
Catalase 600 U/ml
N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline
(HDAOS) 0.42 mmol/l

Substrate 1 x 20 ml Substrate (green cap)

Peroxidase 1000 U/l
4-Aminoantipyrin (4-AA) 1.00 mmol/l
Good’s buffer, pH 7.0 (20°C) 100 mmol/l
Sodium azide 0.05%
Detergents > 1%

Calibrator 1 x 4 ml Calibrator
Cholesterol Concentration see vial label

Reagent preparation and Stability:

Enzymes and Substrate are ready for use.

Stability: after opening the reagents are stable up to 1 month when stored at 2…8°C. Avoid
contamination. Do not freeze. Do not mix caps.

155
Calibrator: Reconstitute the content of the vial with exactly 4 ml dist. germ free water, close
the vial and swirl carefully to dissolve all lyophilisate. Avoid foaming. Let stand for 30 minutes
before use.

Stability: 10 days at 2…8°C. If required, freshly prepared calibrator can be divided into
portions and kept frozen at -20°C for maximum 30 days. Freeze and thaw only once, mix
carefully after thawing.

Specimen:

Serum, plasma.
Stability: we recommend to test directly after sampling, otherwise store the serum at -20°C (up
to several weeks; avoid repeated freezing and thawing).

Assay:

Wavelength : Hg 578 nm, 593 nm, (570 to 610 nm)

Optical path : 1 cm
Temperature : 37°C
Measurement : Against reagent blank, one black per series is sufficient

Procedure: (Manual Procedure)

Warm the reagents and the cuvette to 37°C. Temperature must be kept constant (± 0.5°) for
the duration of the test.

(Table V.9)

Pipette into cuvettes Reagent blank (RB) Calibrator / Sample

Water 10 µl ---
Calibrator / Sample --- 10 µl
Enzyme 750 µl 750 µl

Mix gently and incubate exactly for 5 minutes at 37°C

Substrate 250 µl 250 µl

Mix Gently, incubate at 37°C and read the absorbance ∆A of Calibrator and samples against
RB after 5 minutes.

Calculation:

Calculate the concentration of the sample as follows:

∆Asample
C sample = C Calibrator x (mg/dl)
∆Acalibrator

Conversion factor: C (mg/dl) x 0.02586 = C (mmol/l)

156
 Performance Characteristics:

Linearity: Up to 150 mg/dl HDL

Linearity limit depends on the analyser-specific application. If the serum concentration of HDL
exceeds the measuring range, dilute the sample 1 + 1 with saline (0.9%) and repeat the test.
Multiply the result by 2.

Interference: no interference was observed with triglycerides up to 500 mg/dl, bilirubin up to 30

mg/dl, ascorbic acid up to 50 mg/dl, and slightly turbid samples. Dilute samples with
triglycerides exceeding 1200 mg/dl with phys. saline (0.9%) 1 + 1 and multiply the result by 2.

Reference Values2:

< 35 mg/dl (< 0.9 mmol/l) Risk factor for CHD

> 60 mg/dl (> 1.54 mmol/l) Reduced risk for CHD

This range is given for orientation only; each laboratory should establish its own reference
range, as sex, diet, age, geographical location and other factors affect the expected values.

Quality Control:

All human serum based control sera with HDL values determined by this method can be
employed.

Automation:

The test: can be run in a fixed time kinetic mode on analyzers. Applications for respective
instruments are available on request.

IX. Triglycerides (Enzymatic colorimetric method)

Principle:

Triglycerides determination is based on enzymatic hydrolysis, phosphorylation and oxidation of

triglycerides in the sample. The coloured complex quinoneimine is formed from the reaction of
the liberated hydrogen peroxide and 4-aminoantipyrene in the presence of 4-chlorophenol and
the catalytic influence of peroxidase.

The Absorbance of this coloured compound at 500 nm is proportional to the concentration of

triglycerides.

Lipase
Triglycerides + 3H2O ⎯ ⎯⎯
⎯→ Glycerol + fatty acids
GlycerolKinase
Glycerol + ATP ⎯ ⎯⎯⎯⎯⎯→ Glycerol-3-phosphate + ADP

GPO
Glycerol-3-phosphate + O2 ⎯ ⎯⎯→ Dihydroxyacetone-phosphate + H2O2

Peroxidase
2H2O2 + 4-aminoantipyrine + 4-chlorophenol ⎯ ⎯⎯⎯⎯→ Quinoneimine + HCL + 4H2O
(Coloured complex)

157
Clinical significance:

Serum triglycerides values are found elevated in secondary hyperlipoproteinemia,

atherosclerosis, glycogen storage diseases, nephrotic syndrome and greatly elevated in diabetes
mellitus, chronic hepatitis and alcoholism.

Sample:

Serum, heparinized or EDTA plasma.

Fasting blood sample (12 hr) is required

Procedure: (HUMAN-kit Cat. No. 10164)

One reagent blank per series is required.

(Table V.10)
Reagent Blank Sample or Standard
Sample / standard -- 10 µl
Reagent 1ml 1ml
Mix, incubate for 10 min. at 20 - 25 °C or 5 min. at 37 °C. Measure the absorbance of the
sample (Ab) and the standard (As) against the reagent blank within 60 min. at 500 nm.

Calculation:
Ab
Cb (mg / dl) = x Cs
As

Cb = the concentration of triglycerides in a given sample.

Cs = the concentration of triglycerides in a given standard.
(mg/dl x 0.0115 = mmol/L)

a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.

b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

Men 60 - 160 mg/dl

Women 40 - 140 mg/dl

Reagents:

a) Buffer solution (1)

PIPES buffer (pH 7.5) 40 mmol/l
Magnesium ions 5.0 mmol/l
4-chlorophenol 5.0 mmol/l
Sodium azide 0.1 %

158
 b) Enzyme reagent (2)
4-aminoantipyrine 0.4 mmol/l
ATP 1.0 mmol/l
Lipases > 150 U/ml
Glycerol kinase > 0.4 U/ml
Glycerol-3-phosphate oxidase > 1.5 U/ml
Peroxidase > 0.5 U/ml

c) Triglycerides standard 200 mg/dl (2.28 mmol/l)

Reagent preparation:
Reconstitute the contents of vial-2 with 15 ml buffer from vial 1.
The reconstituted reagent is stable for 21 days when stored at 2-8°C and for 3 days at (15 -
25°C) protected from light.

Quality Control:

The following variances should not be exceeded:

- OCV within 4%
- RCV within 8%

XI. Bilirubin, Direct & Total (Photometric colorimetric method)

Principle:

Bilirubin reacts with diazotized sulphanilic acid (DSA) to form a red azo dye. The absorbance
of this dye at 546 nm is directly proportional to the billirubin concentration in the sample.
Water-soluble bilirubin glucuronides react directly with DSA whereas the albumin conjugated
indirect bilirubin will only react with DSA in the presence of an accelerator:

Total bilirubim = direct + indirect bilirubin.

Sulphanilic acid + sodium nitrite ⎯

⎯→ DSA

Bilirubin + DSA ⎯
⎯→ DIRECT Azobilirubin

Bilirubin + DSA + Accelerator ⎯

⎯→ TOTAL Azobilirubin

Clinical Significance:

Determination of total and conjugated bilirubin is a useful diagnostic tool in a variety of clinical
conditions. Total bilirubin values are used in neonatal jaundice as a guide to intervention for
preventing kernicterus. In adults and children, total and direct (conjugated) bilirubin
determination are used to diagnose liver disease, obstruction of common bile duct, and
haemolytic disorders.

Sample:

Serum or heparinized plasma. Avoid haemolysis, samples must be protected from light.
Bilirubin is stable for 3 days when stored light-protected at 2-8 oC.

159
Procedure: (HUMAN kit Cat. No. 10740)

* For total bilirubin

(Table V.11)
Sample blank Sample
Total bilirubin reagent (1) 1ml 1ml
T-Nitrite reagent (2) --- 1 drop(40 µl)
Mix thoroughly, incubate for 5 min.
Sample 100 µl 100 µl
Mix, incubate at room temperature for 10 to 30 min. Measure the absorbance of sample (As)
against sample blank at 546 nm.

** For direct bilirubin

(Table V.12)
Sample blank Sample
Direct bilirubin reagent (3) 1ml 1ml
D-Nitrite reagent (4) --- 1 drop(40 µl)
Mix thoroughly, add sample within 2 min.
Sample 100 µl 100 µl
Mix, incubate at room temperature for exactly 5 min. Measure the absorbance of sample
(As) against sample blank at 546 nm.

Calculation:

Calculate the concentration of total and direct bilirubin by using the factor 13.0
Bilirubin concentration mg/dl = As x 13.

mg/dl x 17.1 = µmol/l

Linearity

The assay is linear up to 25 mg/dl. For bilirubin concentrations exceeding 25 mg/dl dilute the
sample 1 + 4 with normal saline 0.9% and repeat the assay. Multiply the result by 5.

Reference values:

(Table V.13)
Total bilirubin mg/dl µmol/l
At birth up to 5 up to 85.5
5 days up to 12 up to 205.0
1 month up to 1.5 up to 25.6
Adults up to 1.1 up to 18.8
Direct bilirubin
Adults up to 0.25 up to 4.3

160
 Reagents:

a) Total bilirubin reagent (1)

Sulphanilic acid 14 mmol/l
Hydrochloric acid 250 mmol/l
Caffeine (accelerator) 200 mmol/l
Sodium benzoate 420 mmol/l

b) T-Nitrite reagent (2)

For determination of total bilirubin
Sodium nitrite 14 mmol/l

c) Direct bilirubin reagent (3)

Sulphanilic acid 14 mmol/l
Hydrochloric acid 250 mmol/l

d) D-Nitrite reagent (4)

For determination of direct bilirubin
Sodium nitrite 0.9 mmol/l

Reagent preparation and stability:

Both reagents and nitrite solutions are ready for use.

They are stable up to the given expiry date if unopened and stored at 15 to 25 °C.

XI. Total Protein (Biuret method)

Principle:

Cupric ions (Cu++) react with protein in alkaline solution to form a purple complex. The
absorbance of this complex is proportional to the protein concentration in the sample.

Clinical Significance:

In disease states, both the total protein and the ratio of the individual protein fractions may
change independently of one another. In state of dehydration, total protein may increase some
10 to 15 percent. In multiple myeloma, the total protein may increase to over 10g/dl.

Hypoproteinemia which is characterized by total protein levels below 6g/dl as in nephrotic

syndrome where large amounts of albumin may be lost in the urine.

Sample:

Serum, heparinized or EDTA plasma.

161
Procedure: (HUMAN kit Cat. No. 10570)

One reagent blank per series is required

(Table V.14)
Reagent Blank Sample or Standard
Sample / standard -- 20 µl
Biuret Reagent 1 ml 1 ml
Mix, incubate for 10 min. at 20 - 25 °C. Measure the absorbance of the sample (Ab) and the
standard (As) against the reagent blank within 30 min. at 500 nm.

Calculation:

Ab
Cb (g / dl) = x Cs
As

Cb = Concentration of total protein in a given sample.

Cs = Concentration of total protein in a given standard.
(g/dl x 10 = g/L)

a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.
b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

6-8 g/dl = (60-80 g/L)

Reagents:

a) Color reagent 1
Sodium hydroxide 200 mmol/l
Potassium sodium tartrate 32 mmol/l
Copper sulfate 18 mmol/l
Potassium iodide 30 mmol/l

b) Protein standard
Protein 8 g/dl or (80 g/l)
Sodium azide 0.1 %

Quality Control:

The following variances should be obtainable:

- OCV within 2%.
- RCV within 4%.

162
XII. Albumin (BCG-method)

Principle:

Bromcresol green forms with albumin in citrate buffer a colored complex. The absorbance of
this complex is proportional to the albumin concentration in the sample.

Clinical Significance:

Albumin is synthesized in the liver. Decreased levels of albumin in serum may be found in
certain conditions such as cirrhosis of the liver and other liver disorders. Loss of albumin due to
kidney disorders will also lead to a decline of serum albumin level as also malnutrition,
malignancy, and chronic protracted conditions associated with increased levels of serum
albumin are not known.

Sample:

Serum, heparinized or EDTA plasma.

Procedure: (HUMAN kit Cat. No. 10560)

One reagent blank per series is required

(Table V.15)
Reagent Blank Sample or Standard
Sample / standard -- 10 µl
Color Reagent 1 ml 1 ml
Mix, incubate for 5 min. at 20 - 25 °C. Measure the absorbance of the sample (Ab) and the
standard (As) against the reagent blank within 30 min. at 500 nm.

Calculation:
Ab
Cb (g / dl) = x Cs
As

Cb = Concentration of albumin in a given sample.

Cs = Concentration of albumin in a given standard.
(g/dl x 10 = g/L)

a) Absorption Mode
Same procedure as for glucose by GOD-PAP Method.

b) Concentration Mode
Same procedure as for glucose by GOD-PAP Method.

c) Factor Mode
Same procedure as for glucose by GOD-PAP Method.

Reference values:

(3.8 - 5.1 g/dl) or (38 - 51 g/L)

163
 Reagents:

a) Color reagent 1
Citrate buffer (pH 4.2) 7.5 mmol/l
Bromcresol green ≥ 150 µmol/l
Sodium azide 0.05 %

b) Albumin standard
Albumin (4 g/dl) or (40 g/l)
Sodium azide 0.1 %

Quality Control:

The following variances should not be exceeded:

- OCV within 2%.

- RCV within 4%.

XIII. Aspartate aminotransferase AST - (GOT)

Principle:

Glutamate Oxaloacetate Transaminase Enzyme GOT (AST) catalyzes the following chemical
reaction:

GOT
2-oxoglutarate + L-aspartate ⎯ ⎯⎯→ L-glutamate + oxaloacetate
←
⎯⎯

MDH
Oxaloacetate + NADH + H+ ⎯ ⎯⎯→ L-malate + NAD+
←
⎯⎯

The change in an absorbance of NADH is measured at 340nm which is directly proportional to

the concentration of AST enzyme.

Clinical Significance:

AST is an enzyme present in tissues of high metabolic activity such as the heart, liver, skeletal
muscles and red cells. The enzyme is released into the circulation following injury or death of
cells. Any disease that causes change in these highly metabolic tissues will result in a rise in
AST. Following this, the blood AST level will rise in 12 hours and remain for more than 5 days.

Specimen:

Serum and heparinized or EDTA plasma.

Avoid hemolysis

164
Procedure:

(Table V.16)
Assay temperature 25 °C or 30 °C 37 °C
Sample 200 µl 100 µl
Working reagent 1 ml 1 ml
Mix, read the absorbance after 1 minute and at the same time start the stop-watch. Read the
absorbance again after exactly 1, 2 and 3 minutes at 340 nm.

Calculation:

From the readings, calculate the mean absorbance change per minute (DA/min).

If DA/min exceeds 0.2, dilute 0.1ml of sample with 0.4ml saline, then multiply the result by 5.

Mean DA/min at 340 nm x Factor (as indicated in the used kit) = U/L

Example:

Adjust the spectrophotometer to read 0.0 absorbance against air at 340 nm.

a) Insert the sample in the sample compartment and read absorbance, i.e. R0= 0.25

b) Leave the sample in the sample compartment and read the absorbance after 1, 2 and 3
minutes.

i. e. R1 = 0.20
R2 = 0.14
R3 = 0.10

DA1 = R0 - R1 = 0.25 - 0.20 = 0.05

DA2 = R1 - R2 = 0.20 - 0.14 = 0.06
DA3 = R2 - R3 = 0.14 - 0.10 = 0.04

Result = mean DA/min x Factor (given in your kit for the selected temperature)
= 0.05 x 1745 (37°C)
= 87 U/L

Reference values:

Men = up to 37 U/L at 37°C or up to 18 U/L at 25°C

Women = up to 31 U/L at 37°C or up to 15 U/L at 25°C

Reagents: (HUMAN kit Cat. No. 12001)

a) Buffer - Substrate
Phosphate buffer pH 7.4 80 mmol/l
L-aspartate 200 mmol/l
Sodium azide 0.1 %

165
 b) Enzyme - Substrate
Lactate Dehydrogenase (LDH) 1.2 U/ml
Malate Dehydrogenase MDH) 0.6 U/ml
Reduced form of Nicotinamide- 0.18 mmol/l
Adenine- dinucleotide (NADH)
2-Oxoglutarate 12 mmol/l

Reconstitute the contents of the enzyme-substrate bottle with the recommended quantity of the
buffer-substrate reagent.

Reconstituted reagent is stable for 5 days at 2-8 °C and for 32 hours at 15-25 °C

XIV. Alanine aminotransferase ALT - (GPT)

Principle:

Glutamic - pyruvic transaminase enzyme GPT (ALT) catalyzes the following reaction:

GPT
2-oxoglutarate + L-alanine ⎯ ⎯⎯→ L-glutamate + pyruvate
←
⎯⎯

LDH
pyruvate + NADH + H+ ⎯ ⎯⎯→ L-lactate + NAD+
←
⎯⎯
The change in absorbance of NADH is measured at 340 nm which is directly proportional to the
concentration of GPT enzyme.

Clinical significance:

ALT is an enzyme found in tissues of high metabolic activity such as the heart, liver, skeletal
muscles and red cells. In cases of acute cellular destruction, the enzyme is released into the
blood stream from damaged cells. Elevated values usually appear 8 hours after injury and
remain for more than 5 days.

Specimen:

Serum, heparinized or EDTA plasma. Avoid hemolysis

Procedure:

(Table V.17)
Assay temperature 25 °C or 30 °C 37 °C
Sample 200 µl 100 µl
Working reagent 1 ml 1 ml
Mix, read the absorbance after 1 minute and at the same time start the stop-watch. Read the
absorbance again after exactly 1, 2 and 3 minutes at 340 nm.

166
 Calculation:

From the readings, calculate the mean absorbance change per minute (DA/min).

If DA/min exceeds 0.2, dilute 0.1ml sample with 0.4 ml Saline, then multiply the result by 5.

Reference values:

Men = up to 42 U/L at 37°C or up to 22 U/L at 25°C

Women = up to 32 U/L at 37°C or up to 17 U/L at 25°C

Reagents: (HUMAN kit Cat. No. 12002)

a) Buffer - Substrate
Phosphate Buffer pH 7.4 80 mmol/l
L-alanine 800 mmol/l
Sodium azide 0.095 %

b) Enzyme - Substrate
LDH 1.2 U/ml
NADH 0.18 mmol/l
2-oxoglutarate 8 mmol/L

Reconstitute the contents of the enzyme-substrate bottle with the recommended quantity of the
buffer - substrate reagent.

Rreconstituted reagent is stable for 5 days at 2-8 °C and for 32 hours at 15-25 °C

XV. Alkaline phosphatase

Principle:

Alkaline phosphatase enzyme AP catalyzes the following chemical reaction:

AP
p-nitrophenyl phosphate + H2O ⎯ ⎯→ phosphate + p-nitrophenol
←
⎯⎯

Alkaline phosphatase enzyme hydrolysis p-nitrophenol phosphate (PNPP) into p-nitrophenol

(PNP) and phosphate. At the alkaline pH of the buffered medium, PNP is yellow. The color
developed by hydrolysis is measured at 405 nm at 0, 1, 2 and 3 minutes and the change in the
absorbance is calculated and is proportional to the AP enzyme activity.

Clinical significance:

Increased alkaline phophatase activity may be related to hepatobiliary and bone diseases. Very
high alkaline phosphatase activity in serum is seen in patients with bone cancer and marked
increase also occur in obstructive jaundice and biliary cirrhosis. Moderate elevations have been
noted in case of Hodgkin’s disease, congestive heart failure, infective hepatitis and abdominal
prblems.

167
Specimen:

Serum or heparinized plasma.

Avoid hemolysis

Procedure:

(Table V.18)
Assay temperature 25 °C or 30 °C 37 °C
Sample 20 µl 10 µl
Working reagent 1 ml 1 ml
Mix, read the absorbance immediately and at the same time start the stop-watch. Read the
absorbance again after exactly 1, 2 and 3 minutes at 405 nm.

Calculation:

From the readings, calculate the mean absorbance change per minute (DA/min).

If DA/min exceeds 0.25, dilute 0.1ml of sample with 0.5ml saline and multiply the result by 6.

DA/min at 405 nm x Factor (as indicated in the used kit) = U/L

AP activity U/L = mean DA/min x Factor (given in your kit for the selected temperature)

Reference values:

Men = 80-306 U/L at 37°C or 50-190 U/L at 25°C

Women = 64-306 U/L at 37°C or 40-190 U/L at 25°C
Children = up to 644 U/L at 37°C or up to 400 U/L at 25°C
Reagents: (HUMAN kit Cat. No. 12070)

a) Buffer - Substrate
Diethanolamine buffer pH 9.8 1.0 mol/l
Magnesium chloride 0.5 mmol/l
Sodium azide 0.1 %

b) Substrate
p-Nitrophenyl phosphate (PNPP) 10 mmol/l

Reconstitute the contents of substrate bottle with the recommended quantity of the
buffer solution.

Rreconstituted working reagent is stable for 4 weeks at 2-8 °C and for 1 week at (15-
25°C).

168
169
170
 PART VI - SEROLOGY

I. Antigen-Antibody Reactions

II. Brucella Test

1. Slide method
2. Tube method.

III. C-Reactive Protein (CRP)

IV. Anti Streptolysin - O (ASO)

V. Rheumatoid Factor (RF)

171
172
I. Antigen-Antibody Reactions

Serology tests are those tests based on the antigen-antibody reactions. They include
Agglutination, Complement Fixation, Immunodiffusion, Precipitation, Neutralization
inhibition,Enzyme-Linked Immunosorbent Assay(ELISA), Immunofluorescence and
Radioimmunoassay(RIA) tests.

Antibodies:

All antibodies are globulins that are made up of heavy and light polypeptide chains with the
following characteristics:

a) They are produced in response to antigenic stimulation

b) There are five classes of immunoglobulins. IgG, IgA, IgM, IgE and IgD
c) Antibodies are heterogeneous in structure.
d) All antibodies have the capacity to bind with their respective antigens.

Antigens:

Complete antigens are substances which can induce, in an animal host, either a humoral or a
cellular immune response, or both; they can react with the elicited antibodies or sensitized
lymphocytes. An incomplete antigen, which is known as a hapten, cannot elicit an immune
response by itself but can react with a specific antigen - antibody reaction. Many of the known
complete antigens are proteins, glycoproteins, or lipoproteins. Pure carbohydrate can elicit
immune response only in certain animal species, such as man and mouse, whereas pure lipids
or free DNA are not antigenic, although they can serve as haptens.

Pro-zone Phenomenon:

When the antibody involved in the Ag-Ab reaction is in excess (six to seven for each molecule of
antigen), it inhibits the reaction and results in a false negative result. This can be avoided by
diluting the serum sample.

1. Agglutination Test:

Agglutination is a classic serological reaction that involves clumping of a cell suspension

by specific antibody. This phenomenon may be observed when particular antigens such
as blood cells or bacteria are exposed to specific antibody under appropriate conditions.
Reactions of soluble antigens can be adapted to agglutination tests by the coated or
covalent linking of the antigen on specific antibody to a particulate carrier, e.g. red blood
cells or latex particles.

1.1 Direct Agglutination Assay:

The simple direct agglutination assay involves clumping of a cell or insoluble

particulate suspension, as bacteria, fungi, and other microbial organisms, by
specific antibody. Tests for detection of a specific antibody are performed by
determination of the dilution of serum that will agglutinate a constant amount of
antigen. A titre of a given serum is not considered significantly different from
that of another serum sample unless there is a four-fold difference.
Agglutination may be more visible and sensitive in a medium with a higher
viscosity (5% to 30% bovine serum albumin) or in the presence of some
enzymes like trypsin, papain, ficin, and bromelin.

173
 1.2 Indirect Agglutination:

This technique involves the agglutination of cells or inert particles coated with
soluble antigen or antibody. The cells or inert particles are passive carriers and the
antigens may be physically absorbed or covalently coupled to the surface. Inert
particles such as bentonite, latex, collodion, and charcoal have been used.

2. Haemagglutination Test:

Haemagglutination is the single most important reaction in blood banking. The number
of ABO sites may be close to one million per cell, and they are considered to be
extramembranous; thus, erythrocytes are easily agglutinated with the appropriate
antibodies. On the other hand, the Rh antigens have only about 10,000 to 30,000 sites
per cell and are considered to be intramembranous; thus, they are less easily
agglutinated by the appropriate antibodies. There are four groups of factors that can
influence the outcome of the reaction:

2.1 Erythrocytes:

Type, number and location of antigens on the erythrocytes.

2.2 Antibodies:

IgM antibodies can agglutinate red cells suspended in saline, but IgG usually do
not.

2.3 Medium:

Low pH and low ionic strength medium accelerate the binding of antibodies onto
red blood cells.

2.4 Physical Conditions:

Incubation temperature and time, as well as the uration and speed of

centrifugation, markedly affect the agglutination reaction.

3. Haemagglutination Inhibition Test:

This reaction is used to detect the presence of soluble antigens in saliva, serum, or other
fluids by neutralizing a specific antibody in a two-stage haemagglutination inhibition.
These antigens share the same antigenic specificity as found on the erythrocytes and are
capable of neutralizing the corresponding antibodies in the serum. The neutralized serum
will then no longer agglutinate the erythrocytes. Consequently, the absence of
agglutination indicates a positive test and signifies the presence of a specific antigen in
the fluid tested.

174
4. Complement Fixation Test:

The test procedure depends on the ability of fresh serum complement to interact with
antigen-antibody complexes. In the first step of the reaction, the complement is
incubated with the materials which may contain antigen and antibody. If antigen-
antibody complexes are formed, they will interact with complement in much the same
way as a complex of antibody and a cell surface antigen interact with complement. The
complement is activated, components are fragmented, and the complement is used up or
fixed. In the second stage, sensitized sheep cells are added, and the mixture is incubated
at 37oC for one hour. If the serum contains antibody to the antigen used, complement is
fixed and is, therefore, no longer available to lyse the sensitized sheep cells. Thus, the
absence of lysis indicates a positive reaction, while complete lysis indicates a negative
result.

5. Precipitation Test:

5.1 Tube precipitation

The quantitative precipitin reaction provides a systematic approach to determine

the amount of either antibody or antigen by defining the optimal proportions of
each reactant in the formation of the immune precipitate. The approach usually
followed is to prepare a series of test tubes, each containing a fixed amount of
antisera; to each tube in sequence an increasing quantity of the antigen used for
immunization is added. Following addition of antigen, an appropriate period of time
is allowed for the reaction to occur and the precipitate to form. The amount of
nitrogen or protein in the precipitate is then determined.

5.2 Immunodiffusion:

Immunodiffusion involves incorporation of antibody into the agar gel. Direct

application of the test antigen is then made by diffusion from a well punched in the
agar plates. After a period of time to allow for diffusion and equilibrium, the
formation of precipitin bands in the agar occurs. The distance of the precipitin line
from the point of application of antigen has been shown to be directly proportional
to the concentration of antigen when using a defined quantity of antiserum in the
agar.

6. Neutralization Test:

To test for certain antibodies, the ability of a patient's serum to block the effect of the
antigenic agent can be evaluated. Antibodies to bacterial toxins and other extracelluar
products that display measurable activities can be tested in the same way. The ability of
a patient's serum to neutralize the erythrocyte-lysing capability of streptolysin O (an
extracelluar enzyme produced by Streptococcus pyogenes during infection) has been
used for many years as a test for previous streptococcal infection. The use of particle
agglutination (latex or indirect haemagglutination) tests for the presence of antibody to
many of the streptococcal enzymes has replaced the use of these neutralization tests in
many laboratories.

175
 7. Enzyme-Linked Immunosorbent Assay(ELISA):

The most widely used name is enzyme-linked immunosorbent assay (ELISA). The ELISA
is capable of detecting extremely small quantities of immune reactants. The basic ELISA
detection system consists of antibodies bonded to enzymes that remain able to catalyze a
reaction while attached to the antibody. Furthermore, the antibody-binding sites remain
free to react with their specific antigen. Advantages of ELISA over Radioimmunoassay:

a) Sensitive assays can be developed by the amplification effect of enzymes.

b) Reagents are relatively cheap and can have a long shelf life.
c) Multiple simultaneous assays can be developed.
d) A wide variety of assay configurations can be developed.
e) Equipment can be inexpensive and is widely available.
f) No radiation hazards occur during labelling or disposal of wastes.

8. Radioimmunoassay (RIA):

Radioimmunoassay employed radioisotopes, either tritium (3H) iodine (125I), cobalt (57CO),
or carbon (14C), to label antigen molecules of the same substance being measured in the
assay. RIA, as the method is called, relies on the competitive binding to antibody of
labelled antigen, provided by the assay, and unlabelled antigen, present in the unknown
patient sample. When all three components are present in the system, an equilibrium
exists, dependent on the amount of unlabelled antigen. The more unlabelled (patient)
antigen is present, the less the labelled antigen will bound to the antibody. When the
antigen-antibody complexes are precipitated out of solution and the amount of the
radioactive label in the precipitate is determined, the unlabelled antigen present in the
sample being assayed can be quantified. In practice, a standard curve is first created by
adding known amounts of unlabelled antigen to the system. The amount of radiolabel
present in the precipitate of the test solution is compared with values obtained from the
standard curve to provide quantitative results.

9. Fluorescence Immunoassays:

Because of the inconveniences associated with radioactive substances and scintillation

counters, fluorescent immunoassays (FIA) were developed. Using fluorescent dyes or
molecules as markers instead of radioactive labels, these tests are based on the same
principle as RIA. The primary difference is that in RIA systems the competitive antibody
is labelled with a radioisotope and in FIA the antigen is labelled with a compound that will
fluoresce under the appropriate light rays. Binding of patient antibody to a fluorescent -
labelled antigen can reduce the fluorescence, or binding can cause fluorescence by
allowing conformational change in fluorescent molecule.

II. Brucella Test

Brucellosis is not uncommon disease which must be considered in the differential diagnosis of
fever of unknown origin. Brucellosis is often transmitted to man by infected milk or milk
products.

Blood culture is the best means of isolation, but a special culture medium is required. A
serologic slide agglutination test is the most frequent method of diagnosis.

176
Principle:

This test is used for the detection of different types of Brucella (abortus, melitensis, bovis and
suis). This screening test is a sensitive and quick one. It is necessary to test positive samples
by a quantitative tube method (Wright's test).

1. Slide Method: (Rose Bengal Antigen)

a) Before using, bring the reagent to room temperature.

b) Shake well to homogenize suspension.

c) Mix 30ul of the antigen with 30ul of the serum (use a thin stick for mixing).

d) Observe for any agglutination within 4 minutes.

Results:

Negative = Complete absence of agglutination

Positive = Agglutination (even minimum).

2. Serial Dilutions: Brucella titer – (Figure VI.1)

1 ml

1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

0.1 ml
Serum

Antigenic 1.9 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
Suspension 0.1+1.9 1+1 1+1 1+1 1+1 1+1 1+1 1+1 1+1 Control
1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120

Results:

The titre of the serum is the highest dilution that shows a 2+ reaction (approximately
50% of organism are clumped and the supernatant is slightly cloudy).
Serum is confirmed to be reactive (positive) if agglutination is recognized at a dilution
of 1 in 40 or greater.

Reagents:

a) Brucella antigen suspension (0.5%)

b) Positive control

References:

a) Brad street CM and others: Intradermal test and serological tests in suspected brucella
infection in men. Lancet 2:653, 1970.

b) Roux j, J. Med. Mal. Infect., 1974, 4, 259-266

c) Clinical Diagnosis and Management by Laboratory Methods, Henry, 18th edition, 1991.

177
III. Latex C-Reactive Protein (CRP)

Method:

The HUMATEX CRP lest is based on the immunological reaction between HUMAN C-creative
protein (CRP) of a patient specimen or control serum and the corresponding anti-HUMAN CRP
antibodies bound to latex particles. The positive reaction is indicated by a distinctly visible
agglutination of the latex particles in the test cell of the slide.
Specimen:

Serum
Stability: Up to 24 hours at 2 to 8°C.
Up to 4 weeks at -20°C.

Pipetting Scheme:

a) Qualitative Determination (Screening Test

(Table VI.1)
Bring (LR), (PC), (NC) and serum samples to room temperature. Mix (LR) carefully prior
to use to suspend the latex particles completely.
Pipette / drop onto separate cells of the slide
Serum sample 40 µl
(PC), red cap 1 drop
(NC), green cap 1 drop
(LR), white cap, onto all sample and control cell 1 drop
each
Mix with separate sticks and spread the fluid over the entire area of the particular cell.
Tilt the slide back and forth for 2 minutes so that the mixture rotates slowly inside the
cells or place the slide on an automated rotator at 100 r.p.m.
At the end of the 2 minutes read results under bright artificial light.

Interpretation of results:

Distinct agglutination indicates a CRP content of more than 6 mg/l in the non-diluted
specimen. Sera with positive results in the screening test should be retested in the titration
test (see part b).

b) Semi Quantitative Test:

Dilute specimens with Glycine – NaCl Buffer, as follows:

(Table VI.2)
Dilution CRP (mg/l in non-diluted specimen)
1 + 1 (1 : 2) 12
1 + 3 (1 : 4) 24
1 + 7 (1 : 8) 48
1 + 15 (1 : 16) 96
1 + 31 (1 : 32) 192
Continue test as described in part (a)

178
(Figure VI.2)
100 µl

100 µl 100 µl 100 µl 100 µl

Serum 100 µl

Glycine-NaCl 100 µl 100 µl 100 µl 100 µl 100 µl

Dilution 1+1 1+3 1+7 1+15 1+31

1:2 1:4 1:8 1:16 1:32
Conc. mg/L 12 24 48 96 192

Interpretation of Results:

Read the titre in the last dilution step with visible agglutination and multiply the titre with
the conversation factor 6 (see “Sensitivity”) to get the result in mg/l;
e.g. titre 1 : 16 →CRP concentration
16 x 6 [mg/l] = 96 [mg/l].

Sensitivity:

HUMAN’s HUMATEX CRP is standardised to detect CRP concentrations in non-diluted serum

samples of approximately 6 mg/l or higher.

Diagnostic Value:

The CRP test is a sensitive indicator for inflammatory processes, e.g. for rheumatic fever
and for the acute phase of rheumatic arthritis. The determination of the CRP level can be
used in therapy control.

Notes:

1. Contaminated and markedly lipemic sera may cause non-specific reactions and
should therefore not be tested.
2. A reaction time longer than 2 minutes may lead to false positive results due to a
drying effect.
3. During dispensing hold pipette vertically!
4. As with all diagnostic methods, the final diagnosis should not be made using the
result of a single test, but should be based on a correlation of more than one test
results with other clinical findings.
5. All reagents contain sodium azide: do not swallow. Avoid contact with skin and
mucous membranes.
6. Positive control has been tested for HBsAg and HIV and HCV antibodies and proved
to be negative using FDA approved methods. However, in spite of negative results
all reagents should be treated as potentially infectious.

179
IV. Latex Antistreptolysin O (ASO)

Method:

The HUMATEX ASO test kit contains polystyrene latex particles, coated with stabilized
streptolysin-O as antigen which reacts immunologically with corresponding anti-streptolysin-O
(ASO) antibodies of a patient specimen or control serum. The positive reaction is indicated by a
distinctly visible agglutination of the latex particles in the test cell of the slide.

Specimen:

Serum
Stability: Up to 48 hours at 2 to 8°C.
Up to 4 weeks at -20°C.

Pipetting Scheme:

a) Qualitative Determination (Screening Test

(Table VI.3)
Bring latex reagent, positive control, negative control and serum samples to room
temperature. Mix latex reagent carefully prior to use to suspend the latex particles
completely.
Pipette / drop onto separate cells of the slide
Serum sample 40 µl
Positive control, red cap 1 drop
Negative control, green cap 1 drop
Latex reagent, white cap, onto all sample and control cell 1 drop
each
Mix with separate sticks and spread the fluid over the entire area of the particular cell.
Tilt the slide back and forth for 2 minutes so that the mixture rotates slowly inside the
cells or place the slide on an automated rotator at 100 r.p.m.
At the end of the 2 minutes read results under bright artificial light.

Interpretation of results:

Distinct agglutination indicates an ASO content of more than 200 IU/ml in the non-diluted
specimen. Sera with positive results in the screening test should be retested in the titration
test.

b) Semi Quantitative Test:

Dilute specimens with Glycine – NaCl Buffer solution, as follows:

180
(Table VI.4)
Dilution CRP (mg/l in non-diluted specimen)
1 + 1 (1 : 2) 400
1 + 2 (1 : 3) 600
1 + 3 (1 : 4) 800
1 + 4 (1 : 5) 1000

Continue test as described in part (a)

Interpretation of Results:

Read the titre in the last dilution step with visible agglutination and multiply the titre with
the conversation factor 200 (see “Sensitivity”) to get the result in IU/ml;
e.g. titre 1 : 5 → ASO concentration
5 x 200 [IU/ml] = 1000 [IU/ml].

Sensitivity:

HUMATEX ASO is standardised to detect ASO concentrations in non-diluted serum samples

of approximately 200 IU/ml or higher in accordance with the “International Reference
Preparation” of the WHO.

Diagnostic Value:

The ASO titres may be associated with rheumatoid fever and glomerulonephritis. An
elevated ASO titre of more than 200 IU/ml may indicate an acute streptococcal infection.
The titre of ASO should be monitored every 2 weeks over a period of 4 to 6 weeks.

Notes:

1. Contaminated and markedly lipemic sera may cause non-specific reactions and
should therefore not be tested.

2. A reaction time longer than 2 minutes may lead to false positive results due to a
drying effect.

3. During dispensing hold pipette vertically.

4. As with all diagnostic methods, the final diagnosis should not be made using the
result of a single test, but should be based on a correlation of more than one test
results with other clinical findings.

5. All reagents contain sodium azide: do not swallow. Avoid contact with skin and
mucous membranes.

6. Positive control has been tested for HBsAg and HIV and HCV antibodies and proved
to be negative using FDA approved methods. However, in spite of negative results
all reagents should be treated as potentially infectious.

181
V. Latex Rheumatoid Factor (RF)

Method:

The HUMATEX RF is based upon the agglutination reaction between Rheumatoid Factors (RF)
of a patient specimen or control serum and human immunoglobulin G (IgG) coated onto
polystyrene latex particles.

The positive reaction is indicated by a distinctly visible agglutination of the latex particles in the
test cell of the slide.

Specimen:

Serum
Stability: Storage at 2 to 8°C up to 24 hours
Storage at -20°C up to 4 weeks

Pipetting Scheme:

a) Qualitative Determination (Screening Test) Pepetting Scheme

(Table VI.5)
Bring latex reagent, positive control, negative control and serum samples to room
temperature. Mix latex reagent carefully prior to use to suspend the latex particles
completely.
Pipette / drop onto separate cells of the slide
Serum sample 40 µl
Positive control, red cap 1 drop
Negative control, green cap 1 drop
Latex reagent, white cap, onto all sample and control cell 1 drop
each
Mix with separate sticks and spread the fluid over the entire area of the particular cell.
Tilt the slide back and forth for 2 minutes so that the mixture rotates slowly inside the
cells or place the slide on an automated rotator at 100 r.p.m.
At the end of the 2 minutes read results under bright artificial light.

Interpretation of results:

Distinct agglutination indicates an ASO content of more than 200 IU/ml in the non-diluted
specimen. Sera with positive results in the screening test should be retested in the titration
test (see part b).

b) Semi Quantitative Test:

Dilute specimens with (GBS) [(REF) 40037], as follows:

182
 (Table VI.6)
Dilution CRP (mg/l in non-diluted specimen)
1+1 (1 : 2) 24
1+3 (1 : 4) 48
1+7 (1 : 8) 986
1 + 15 (1 : 16) 192
1 + 31 (1 : 32) 384
Continue test as described in part (a)

Interpretation of Results:

Read the titre in the last dilution step with visible agglutination and multiply the titre with
the conversation factor 12 (see “Sensitivity”) to get the result in IU/ml;
e.g. titre 1 : 16 → RF concentration
16 x 12 [IU/ml] = 192 [IU/ml].

Sensitivity:

The sensitivity of the product is 12 IU/ml when samples are diluted in glycine buffer saline.
Standardization is in accordance with the “International Reference Preparation of
Rheumatoid Arthritis Serum” (WHO).

Diagnostic Value:

The clinical significance of RF determinations consists in differentiating between rheumatoid

arthritis, in which the rheumatoid factor has been demonstrated in the serum of
approximately 80% of the cases examined, and rheumatic fever, in which the rheumatoid
factor is almost always absent. The RF test is more frequently positive in long term active
processes than in diseases which are less active or are still in early stages. RF are
occasionally found in the serum of patients with polyarthritis nodosa, systemic lupus
erythematosus, hepatitis and certain other diseases.

Notes:

1. Contaminated and markedly lipemic sera may cause non-specific reactions and
should therefore not be tested.

2. A reaction time longer than 2 minutes may lead to false positive results due to a
drying effect.

3. During dispensing hold pipette vertically!

4. As with all diagnostic methods, the final diagnosis should not be made using the
result of a single test, but should be based on a correlation of more than one test
results with other clinical findings.

5. All reagents contain sodium azide: do not swallow. Avoid contact with skin and
mucous membranes.

6. Positive control has been tested for HBsAg and HIV and HCV antibodies and proved
to be negative using FDA approved methods. However, in spite of negative results
all reagents should be treated as potentially infectious.

183
184
 PART VII – BACTERIOLOGY

I. Classification of Bacteria.

II. Growth of Bacteria.

III. Pathogenicity of Bacteria.

IV. Sterilization.

V. Decontamination.

VI. Safety Measures.

VII. Artificial Culture Media.

VIII. Identification of Common Bacterial Pathogens.

IX. Bacteriological Examinations.

X. Sensitivity Tests.

XI. Gram Stain.

XII. Ziehl-Neelsen Stain.

XIII. Direct Microscopic Examination for Fungi.

185
186
I. Classification of Bacteria

Microorganisms are placed in a kingdom called the Protista, which is subdivided into two cell
classes:

1. The higher Protista (Eukaryotic):

Algae, Protozoa, Simple Moulds and Fungi.

2. The lower Protista (Prokaryotic):

Bacteria, Rickettsiae, Mycoplasma and Viruses.

Bacteria are unicellular, spherical, rod-shaped (straight or curved), or spinal in form.

Motile, by means of flagella, or non-motile. The average size range is 0.2-5 µ. They are
mainly classified into:

2.1 Saprophytic Bacteria:

Those, which are found in soil water and multiply on dead and decaying, matter.

2.2 Parasitic Bacteria:

Are those which include the bacteria of medical importance. They multiply on the
living tissue and are classified into commensal and pathogenic:

a. Commensal Bacteria:

Are those which constitute the normal flora of the healthy body. They live on
skin, mucous membrane of the nose, pharynx, throat ...etc, in the intestine,
genito-urinary tract, vagina... etc and obtain nourishment from the secretion
and food residue. When the body's defence is impaired, they may invade the
tissue and cause disease.

b. Pathogenic Bacteria:

Are those that overcome the normal defence of the body and invade the tissue
and cause disease.

II. Growth of Bacteria

When bacteria are inoculated in a suitable medium and placed in a correct environment, they
will grow at a rapid rate in numbers rather than size. There are four phases of growth in a
typical growth curve:

187
a. Lag Phase: Bacterial Growth Curve
STATIONARY
It is the period when the bacteria adapt PHASE DEATH
themselves to their new environment.
LOG
PHASE

LOG OF COUNT
PHASE

b. Log Phase:

At this stage the cells divide at a constant LAG

rate and reproduce by binary fission, the PHASE

relationship of time and number of cells is

linear and the cells are most active
metabolically. TIME

Figure VII.1
c. Stationary Phase:

The production of toxic waste products as well as depletion of certain nutrient substances cause
a decrease in the rate of growth to such an extent that the number of cells remains relatively
constant.

d. Death Phase:

At this stage cells begin to die due to a continuation of events leading to the stationary phase.

III. Pathogenicity of Bacteria:

The pathogenicity of bacteria depends upon various factors such as invasiveness, liberation of
exotoxin, presence of endotoxin, presence of capsule, presence of coagulase factor and other
bacterial products.

1. Sources of Infection:

Exogenous Infection: from patients, animals, insects, soil, food, water and cross infection.

Endogenous Infection: E. coli from bowel may cause urinary tract infection and
Pneumococci from naso-pharynx may cause bronchitis and broncho-pneumonia....etc.

2. Route of Infection:

a. Inhalation : respiratory infection, influenza, tuberculosis … etc.

b. Ingestion : cholera, food poisoning and hepatitis.

c. Inoculation : tetanus and hepatitis.

d. Insects : malaria, yellow fever and virus diseases.

e. Iatrogenic : induced by doctors and nurses during investigations like catheterization,

transfusion and lumbar puncture.

f. Contact : gonorrhoea and other venereal diseases.

g. Congenital : syphilis, rubella and toxoplasmosis.

188
 3. Laboratory Infections:

From bacterial culture and infected diagnostic material collected from patient or
experimental animals. Common organisms of Laboratory infection are tuberculosis,
brucellosis, anthrax, typhoid, dysentery, hepatitis virus and AIDs.

IV. Sterilization Methods:

The common methods of sterilization include the use of heat, filtration, gas and irradiation.

1. Dry-heat:

For equipment such as glassware dry-heat sterilization is used. Keep the temperature at
170°C for one hour while the equipment is in the hot air sterilizer.

2. Steam-under-pressure:

For most types of media, cloth, rubber, and other materials that would be destroyed by
dry heat, steam-under-pressure sterilization is used. Such material is autoclaved at 121°C
for 15 minutes using steam under 15 pounds pressure. The time necessary for complete
sterilization will vary somewhat, according to the kind and amount of material and if they
are in packs or not.

3. Filtration:

Many materials such as certain sugars and blood sera are destroyed by heating at
temperatures used normally for sterilization. To sterilize such heat-labile materials that
are liquids or substances in solution, filtration may be used. Filters in this method remove
bacteria by two ways:

a. by mechanical sievelike action of the minute pores of the filter, and ..

b. by the adsorption of the microbes to the filter due to the difference in their electrical
charges

4. Gaseous:

Ethylene oxide and other gaseous vapours are becoming increasingly important as
sterilizing agents. Although many laboratories do not have the specialized equipment
necessary to demonstrate this method of gaseous sterilization, you should be aware of its
use and advantages. The use of ethylene oxide vapours under pressure in special
equipment resembling a modified autoclave is becoming a common method of "cold
sterilization". Ethylene oxide is highly toxic for most heat resistant bacterial endospores.
As a sterilizing agent, it is very easy to handle with proper equipment and it’s relatively
inexpensive.

5. Irradiation:

Another method for the sterilization of certain materials (for example, pharmaceuticals) is
irradiation. Irradiation with ultraviolet light is not a satisfactory means of sterilization
because of the low penetratability of the ultraviolet wavelengths of the spectrum.

189
V. Decontamination

All potentially infectious agents must be decontaminated before disposal. These include unused
patient specimens as well as media that have been inoculated, whether pathogens have grown
or not. Infectious material to be disposed, should be placed into containers that are labelled as
to their biohazard risk.

Certain instruments such as scissors, forceps, and scalpel blade holders, should be placed into a
closed metal container until they can be autoclaved.

Decontamination of potentially infectious material to be discarded from the microbiology

laboratory is most often accomplished by steam sterilization in an autoclave.

Workbenches and other horizontal surfaces should be decontaminated at least after every shift
and immediately after every spill by washing with a liquid antiseptic agent, such as a phenolic
compound, 70% ethanol, or a 0.5% solution of sodium hypochlorite (10% solution of
household bleach in water), diluted bleach is more effective agent against viral contamination,
such as hepatitis B virus.

It is a good practice to wipe down the outside surfaces of laboratory equipment, such as
centrifuges, vortex mixers, telephones, with disinfectant on a regular basis.

VI. Safety Measures

1. Careful handling of infectious materials to prevent aerosol formation is an important

aspect of microbiology technique. If open flame burners are used care must be taken to
prevent spattering of material from inoculating needles and loops. The burner should be
in a protective container to prevent accidental burns.

2. Biological safety cabinet or hood should be used for inoculation procedures.

3. Personnel should wear gloves, masks and laboratory coat when handling material
containing organisms that might cause disease if they penetrate through the skin, when
performing procedures that require a significant amount of manipulation (such as grinding
tissue or filtering faeces), or when handling materials from patients with particularly
virulent or lethal infections.

4. Personnel should not touch their eyes, nose, exposed membranes or skin and should not
leave the work place or walk around the laboratory with gloved hands.

5. All used needles should be placed in the needle destroyer, LYM-03-1 Terminator. The
machine will render the needles harmless. The syringes must then be put in the "Syringe
Safety Box". When the box is full, it must be sealed according to the instructions provided
by the manufacturer and placed in the garbage bag. The destroyed needles must be
emptied directly from the box of the needle destroyer into Syringe Safety Box.

6. Work place should be kept clean, neat and free from any extraneous materials and
equipment.

7. All materials contaminated with potential pathogens must be decontaminated before

disposal.

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 8. Disinfect work surfaces when procedures are completed and at the end of each working
day. An effective all-purpose disinfectant is sodium hypochlorite solution 0.1% if prepared
with potable water and 0.5% if prepared with impure water.

9. Mouth pipetting is strictly prohibited.

10. Eating, drinking, smoking, and applying cosmetics are strictly forbidden in work areas.

11. All personnel should wash their hands with soap and water after handling infectious
material and before leaving the laboratory area.

11. Effective insect and rodent control programme should be made available.

VII. Artificial Culture Media

Over the years various strategies have been developed for cultivation of pathogens. Ingredients
necessary for the growth of pathogens can be supplied by the living system, as in the human or
animal host or in cell culture, or by mixing together the required nutrients in an artificial system.

1. Artificial media:

1.1 Enrichment media

To encourage the growth of particular organisms from a milieu containing only a

few of the desired organisms among large number of normal flora, various kinds of
laboratory prepared nutrient-containing solutions were collected, and called
enrichment media, an example of such media is selenite broth, which encourages
the growth of small numbers of stool pathogens and suppresses the growth of the
much larger numbers of normal stool organisms.

1.2 Supportive media

A second class of artificial media is called supportive media, These media contain
nutrients that allow the most nonfastidious organisms to grow at their natural rates,
without affording any particular organism a growth advantage (except for the
organism's own metabolism) on the medium. Examples of supportive media are
nutrient agar and brain heart infusion agar.

1.3 Selective media

Media containing one or more agents inhibitory to all organisms except the
organism being sought were developed. Such media are known as selective media,
since they select for certain organisms to the disadvantage of others. An example of
a selective medium is phenylethyl alcohol agar, which inhibits the growth of
aerobic and facultative anaerobic gram-negative rods and allow gram-positive cocci
to grow and also S S agar.

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1.4 Differential media

The fourth type of media is called differential medial. These media contain a factor
or factors that allow colonies of organisms that possess certain metabolic or cultural
characteristics to be morphologically distinguished from those organisms that have
different characteristics. The most supportive differential medium is sheep blood
agar, which allows many organisms to grow and additionally allows different
organisms to grow and to be distinguished on the basis of their haemolytic reaction
against the sheep red blood cells, production of pigment, etc.

(Table VII 3) lists a number of media that are commonly used in the clinical
microbiology, along with the ingredients that allow for differential and selective
abilities.

In order for bacteria to multiply on or in artificial media they must have available
the required nutrients, temperature, enough moisture in the medium and in the
atmosphere, proper salt concentration, an appropriate pH, and there must be no
growth-inhibiting factors.

1.5 Transport media – (Available commercially)

Transport media swab is used for sampling and transport of biological samples, as
follows:

a. Collect the specimen using the sterile swab.

b. Insert the swab into the tube containing the transport media.

c. Seal the cap with a suitable adhesive and sent directly to the laboratory.

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 2. Commonly used media

(Table VII.1)

Medium Component/Comments Primary Purpose

Blood agar Trypticase soy agar base or beef heart infusion base with 5% sheep Cultivation of fastidious microorganisms,
blood. determination of haemolytic reactions.

Chocolate agar Peptone base, enriched with solution of 2% haemoglobin or Cultivation of Haemophilus & Neisseria sp.
IsoVitalex (BBL).

Cystine-Lactose-Electrolyte- Peptone base agar with lactose & L-cystine; bromthymol blue Isolation & enumeration of bacteria in
Deficient (CLED) Agar indicator inhibits swarming of Proteus sp. urine.

Hektoen enteric (HE) agar Peptone base agar with bile salts, lactose, sucrose, salicin & ferric Differential, selective media for the isolation
ammonium citrate. Indicators include bromthymol blue & acid & differentiation of Salmonella & Shigella
fuchsin. from other gram-negative enteric bacilli.

Lowenstein 4 Jensen (L-J) Egg-based medium; contaminants inhibited by malachite green. Isolation of mycobacteria.
Agar

MacConkey agar Peptone base with lactose. Gram-positive organisms inhibited by Isolation & differentiation of lactose
crystal violet & bile salts. Neutral red acts as indicator. fermenting & non-lactose fermenting bacilli.

Sabouraud dextrose agar Peptone base agar. Final pH of medium (5.6) favours growth of Isolation of dermatophytes.
fungi over bacteria.

Salmonella-Shigella (SS) agar Peptone base with lactose, ferric citrate & sodium citrate. Neutral red Selective for Salmonella & Shigella species.
acts as indicator, indicator inhibition of coliforms by brilliant green,
bile salts.

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 Medium Component/Comments Primary Purpose

Selenite broth Peptone base broth. Sodium selenite toxic for most Enrichment for isolation of Salmonella.
Enterobacteriaceae.

Thayer-Martin agar Blood agar base enriched haemoglobin & supplement B; Selective for Neisseria gonorrhoea & N.
contaminating organisms inhibited by colistin, nystatin, vancomycin & meningitides.
trimethoprime

Thioglycolate broth Peptone 20g, L-Cystine 0.25g, Glucose 6g, Sodium chloride 2.5g, Support growth of anaerobes, aerobes,
Sodium thioglycollate 0.5g, sodium Sulfite 0.1g, Agar 0.7g and microaerophilic and fastidious
Distilled water 1000ml (Final pH 7.2). microorganisms.

Oxidase Reagent (Kovacs Tetramethyl-p-phenylenediamine dihydrochloride. To differentiate between oxidase positive

Method) and negative bacteria.

Indole Test 1- p-Dimethylaminocinnamaldehyde : 10 mg To differentiate between Indole positive

2- Amyl alcohol : 150 ml and negative bacteria.
3- Conc. HCl : 50 ml

Urea agar Peptone 1g, Glucose 1g, NaCl 5g, Monopotassium phosphate 2g, To differentiate between Urease positive
Phenol red 0.012g, Agar 20g, Distilled water 1000 ml (final pH 6.8). and negative bacteria.

Citrate agar Agar 20g, NaCl 5g, Magnesium sulphate 0.2g, Ammonium To differentiate between Citrate positive
dihydrogen phosphate 1g, Dipotassium phosphate 1g, Sodium citrate and negative bacteria.
2g, Bromthymol blue 0.08g, Distilled water 1000ml, (Final pH 6.9)

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 3. Recommended media commonly used for the identification of different kinds of bacteria:

(Table VII.2)

Medium Incubation Control Organism Expected Results

Bile esculin agar 24 hrs. Enterococcus faecalis Growth and blackening

Streptococcus pyogenes No growth

Blood agar 24 hrs, CO2, Candle jar Streptococcus pyogenes Growth and B-haemolysis

Chocolate agar 24h, CO2 Streptococcus pneumoniae Growth and a-haemolysis

Haemophilus influenza

MacConkey agar 24h, CO2 Escherichia coli Red colonies

Proteus mirabilis Colourless colonies

Mannitol salt agar 24h. Staphylococcus aureus Yellow colonies

Staph. epidermides Rose colonies
E. coli No growth

Peptone water-Indole 24h. Escherichia coli Positive

Klebsiella pneumoniae Negative

Salmonella-Shigella 24h. E. coli No growth

Agar or DCA agar Salmonella typhimurium Black metallic shain colonies
Shigella flexneri Black metallic shain colonies

Triple Sugar Iron (TSI) agar (Kligler agar) 24h. Citrobacter freundii * A/A gas + H2S
Salmonella typhimurium K/A gas + H2S
Shigella K/A gas

Urea medium 24h. E. coli Negative

Proteus mirabilis Positive (pink)
* A/A: acid slant; K/A: alkaline slant.

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VIII. Identification of Common Bacterial Pathogens

1. Identification Methods:

1.1 Staining Methods:

Smears are prepared from the specimen and stained with Simple Stain such as
methylene blue or carbol fuchsin. Gram's Stain to differentiate gram positive from
gram negative and with Special Stain such as Alberts Stain for Diphtheria bacilli
and Acid Fast Stain for Mycobacteria.

1.2 Cultural Methods:

Bacteria can be cultured on Liquid Media like Alkaline peptone water for Vibrio
Cholera and Selenite F broth for intestinal pathogen, but a single colony can not be
obtained from a liquid Media. On Solid Media colony morphology such as size,
shape, margin, surface, colour and consistency can be studied, also pure culture
can be isolated.

1.3 Biochemical Reactions / API 20 ∈:

The isolated organisms can be further identified by their Biochemical reactions such
as Sugar fermentation, Indole test, Oxidase test, Catalase test, Methyl red, Voges-
Proskauer, Citrate, Nitrate reduction test, Hydrogen sulphide production test.

API 20 ∈ is a standardized identification system for Enterobacteriaceae and other

Gram-negative rods which uses 23 miniaturized biochemical tests and a
database. The complete list of those organisms that it is possible to identify with
this system is given in the identification table of the instruction manual.

The API 20 ∈ strip consists of 20 micro-tubes containing dehydrated substrates.

These tests are inoculated with a bacterial suspension, which reconstitutes the
media. During incubation, metabolism produces colour changes that are either
spontaneous or revealed by the addition of reagents.

The reactions are read according to the Interpretation Table and the identification
is obtained by referring to the Identification Table (Analytical Profile Index).

Instructions for use

a. Preparation of the strip

Prepare the strip as indicated in the instruction manual.

b. Preparation of the inoculum

- Open an ampoule of Suspension Medium.

- With the aid of a pipette, remove a single well-isolated colony from and
isolation plate.

- Carefully emulsify to achieve a homogeneous bacterial suspension.

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 c. Inoculation of the strip:

- Fill the tubes as indicated in the instruction manual.

- Close the incubation box and incubate at 35-37 °C for 18-25 hours.

d. Reading of the strip

- After 18-24 hours at 35-37 °C, react the strip by referring to the
interpretation table.

- Record all spontaneous reactions on the report sheet.

- Continue as indicated in the instruction manual.

e. Identification:

- Using the Identification Table: compare the results recorded on the

report sheet with those given in the table.

- With the Analytical Profile Index: the pattern of the reactions obtained
must be coded into a NUMERICAL PROFILE.

1.4 Antigenic Reactions:

The isolated organism can be confirmed by agglutination reaction with specific

antisera.

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2. Identification of Clinically Important Enterobacteriaceae – (Table VII.3)

D-MANNITOL FERMENTATION
PHENYLALANINE DEAMINASE

ORNITHINE DECARBOXYLASE

GELATIN HYDROLYSIS (22°C)

D-SORBITOL FERMENTATION

RAFFINOSE-FERMENTATION

MELIBOISE FERMENTATION
DULICITOL FERMENTATION

ADONITOL FERMENTATION

D-XYLOSE FERMENTATION
HYDROGEN SULFIDE (TSI)

SUCROSE FERMENTATION
ARGININE DIHYDROLASE

LACTOSE FERMENTATION
LYSINE DECARBOXYLASE
INDOLE PRODUCTION

CITRATE (SIMMONS)
VOGES-PROSKAUER

galactopyranoside
UREA HYDROLYSIS

o-Nitrophenyl-D-
MOTILITY (36°C)

D-GLUCOSE, GAS

FERMENTATION

FERMENTATION

D NASE (25°C)
L-ARABINOSE

L-RHAMNOSE
METHYL RED
Species
Escherichia coli 98 99 0 1 1 1 0 90 17 65 95 0 95 95 50 98 60 5 94 99 50 80 95 75 0 95
Shigella serogroups 50 100 0 0 0 0 0 0 5 1 0 0 2 0 0 93 2 0 30 60 50 5 2 50 0 2
A, B, and C
Shigella Sonnei 0 100 0 0 0 0 0 0 2 98 0 0 0 2 1 99 0 0 2 95 3 75 2 25 0 90
Salmonella, most 1 100 0 95 95 1 0 98 70 97 95 0 96 1 1 100 96 0 95 99 2 95 97 95 2 2
Serotypes
Salmonella typhi 0 100 0 0 97 0 0 98 3 0 97 0 0 1 0 100 0 0 99 2 0 0 82 100 0 0
Salmonella paralyphi A 0 100 0 0 10 0 0 0 15 95 95 0 99 0 0 100 90 0 95 100 0 100 0 95 0 0
Citrobacter freundii 5 100 0 95 80 70 0 0 65 20 95 0 95 50 30 99 55 0 98 100 30 99 99 50 0 95
Citrobacter diversus 99 100 0 99 0 75 0 0 65 99 95 0 98 35 45 100 50 98 99 100 0 100 100 0 0 96
Edwardsiella tards 99 100 0 1 100 0 0 100 0 100 98 0 100 0 0 0 0 0 0 9 0 0 0 0 0 0
Klebsiella pneumoniae 0 10 98 98 0 95 0 98 0 0 0 0 97 98 99 99 30 90 99 99 99 99 99 99 0 99
Klebsiella oxytoca 99 20 95 95 0 90 1 99 0 0 0 0 97 100 100 99 55 99 99 98 100 100 100 99 0 100
Enterobacter aerogenes 0 5 98 95 0 2 0 98 0 98 97 0 100 95 100 100 5 98 100 100 96 99 100 99 0 100
Enterobacter cloacae 0 5 100 100 0 65 0 0 97 96 95 0 100 93 97 100 15 25 95 100 97 92 99 90 0 99
Hafnia alvei 0 40 85 10 0 4 0 100 6 98 85 0 98 5 10 99 0 0 0 95 2 97 98 0 0 90
Serratia marcescens 1 20 98 98 0 15 0 99 0 99 97 90 55 2 99 99 0 40 99 0 2 0 7 0 98 95
Proteus mirabilis 2 97 50 65 98 98 98 0 0 99 95 90 96 2 15 0 0 0 0 0 1 1 98 0 50 0
Proteus vulgaris 98 95 0 15 95 95 99 0 0 0 95 91 85 2 97 0 0 0 0 0 1 5 95 0 80 1
Providencia rettgeri 99 93 0 95 0 98 98 0 0 0 94 0 10 5 15 100 0 100 1 0 5 70 10 5 0 5
Providencia stuartii 98 100 0 93 0 30 95 0 0 0 85 0 0 2 50 10 0 5 1 1 7 0 7 0 10 10
Providencia alcalifaciens 99 99 0 98 0 0 98 0 0 1 96 0 85 0 15 2 0 98 1 1 1 0 1 0 0 1
Morganella morganii 98 97 0 0 5 98 95 0 0 98 95 0 90 1 0 0 0 0 0 0 0 0 0 0 0 5
Yersinia enterocolitica 50 97 2 0 0 75 0 0 0 95 2 0 5 5 95 98 0 0 99 98 5 1 70 1 5 95
Yersinia pestis 0 80 0 0 0 5 0 0 0 0 0 0 0 0 0 97 0 0 50 100 0 1 90 20 0 50
Yers. pseudotuberculosis 0 100 0 0 0 95 0 0 0 0 0 0 0 0 0 100 0 0 0 50 15 70 100 70 0 70
Each number gives the percentage of positive reactions after 2 days of incubation at 36°C. The vast majority of these positive reactions occur within 24 hours.
Reactions that become positive after 2 days are not considered.
198
 3. Identification of Gram-positive cocci bacteria

Figure VII.2
Gram +ve cocci

Catalase

(-ve) (+ve)

Streptococcus Staphylococcus

Coagulase
Optochin Bacitracin
disc (+ve) disc (+ve)

Streptococcus β-haemolytic group A (-ve) (+ve)

Pneumoniae Streptococcus Other spp S. aureus

4. Basic reactions of Enterobacteriaceae on Kligler’s agar

Figure VII.3
Alk/A Alk/A A/A Alk/A A/A Possible species
Gas No gas Gas H2S H2S

+ + + - - Escherichia coli
+ - + - - Enterobacter aerogenes
+ - + + + Citrobacter sp.
+ + - + - Salmonella sp.
- + - - - Shigella sp.
- + + - - Yersinia sp.
- - + - - Klebsiella sp.
- - - + + Proteus mirabilis
Proteus vulgaris

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 5. Special tests recommended for identification of certain species:

(Table VII.4)

Positive Negative Media

Catalase Staphylococci Streptococci Non-blood containing

media
Oxidase Pseudomonas spp Enterobacteriaceae TSA
Blood agar
Bacitracin disc β-haemolytic group A Other Strep. Spp Blood agar
Streptococcus
Optochin discs Strep.pneumoniae Other Strep. Spp Blood agar

Coagulase plasma Staph.aureus Other Staph. Spp Blood agar

Germ tube test Candida albicans Candida tropicalis Blood agar

plasma

5.1 Catalase Test

a. With a loop or sterile wooden stick, transfer a small amount of pure growth
from the agar onto the surface of clean, dry glass slide.

b. Immediately place a drop of 3% hydrogen peroxide (H2O2) onto a portion of a

colony on the slide.

c. Observe for the evolution of bubbles of gas, indicating a positive test.

5.2 Spot Oxidase Test

a. Prepare a solution of 1% tetramethyl-p-phenylenediamine dihydrochloride in

sterile distilled water each day.

b. Place a filter paper circle into a sterile plastic disposable Petri dish and moisten
the filter paper with several drops of the fresh reagent.

c. Remove a small portion of the colony to be tested (preferably not more than
24 hr old) with a platinum wire or wooden stick and rub the growth on the
moistened filter paper.

d. Observe for a colour change to blue or purple within 10 seconds (timing is

critical).

5.3 Bacitracin Susceptibility Test

Inoculate one quadrant or one half of a 5% sheep blood agar plate with a pure
culture of the organism to be tested by heavily streaking the entire surface.

Place 0.04 U bacitracin disk (Taxo A, BBL Microbiology Systems or Bacto Bacitracin
Differentiation disk, Difco Laboratories) in the centre of the inoculum.

200
 Incubate overnight in air or 5%-10% CO2 at 35°C and examine for a zone of
inhibition of growth around the bacitracin disk.

Any zone of inhibitions is indicative of bacitracin susceptibility. In addition to S.

pyogenes, essentially all of which are susceptible, a small percentage of group B (S.
agalactiae) and 10%-20% of group C and G are susceptible. Since group B
streptococci can be differentiated morphologically and biochemically, and since
isolates of groups C and G occur infrequently, bacitracin serves as an adequate
presumptive test from group A streptococci.

5.4 Optochin Susceptibility Test

a. Streak one quadrant or one half of a 5% sheep blood agar plate with an
inoculum from a pure isolate of the organism to be tested.

b. Place an optochin disk (Taxo P, BBL Microbiology Systems, or Bacto Optochin

disk, Difco Laboratories) in the centre of the inoculum.

c. Incubate overnight at 37°C in 5%-10% CO2 or a candle jar. Observe for zones
of inhibition surrounding the disk. Zones of equal to or greater than 14mm
surrounding a 6-mm diameter disk and zones equal to or greater than 16mm
surrounding a 10-mm diameter disk are considered positive, presumptive
identification of S. pneumoniae. Zone sizes between 6 and 14mm (6mm disk)
and 10 and 16mm (10mm disk) are equivocal, and those isolates should be
tested for bile solubility.

5.5 Coagulase Test:

a. Slide Coagulase (Clumping Factor) Test

i. Place a drop of coagulase plasma (rabbit plasma with EDTA or Citrate,

available commercially from BBL Microbiology Systems, Difco Laboratories,
GIBCO Laboratories, and others) on a clean, dry glass slide.

ii. Place a drop of distilled water or saline next to the drop of plasma as a
control.

iii. With a loop, straight wire, or wooden stick, emulsify an amount of the
isolated colony being tested in each drop, inoculating the water or saline
first. Try to create a smooth suspension.

iv. Observe for clumping in the coagulase plasma drop and a smooth,
homogeneous suspension in the control. Clumping in both drops indicates
that the organism autoagglutinates and is unsuitable for the slide
coagulase test.

b. Tube Coagulase Test

i. Prepare coagulase reagent, rabbit plasma with EDTA (Difco Laboratories

and BBL Microbiology Systems) in 0.5 ml amounts in 13 x 100mm glass or
plastic tubes. The tubes can be prepared in large numbers and
refrigerated for 10 days, or frozen at - 20°C for several months.

201
 ii. Emulsify a visible portion of growth from isolated colonies in the plasma by
rubbing the material on the side of the tube while holding the tube,
causing the plasma level to cover the site of inoculation.

iii. Incubate the suspension for 1-4 h at 35-37°C and observe for the
presence of a gel or clot that cannot be re-suspended by gentle shaking. If
no clot forms after 4 h, the tube should be incubated at room temperature
overnight. Rare isolates require such extended incubation.

iv. Organisms that fail to clot the plasma within 24 h are considered
coagulase negative, and must be identified by other methods.

5.6 Germ-tube Test

Principle:

Strains of Candida albicans produce germ tubes from their yeast cells when placed
in a liquid nutrient environment and incubated at 35 oC for 3 hours (similar to the in
vivo state).

Method:

a. Suspend a very small inoculum of yeast cells obtained from an isolated

colony in 0.5 ml of sheep serum (or rabbit plasma).

b. Incubate the tubes at 35° to 37° C for no longer than 3 hours.

c. After incubation, remove a drop of the suspension and place on a

microscope slide. Examine under low-power magnification for the presence
of germ tubes. A germ tube is defined as an appendage that is one half the
width and three to four times the length of the yeast cell from which it
arises.

Expected results:

Candida albicans will produce germ tubes, usually within 2 hours, whereas
Candida tropicalis will not.

IX. Bacteriological Examination:

1. Urine

1.1 Common pathogens:

Pathogens with high priority

- Escherichia coli
- Other gram-negative rods
- Enterococci
- Staphyloccus saprophytics

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 Pathogens with intermediate priority

- Pseudomonas and other non-fermenters

- Other staphycococci

Pathogens with low priority

- Candida albicans

- Mycobacterium tuberculosis

1.2 Isolation media and diagnostic reagent:

Isolation media:

- MacConkey agar without crystal violet

Identification media and reagents:

- Bile-esculin agar (for enterococci)

- Kligler iron agar

- Motility indol urease medium

- Kovac’s reagent for indole

- Oxidase reagent

- Simmon’s citrate agar

For Staphylococci and enterococci:

- Coagulase plasma

- Catalase test

- Oxidase test

1.3 Direct microscopy:

Microscopy is valuable adjunct in the diagnosis of urinary infections. The presence of

bacteria detected by direct microscopy of unstained, un-centrifuged samples of urine
can be correlated with infection. Bacteria which enter the urine as contaminates are
usually firmly attached to epithelial cells and therefore not easily seen. The presence of
large numbers of white cells provides indirect evidence of infection. Detection of
epithelial cells helps in establishing the quality of the specimen and, indirectly the
clinical significance of any isolate, their presence indicate that organisms normally
resident in the perineum or vagina are likely to have contaminated the specimen.

203
1.4 Specimen Collection:

Early morning urine which contains concentrated urine should be obtained if possible.

Every effort must be made to collect a clean-catch urine specimen in a sterile container
and to ensure that it is delivered promptly to the laboratory together with information
on the patient, clinical diagnosis, and the requested procedures.

The urethra is flushed by the passage of the first portion of the voiding, which is
discarded. The subsequent midstream urine voided directly into sterile container is
used for culturing and colony count.

1.5 Media:

As discrete colonies are required for accurate enumeration of bacterial count, Blood
agar and MacConkey media are used in the primary isolation of urinary pathogens.

1.6 Culture and interpretation

All urine specimens brought to the microbiology laboratory should be examined at

once, or placed in a refrigerator at 4 oC until they can be examined.

The examination procedure includes the following steps:

a. A screening test for significant bacteruria (test strip for leukocyte esterase/nitrate
reduction).

b. A definitive culture for urine specimens found to be positive in the screening test.

c. Susceptibility tests on clinically significant bacterial isolates.

Urine samples that are positive in the screening test should be cultured as soon as
possible to prevent possible overgrowth by nonsignificant bacteria. If the strip does not
develop a pink color it is interpreted as a negative screening test, is so reported, and
no culture is indicated.

The test strip may not be sensitive enough to detect bacterial counts of less than 105
per ml of urine.

Streaking plates with a measured amount of inoculum, such as that found in a

standard calibrated loop (1µl) should be done to facilitate counting colonies.

For this purpose, the inoculum should be spread out more evenly over the entire plate
(Figure VII.4).

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 Figure VII.4

Loop is touched to the centre of the

plate, from which the inoculum is
spread in a line across the diameter
of the plate.

Without flaming or re-entering urine,

Method for inserting a calibrated loop is drawn across the entire plate,
loop into urine to ensure that the crossing the first inoculum streak
proper amount of specimen numerous time to produce isolated
adheres to the loop. colonies.

Method for streaking with calibrated urine loop to produce isolated colonies and
countable colony forming units (CFUs) of organisms.

Inoculate a plate of MacConkey agar (with crystal violet) with the urine specimen using
a sterile loop. The inclusion of a blood agar facilitates the rapid identification of Gram-
positive cocci. Plates should be incubated at 35 -37 °C overnight, and examined on the
following day for growth. Identification procedures may then be initiated using well-
separated colonies of similar appearance. If required, the inoculum for performing the
disc-diffusion susceptibility test can be prepared from either of these plates. In this
way, the results of both identification and susceptibility test will be available on the
next day.

Susceptibility tests should only be performed on well-isolated colonies of similar

appearance that are considered significant according to the guidelines presented
above. Susceptibility tests are generally more important on cultures obtained from
patients who are hospitalized or have a history of recurring UTI. Cultures from
patients seen in the clinic with a primary UTI may not require a susceptibility test.

1.7 Reporting

The results of urine examination must be carefully reported. Guidance must be given
to the clinician about the significance of individual isolates and appropriate antibiotics,
if any to be prescribed. When reporting, the delay in transport of the specimen must
be taken into account when deciding the clinical relevance of each isolate, Numerical
criteria have been set up to establish significance but are not the sole determinant.

205
 The purity of the culture is also important as a pure culture of E.coli of 104 Colony
Forming Units of bacteria per milliliter (CFUs/ml) may be significant whereas isolation
of 105 CFU/ml E. coli in mixed culture and where epithelial cells have been seen on the
direct microscopy may not. Mid-stream specimens in which more than one organism is
cultured are likely to have been contaminated by urethral flora. When a specimen is
suspected of being unsuitable by virtue of contamination or delay in transport a repeat
specimen should be requested. A reporting protocol is set out in the following table:

Reporting Protocol

(Table VII.5)
Culture Result Report
No bacterial growth No bacterial growth
single organism
1. < 104 CFU/ml No significant growth
4 5
2. 10 -10 CFU/ml Report no sensitivities ; ? significance
5
3. > 10 CFU /ml Report organism and sensitivity
Tow organisms
1. Both < 105 CFU/ml No significant growth, please repeat.
5
2. One > 10 CFU Mixed growth including >105 isolate . please repeat.
If no epithelial cells
3.Both >105 CFU/ml Report both organism and sensitivity
If no epithelial cells
> + epithelial cells Specimen contaminated, please repeat
More than two organisms No significant growth
>10 5 CFU/ml

2. Purulent exudates, Wounds and Abscesses

2.1 Common pathogens:

Pathogens with high priority

- Streptococcus pyogenes

- Staphyloccus aureus

Pathogens of intermediate priority

- Enterobactericeae

- Pseudomonas and other non-fermenters

- Clostridium perfringens

- Bacteroides and other strict anaerobes

- Streptococcus (other species)

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2.2 Culture media and diagnostic reagents:

Isolation media:

- Blood agar

- MacConey agar

- Thioglycollate broth

Diagnostic reagents:

- Coagulase plasma

- Catalase test (H2O2)

- Oxidase reagent

2.3 Collection of specimens:

It is not possible, to describe in detail the procedures for specimen collection

from each type of wound, abscess, etc. It should be obvious that this is a task
that requires close cooperation between laboratory and the physician. In many,
instances, there is only one opportunity to obtain a specimen-second specimens
are non-existent in many cases. Therefore, proper collection, transport and
storage of these specimens are of the greatest importance, and compromises
should be avoided.

The specimen should be processed as soon as possible. After the preliminary

examinations have been completed and the cultures made the rest of the
specimen should be properly labeled, stoppered and refrigerated, until it is
certain that no additional tests are needed.

Abscess:

A syringe and needle are used to aspirate as much as possible from purulent
material, which is then aseptically, transferred to sterile specimen containers. If
such containers are not available, the specimen should be kept in the syringe
with the needle capped, and the syringe itself should be transported to the
laboratory.

Infected lacerations, penetrating wounds, postoperative wounds, burns and

ulcers:

No single standard procedure for specimen collection can be formulated.

However, certain fundamental guidelines should be followed to obtain the best
possible specimen for laboratory analysis.

After carefully cleaning the site, the surgeon should look beneath the surface for
collection of pus, devitalized tissue, the oozing of gas, or any other abnormal
sign. Pus or other exudate should be carefully collected and placed in sterile
tube. Swabs may be used if necessary.

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2.4 Macroscopic evaluation:

Specimens of pus or wound discharge collected on swabs are difficult to evaluate

macroscopically, particularly when the swab is immersed in a transport medium.
Specimens of pus, received in a syringe or in a needle container, should be
evaluated carefully by experienced technician for color, consistency and odor.

2.5 Microscopic examination:

Gram-stained smear

Using bacteriological loop, make an even smear of the most purulent part of the
specimen in a clean slide. If only one swab is available, the slide should first be
sterilized by being passed through the flame of a Bunsen burner and allowed to
cool.

The cotton swab should be gently rolled over the glass surface, without rubbing
or excessive pressure. Allow the slide to air-dry. Fix by heat, stain and examine
the smear under the oil immersion objective. Inspect carefully and note the
presence and the quantity of:

- Polymorphonuclear granulocytes ( pus cells).

- Gram-positive cocci arranged in cluster, suggestive of staphylococci.

- Gram-positive cocci arranged in chains, suggestive of streptococci.

- Gram-negative rods resembling coliforms (E.coli, Klebseiella, ect). Other

enterobacteriaceae (Proteus,serratia, ect), nonfermentative rods
(Pseudomonas spp) or obligate anaerobes (Bacteriods spps);

- Large straight Gram positive rods with square end suggestive of clostridium
perfringes, the principle agent of gas gangrene, or bacillus anthracis, the
agent of anthrax;

- An extremely heavy and polymorphic mixture of bacteria, including

streptococci, gram negative and gram positive rods of various sizes including
fusiform rods; such a picture is suggestive of “anaerobic mixed flora “ and
should be reported as such;

- Candida or other yeast cells, which as seen as ovoid Gram-positive budding

spheres, often forming branched pseudomycelia.

Direct Microscopy

When requested, or when fungal or parasitic infection is suspected, a wet

preparation should be examined .If the pus is thick, a loopful should be mixed in
a drop of saline. When looking for fungi, a drop of 10% potassium hydroxide
should be used to clear the specimen.

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 2.6 Culture:

If bacteria or fungi are seen on microscopic examination. Appropriate culture

media should be inoculated on to a minimum of three culture media:

- A blood agar for the isolation of staphylococcus and streptococci;

- A MacConky agar plate for the isolation of Gram-negative rods; and

- A tube of broth that can serve as enrichment medium for both aerobes and
anaerobes, e.g., thioglycollate broth.

The size of the inoculum should be determined according to the result of

microscopic examination, and may vary from one loopful to a few drops. If
massive numbers of organisms is seen on the gram stained smear, the specimen
may even have to be diluted in a small amount of sterile broth before plating out.

If a swab is used for the inoculation, it should be applied to a small area of the
plate and the rest of the surface streaked out with a loop. If the swab is dry, It
should first be moistened in a small quantity of sterile broth or saline. In all
cases, the technique of inoculated should provide single colonies for identification
and susceptibility test.

Prior to inoculation, the blood agar plate should be dried for 20 minutes in an
incubator, to minimize the risk of overgrowth by spreading Proteus spp.. The
inoculated plate should be incubated at 35 in a candle jar. Routinely, all media
should be incubated for two days and inspected daily of growth. If the culture for
fastidious organisms is requested, longer incubation (1 – 2 weeks or more) will
be necessary. If growth appears in the broth, it should be gram-stained and
subculture on appropriate culture media. Additional culture media should be used
if specially requested, or if indicated by the results of the microscopic
examination.

3. Upper respiratory tract (Throat and Nasal swabs)

3.1 Common pathogens:

Pathogens with high priority

- Streptococcus pyogenes (group A) (Throat)

- Streptococcus pneumonia

- Hemophilus influenza

Pathogens of intermediate priority

- Candida albicans

- Staphylococcus aureus (ear and sinus)

- Branhamella catarrhalis (ear and sinus)

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 Pathogens with low priority

- Gram-negative rods and Pseudomonas (ear)

- Neisseria memningitidis (nasopharyngeal carriers)

3.2 Culture media and diagnostic reagents:

Isolation media:

- Blood agar

- Chocolate agar

Identification reagents:

- Bacitrcin disc, optochin disc

- Catalase test

Throat and nasal swabs can be used for several diagnostic purposes. The
diagnostic aim in taking throat swab must be clearly followed. Streptococcus
pyogens is the bacteria most clearly associated with acute pharyngitis, diagnostic
endeavor should be directed toward isolation and grouping of beta hemolytic
streptococci.

3.3 Specimen Collection

Ideally, a physician should collect specimen or other well-trained personnel. The

patient should sit in front of a light source, While the tongue is kept down with a
tongue depressor, a sterile-cotton swab is rubbed vigorously over each tonsil,
over the back wall of the pharynx, and over any other inflamed area. Care should
be taken not to touch the tongue or buccal surfaces. If the specimen cannot be
processed within 4 hours, the swab should be placed in transport medium
(e.g.,Amies or Stuart).

3.4 Direct Examination

The Gram-stained smear is not useful for the detection of streptococci or

Neisseria spp. Moreover, the direct smear has poor sensitivity and specificity for
the detection of the diphtheria bacillus, unless the specimen has been collected
with care and is examined by an experienced microbiologist. Candida is best
recognized on a Gram-stained smear, which should be prepared if the physician
makes a special request. In the absence of such a request or clinical information,
a Gram-stained smear should not be made for throat swabs.

3.5 Culture and identification

Immediately upon receipt in the laboratory, the swab should be rubbed over one-
quarter of a blood agar plate, and the rest of the plate streaked with a sterile
wire loop. The blood agar should be prepared from a basal agar medium without
glucose. Blood from any species, even human blood (fresh donor blood) can be
used at a concentration of 5%.

210
 The plate should be filled to a depth of 4-5 mm. Sheep’s blood is preferred
because it does not permit growth of haemolytic Haemophilus spp.

Inoculua are usually spread over the surface of agar plates in a standard pattern
to achieve isolated colonies (Figure VII.5).

Figure VII.5

Streaking Pattern for Primary Inoculation of Plates to Achieve Isolated Colonies

First streak First quadrant Second quadrant

Initial innoculum

Second
streak

Third
Streak Fourth Third
Quadrant quadrant 4+ growth
Streaking pattern 1-2 + growth

Incubation in a candle-jar will detect most β-haemolytic streptococci.

All colonies, which morphologically resemble staphylococci, should be

investigated for coagulase.

After gram staining to verify that they are Gram-positive cocci, the colonies
should be submitted to specific identification tests for S. Pyogenes. Identification
of S. Pyogenes is based on its susceptibility to a low concentration of bacitracin.

For this purpose, a special differential disc is used containing 0.02-0.05 IU of

bacitracin. The ordinary discs used in the susceptibility test, with a content of 10
units, are not suitable for identification. A (β-haemolytic streptococcus) showing
any zone of inhibition around the disc should be reported as S. Pyogenes.

3.6 Susceptibility Testing

Routine susceptibility tests on throat or pharyngeal isolates are most often not
required, and may even be misleading. The major pathogen involved in bacterial
pharyngitis is S. pyogenes. Benzylpenicillin and erythromycin are considered as
the antibiotics of choice to treat.

211
4. Lower respiratory tract samples(Sputum)

4.1 Common pathogens:

Pathogens with high priority

- Mycobacterium tuberculosis

- Streptococcus pneumonia

- Hemophils influenzae

- Staphylcoccus aureus

- Klebsiella pneumoniae

Pathogens of intermediate priority

- Enterobacteriacae

- Candida albicans

- Branhamella catarrhalis

4.2 Culture media and diagnostic reagents:

Isolation media

- Blood agar

- Chocolate agar

- MacConkey agar

Identification reagents

- Coagulase plasma

- Catalase test

- Optochin disc

- V and XV factors

Lower respiratory tract infections (LRTI) are infections occurring below the level
of larynx, i.e. in the trachea, the bronchi, or in lung tissue (tracheitis, bronchitis,
lung abscess, and pneumonia).

Many patients with LRTI cough up purulent (pus containing) sputum that
generally green or yellowish in color; this sputum may be cultured and examined
grossly and microscopically.

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4.3 Collection of sputum specimens

Examination of a badly collected sputum specimen can give misleading results

because of contamination with the normal bacterial flora present in the mouth
and throat; “sputum” consisting of saliva and food particles should not be
examined.

The sputum should be collected in a sterile wide-mouthed container with a

secure, tight-fitting cover and sent to the laboratory without delay. If the sputum
is allowed to stand after collection, overgrowth of contaminating bacteria may
take place before the examination is carried out and the results of smears and
cultures will be highly misleading.

4.4 Processing of sputum

Sputum should not be allowed to remain longer than approximately 1 hour at

room temperature before being processed.

Macroscopic evaluation

The macroscopic appearance of the sputum should be recorded. Possible

descriptions include:

- Purulent, green
- Purulent, yellow
- Mucopurulent (i.e., partially mucoid and partially purulent)
- Blood-stained
- Blood -stained, with green floccules
- Grey, mucoid
- Grey, frothy
- White mucoid
- White, frothy
- White, mucoid, with some food particles
- Watery (i.e., only saliva present)
- Watery, with some food particles

Microscopic Examination

A portion of purulent or mucoprulent sputum should be used for preparation of

Gram-stained smear.

If no floccules of pus can be seen (e.g., in a Grey mucoid sputum sample), the
Gram-stained smear may show only the presence of large, rather square,
squamous epithelial cells, frequently covered with masses of adherent bacteria.
This is an indication that the specimen consists mainly mouth or throat
secretions, and culture should not be carried out as it is not relevant, and
usually highly misleading. An accepted guideline is to reject, for culture, any
specimen that contains fewer than 10 polymorphonuclear neutrophils per
epithelial cell. The presence of polymorphonuclear leukocytes (PMNs) and
alveolar macrophages suggests the presence of material of alveolar origin, and
squamous epithelial cells indicate salivary contamination. Specimens with < 10
squamous cells and > 25 PMNs per X10 field are likely to represent alveolar
specimens.

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 Possible results of Gram-stained smear include:

- Gram-positive diplococci surrounded by an empty space from the unstained

capsules (suggestive of S. pneumoniae);
- Small Gram--negative coccobacilli (probably H. influenzae);
- Gram-negative coccobacilli (probably H. Influenzae);
- Gram-positive cocci in grape-like clusters (suggestive of S. aureus);
- Gram-negative rods (suggestive of the presence of Enterobacteriaceae or
Pseudomonas spp);
- Large Gram-positive yeast-like cells, often with mycelia (suggestive of the
presence of Candida spp).

4.5 Culture and interpretation

A routine set of culture media is as follows:

- Blood agar
- Chocolate agar
- MacConkey Agar

The blood agar and chocolate agar plates are incubated at 30-36 oC in an
atmosphere containing extra carbon dioxide (e.g. in a candle jar) and MacConkey
plate is incubated in air. Cultures should be inspected after incubation overnight
(18 hours) but reincubation for an extra 24 hours may be indicated when growth
is less than expected from the microscopic findings, or when only tiny colonies
are present.

Typical findings include the following:

- Flat, clear colonies with concave centers and zone of green (alpha)
haemolysis, may be S. pneumonia, subculture and do optochin test. It
should be not to be forgotten that other α-haemolytic colonies (the so-
called viridans streptococci) are normally present in the flora of mouth and
throat.

- Tiny, water drop colonies growing as non-haemolytic satellite colonies on

the blood agar plate, but much larger clear colonies on the chocolate agar
or enriched blood agar plates, suggest the presence of H. influenzae. These
colonies are usually present in large numbers, generally more that 20 per
plate.

- Medium-sized, golden-buff colonies are formed by S. aureus. The

coagulase and mannitol fermentation tests are positive, although the slide
coagulase test (“bound” coagulase test) is occasionally negative. If there is
a contradiction between the appearance of the colonies and the slide test,
then a tube coagulase (“free” coagulase) test should be performed.

- Colonies on MacConkey agar suggest that Enterobaceriaceae or

Pseudomonas spp.

- Whitish, round matt colonies on blood agar and chocolate agar plates may
be Candida albicans.

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 4.6 Susceptibility testing

Susceptibility test should be performed only when the amount of growth is

considered significant, and not on every bacterial species present in small
numbers in the culture.

For Enterobacteriaceae and staphylococci the standardized disc-diffusion method

should be used. Strains of S. pneumoniae should be tested on Mueller-hinton
agar.

H. influenzae strains should be tested (on chocolate agar).

5. Stool Culture

5.1 Common pathogens:

Pathogens with high priority

- Salmonella typhi and S. paratyphi

- Shigella
- Vibrio cholerae

Pathogens of intermediate priority

- Non-typhoid salmonellae
- Campylobacter jejuni

Pathogens with low priority

- Echerichia coli (enteropathogenic, enterotoxigenic,enteroinvasive, and

enterohaemorrhagic)

5.2 Culture media and diagnostic reagents:

a. Culture media:

Enrichment media:

- selenite F broth
- alkaline peptone water

Isolation media:

- MacConkey agar
- Salmonella-Shigella agar
- TCBS agar

b. Identification media and reagents

- Kligler iron agar

- Kovac’s reagent for indole
- Oxidase reagent

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5.3 Collection of faecal specimens

Faecal specimens should be collected in early stages of a disease, before

antibiotic treatment is started, when the pathogens are likely to be present in the
stool in high numbers.

The collected stool should be processed as soon as possible upon receipt in the
laboratory, and no longer than 2 hours after collection. Immediate inoculation is
particularly helpful for the identification of Shigella. If it is not possible to process
the specimen within 2 hours, a swab inserted into the stool and rotated together
with any mucus and shreds of epithelium present is inoculated into a suitable
transport medium. Cary-Blair transport medium is appropriate for all enteric
pathogens; alkaline peptone water is suitable for Vibrio spp. If immediate
inoculation is not possible, the specimen should be stored at 4 oC.

5.4 Examination of specimens:

a. Introduction:

Salmonella is generally identified as being a non-lactose fermenting, gram

negative rod shaped organism. With the exception of Salmonella pullorm and
Salmonella gallinarum, they are motile.

Salmonella is oxidase negative, indole negative, urea negative, catalase

positive, citrate positive, lysine positive, ornithine positive, H2S producing
and Gas producing organism.

b. Isolation of Salmonella species:

i. Using a swab inoculate a stool swab on SS agar and place the same swab
in a selenite broth tube.

Incubate at 37o C
For 18-24 hrs.

Inspect SS agar for Salmonella suspected colonies, colorless or yellowish

colonies (non-lactose fermenting) or colonies with black color (H2S
producing).

ii. If no Salmonella suspected colonies on SS agar were observed, subculture

the inoculated selenite broth on SS agar.

Incubate at 37o C
For 18-24 hrs.

Inspect SS agar again for suspected Salmonella colonies

iii. If suspected Salmonella colonies are found confirm by biochemical tests

and perform the serotyping.

iv. If suspected Salmonella colonies are not observed give the result as no
Salmonella growth.

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 c. Identification of Salmonella species:

If suspected Salmonella colonies are observed, perform the API 20E

biochemical tests or the following set of biochemical confirmation tests:

(Table VII.6)
Reaction / Results
Medium
enzymes Negative Positive
TSI, Acid Other reactions A/K
agar slant production

Gas production No air bubbles in butt Air bubbles in butt

H2S production No black color Black color

Urea, Urease Yellow Rose pink
agar slant
Lysine, Lysine Yellow/brown color A purple color
agar slant decarboxylase
MIO:
Indole Indole Yellow ring Red / pink ring
production
Ornithine Yellow Purple

Motility Growth is limited to Growth all over the

the point of inoculation tube

i. Inoculate all of the above mentioned media using a single suspected

colony.

ii. Incubate at 37o C for 18-24 hrs.

iii. Read the biochemical test results noting that Salmonella is oxidase
negative, indole negative, urea negative, catalase positive, citrate
positive, lysine positive, ornithine positive H2S producing and Gas
producing organism.

iv. Perform the serotyping for isolates with biochemical reactions mostly
similar to Salmonella species (Table 2, page 9 of the WHO Laboratory
Protocol refers, copy attached).

d. Serotyping of Suspected Salmonella colonies:

Perform the serotyping using single colonies

e. Reporting of result:

Report your final result as Growth of Salmonella species, or No growth of

Salmonella species depending on your serotyping findings.

5.5 Antibiotic susceptibility

Salmonella are usually susceptible to chloramphenicol, ampicillin and

trimethoprim.

217
6. Vaginal swab:

6.1 Common pathogens:

The vaginal flora of premenopasual women normally consist predominantly of

lactobacilli, and of a wide variety of facultative aerobic and anaerobic bacteria
Abnormal vaginal discharge may be due:

- Vaginitis : Trichomonas vaginalis, Candida albicans

- Bacterial vaginosis : overgrowth of anaerobes and Gardnella vaginalis;
- Cervicitis : Neisseria gonorrhoeae, Chlamydia trachomatis

Other bacteria such as Enterobacteriaceae are not proven to causes vaginitis.

A minimum diagnostic requirement for bacterial vaginosis is the presence of at

least three of the following signs: abnormal vaginal discharge, vaginal pH > 4.5,
clue cells (epithelial cells with so many bacteria attached that the cell border
becomes obscured), and a fishy, amine like odor when a drop of 10% potassium
hydroxide is added to the vaginal secretions. Urethritis in women is also often
caused by N. gonorrhoeae and C. trachomatis.

On special request, cervicovaginal specimens may be cultured for bacterial

species, such as S. aureus (toxic shock syndrome), S. agalactiae ( Group B
streptococci neonatal infection ) , Listetria monocytogenes (neonatal infection )
and clostridium spp ( septic abortion )

6.2 Collection and transport of specimens:

All specimens should be collected during pelvic examination using speculum.

For examination for yeast, T. vaginalis, and bacterial vaginosis, samples of
vaginal discharge may be obtained with a swab from posterior fornix of
the vagina. Amies transport media are convenient for transport of cervical
and vaginal samples.

6.3 Direct examination:

Direct wet mount preparation:

A wet mount is prepared by mixing the vaginal sample with saline on a glass
slide, after which a cover slip is added. A diluted preparation is preferred to
ensure separation of cells, which may be otherwise clumped together.
Examine at magnification of X400 for the presence of T. vaginalis with typical
movement, budding yeast, and clue cells. C. albicans may form pseudomycelia,
which may be observed occasionally in vaginal material. Clue cells are found in
most women with bacterial vaginosis. A granular or dirty appearance of the
epithelial cells cytoplasm is a less objective criterion than the loss of the cell
border. Microscopic examination of a wet mount of cervical specimens is not
recommended.

218
Gram-stained smear:

Preparation of gram stained smear is the method of choice for the diagnosis of
bacterial viginosis. The smear should be prepared by gently rolling, rather than
smearing a swab over the glass slide. A normal vaginal smear contains
predominantly lactobacilli (large gram-positive rods) and fewer than 5 leukocytes
per field. In typical smears from woman with bacterial vaginosis, clue cells
covered with small gram negative rods are accompanied by a mixed flora
consisting a very large numbers of Gram-negative and Gram-variable rods and
coccobacilli, and often a gram-negative rods, in the absence of larger gram-
positive rods, only few (< 5 leukocytes are found per field. This picture is a
sensitive and specific diagnostic indicator for bacterial vaginosis caused by
Gardnella vaginalis. A large number of white blood cells (>10 per field) on the
gram-stained vaginal smear suggests trichomoniasis or cervicitis. Gram staining
is not particularly helpful for the diagnosis of gonococcal infection in female
patients.

219
(Figure VII.6)

1. Clue cells in vaginal wet mount (x 400) 2. Gram-stained smear of vaginal discharge
showing budding yeasts (x 1000)

3. Gram-stained smear of vaginal discharge showing 4. Gram-stained vaginal smear showing a pure flora
Gram-positive yeasts and mycelia (x 1000) of lactobacilli (x 1000)

220
5. Methylene blue stain of a male urethal exudate 6. Potassium hydroxide preparation of vaginal fluid
showing intracellular diplococci (x1000) showing budding yeasts and mycelia (x 400)

7. Trichomonas vaginalis in a wet mount of vaginal 8. Typical clue cell in a Gram-stained vaginal smear
discharge (x 400) (x 1000)

221
7. Ear Swab

7.1 Common pathogens:

Pseudomonas aeruginosa
Staphylococcus aureus
Proteus species
Alpha and beta-hemolytic streptoccoci
Streptococcus pneumonia
Hemophils influenzae

7.2 Culture media and diagnostic reagents:

Isolation media
Blood agar
Chocolate agar
MacConkey agar

Identification reagents
Coagulase plasma
Catalase test
Optochin disc

7.3 Collection of specimen:

Material from the ear, especially that obtained after perforation of the eardrum is
best collected by an otolarngologist, using sterile equipment and a sterile cotton
or polystester swab. Discharges from the ear in chronic otitis media usually
reveal the presence of pseudomonas and proteus species, but often the major
pathogens in chronic otitis media are anaerobes and enteric bacilli. Acute or
subacute otitis usually yields pyogenic cocci.

8. Eye Culture

8.1 Common pathogens:

Staphylococcus aureus
Haemophilus spp.
Streptococcus pneumonia
Neisseria gonorrhea
Alpha and beta-hemolytic streptoccoci

8.2 Culture media and diagnostic reagents:

Isolation media
Blood agar
Chocolate agar (5-10 % CO2)
MacConkey agar

Identification reagents
Coagulase plasma
Catalase test
Optochin disc
X. Sensitivity test:

1. Introduction

Sensitivity test measures the ability of an antibiotic to inhibit bacterial growth in vitro. The
result of the sensitivity test is reported as sensitive or resistant.

A WHO meeting considered that the modified disc technique of Kirby could be
recommended for clinical and surveillance purposes in view of its technical simplicity and
reproducibility. The method is particularly suitable for use with bacteria belonging to the
family Enterobacteriaceae, but it can also be recommended as a general-purpose method
for all rapidly growing pathogens, except strict anaerobes. It was therefore
recommended that the details of this test be mad available for laboratory workers.

2. General principles of antimicrobial susceptibility testing:

Antimicrobial susceptibility tests measure the ability of an antibiotic or other

antimicrobial agent to inhibit bacterial growth in vitro. This ability may be estimated by
either the dilution method or the diffusion method.

3. Clinical definition of terms “resistant” and “susceptible”:

(The three-category system)

The Kirby-Bauer method and its modifications recognize three categories of susceptibility
and it is important that both the clinician and the laboratory worker understand the exact
definitions and the clinical significance of these categories.

a. Susceptible:

An organism is called “susceptible” to a drug when the infection caused by it is likely

to respond to treatment with this drug, at the recommended dosage.

b. Intermediate susceptibility:

It is applicable to strains that are “moderately susceptible” to an antibiotic that can

be used for treatment at a higher dosage. The classification also applies to strains
that show “intermediate susceptibility” to a more toxic antibiotic that cannot be used
at a higher dosage.

c. Resistant:

This term implies that the organism is expected not to respond to a given drug.
Irrespective of the dosage and of the location of the infection.

4. Indications for routine susceptibility tests

A susceptibility test may be performed in the clinical laboratory for two main purposes:

a. To guide the clinician in selecting the best antimicrobial agent for an individual
patient.

b. to accumulate epidemiological information on the resistance of microorganisms of

public health importance within the community.

223
5. Disc Diffusion Method ( the modified Kirby-Bauer method):

This method is well standardized and has been widely evaluated. Official agencies have
recommended it, with minor modification as a reference method, which could be used as
a routing technique in the clinical laboratory.

5.1 Reagents:

a. Mueller-Hinton agar.

b. Antibiotic discs:

Any commercially available discs with the proper diameter and potency can
be used. Stocks of antibiotic discs should preferably be kept at -20oC.

c. Turbidity Standard:

Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 g/liter)

solution of barium chloride dihydrate into a 100-ml graduated cylinder, and
filling to 100 ml with 1% (10 ml/liter) sulfuric acid. The turbidity standard
solution should be placed in a tube identical to the one used for the broth
sample. It can be stored in the dark at room temperature for 6 months,
provided it is sealed to prevent evaporation.

d. Swabs:

A supply of cotton wool swabs on wooden applicator sticks should be

prepared. They can be sterilized in fins, culture tubes, or on paper, either in
the autoclave or by dry heat.

5.2 Procedure:

To prepare the inoculum from the primary culture plate, touch with a loop the
tops of each of 3-5 colonies, of similar appearance, of the organism to be tested.

224
Transfer this growth to a tube of saline.

When the inoculum has to be made from a pure culture, a loopful of the
confluent growth is similarly suspended in saline.

Compare the tube with the turbidity standard and adjust the density of the test
suspension to that of the standard by adding more bacteria or more sterile
saline.

Proper adjustment of the turbidity of the inoculum is essential to ensure that the
resulting lawn of growth is confluent or almost confluent.

Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess
inoculum by pressing and rotating the swab firmly against the side of the tube
above the level of the liquid.

225
Streak the swab all over the surface of the medium three times, rotating the
plate through an angle of 60° after each application. Finally, pass the swab round
the edge of the agar surface. Leave the inoculum to dry for a few minutes at
room temperature with the lid closed.

The antibiotic discs may be placed on the inoculated plates using a pair of sterile
forceps. It is convenient to use a template to place the discs uniformly.

A sterile needle tip may also be used to place the antibiotic discs on the plate.

Alternatively, an antibiotic disc dispenser can be used to apply the discs to the
inoculated plate

226
A maximum of seven discs can be place on a 9-10 cm plate. Six discs may be
spaced evenly, approximately 15 mm from the edge of the plate, and I disc place
in the center of the plate. Each disc should be gently pressed down to ensure
even contact with the medium. The plates should be placed in an incubator at
35° C invalidate results for oxacillin/meticillin. Do not incubate in an atmosphere
of carbon dioxide.

After overnight incubation, the diameter of each zone (including the diameter of
the disc) should be measured and recorded in mm. The results should then be
interpreted according to the critical diameters shown in table. The
measurements can be made with a ruler on the under-surface of the plate
without opening the lid.

If the medium is opaque, the zone can be measured by means of a pair of

calipers.

227
 A template may be used to assess the final result of the susceptibility

5.3 Interpretation of the zone sizes:

As recommended in the manufacturer instruction.

5.4 Technical factors influencing the size of the zone in the disc diffusion method:

a. Inoculum density: If the inoculum is too light, the inhibition zones will be
larger although the sensitivity of the organism is unchanged. Relatively
resistant strain may then be reported as susceptible. Conversely, if the
inoculum is too heavy, the zone size will be reduced and susceptible strains
may be reported as resistant. Usually optimal results are obtained with an
inoculum size that produces near confluent growth.

b. Timing of disc application: If the plates, after being seeded with the test
strain, are left at room temperature for periods longer than the standard
time, multiplication of the inoculum may take place before the discs are
applied. This causes a reduction in the zone diameter and may result in a
susceptible strain being reported as resistant.

c. Temperature of incubation: Susceptibility tests are normally incubated at 35

C for optimal growth. If the temperature is lowered, the time required for
effective growth is extended and larger zones result. When a heterogeneous
resistant strain of staphylococcus is being tested against meticillin (oxacillin),
the resistant portion of the population can be detected at 35 C. At higher
temperatures the entire culture appears to be susceptible. At 35 C or lower
temperatures, resistant colonies develop within the zone of inhibition. These
resistant colonies can be seen more easily if the plate is left for several hours
at room temperature before the result is read. Such colonies should always
be identified to check whether they are contaminants.

d. Incubation time: Most techniques adopt an incubation period of between 16

and 18 hours. In emergencies, however, a provisional report may be made
after 6 hours. This is not recommended as a routine and the result should
always be confirmed after the conventional incubation time.

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e. Size of plate, depth of agar medium, and spacing of antibiotic discs:
Susceptibility tests are usually carried out with 9-10 cm plates and no more
than 6 or 7 antibiotic disc on each plate. If larger numbers of antibiotics
have to be tested, two plates, or one 14-cm diameter plate, is to be
preferred. Excessively large inhibition zones may be formed on very thin
media; the converse is true for thick media. Minor changes in the depth of
the agar layer have negligible effect. Proper spacing of the disc is essential
to avoid overlapping of the inhibition zones or deformation near the edge of
the plates.

f. Potency of the antibiotic discs: The diameter of the inhibition zone is

related to the amount of drug in the disc. If the potency of the drug is
reduced owing to deterioration during storage, the inhibition zone will show
a corresponding reduction in size.

g. Composition of the medium: The medium influences the size of the zone by
its effect on the rate of growth of the organism , the rate of diffusion of the
antibiotic, and the activity of the agent. It is essential to use the medium
appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for
some test organism clearly demonstrate the need for standardization of disc
diffusion methods. Only if the conditions laid down in a particular method
are closely followed can valid results be obtained. Alteration of any of the
factors affecting the test can result in grossly misleading reports for the
clinician.

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5.5 Basic Sets of Antibiotics for Routine Susceptibility Testing

Urine Samples:
Gram -ve isolates Gram +ve isolates
Cefuroxime Cefuroxime
Cephalexin Cephalexin
Ciprofloxacin Ciprofloxacin
Co-amoxiclav Co-amoxiclav
Co-trimoxazole Co-trimoxazole
Gentamicin Erythromycin
Norfloxacin
Doxycyclin

Throat Swabs: (S. Pneumonia, Non-Hemolytic Streptococcus, H. Influenza)

Amoxicillin
Azethromycin
Cefuroxime
Cephalexin
Co-amoxiclav
Co-trimoxazole
Erythromycin
Doxycyclin

Pus Specimens: (Staphylococcus, Streptococcus, Enterobacter)

Amoxicillin
Azithromycin
Cephalexin
Ciprofloxacin
Co-amoxiclav
Co-trimoxazole
Erythromycin
Penicillin

Ear Swabs: (Pseudomonas, Streptococcus, Staphylococcus, Enterobacter,

Proteus, Klebsiella)
Amoxicillin
Azithromycin
Ciprofloxacin
Co-amoxiclav
Co-trimoxazole
Erythromycin
Gentamicin

Stool Specimens: (Salmonella, Shigella)

Azithromycin
Cefuroxime
Ciprofloxacin
Co-trimoxazole
Erythromycin
Gentamicin

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XI. Gram Stain

This is the most widely used staining method, and most organisms are classified by their
reaction to Gram's stain. The basic dye (crystal violet) diffuses into the organism and forms a
water-insoluble lake with the iodine. The decolorizing alcohol dehydrates the walls of gram-
positive organism, forming a barrier through which the Dye Lake cannot pass. In gram-
negative cells, the liquids in the wall may be dissolved, allowing the crystal-iodine complex to
escape.

1. Procedure

a. Prepare and fix film as follows: a thin emulsion is made in a drop of saline from the
examined sample. Smear thinly on a slide with a loop. The air-dried film is fixed by
gentle heating above a Bunsen burner flame by passing through the flame.
b. Stain with crystal violet for 1 minute
c. Wash with tap water
d. Cover the slide with iodine for 1 minute
e. Decolourise with alcohol until no more blue washes out
f. Wash with tap water
g. Counter stain with safranine for 30 seconds
h. Rinse with tap water and dry
i. Examine microscopically using the oiled 100 X objective.

2. Results

The gram-stained smear may show:

a. A gram-positive organism which may appear with blue colour.

b. A gram-negative organism which may appear with red colour.

3. Reagents

a. Crystal violet 0.5% in distilled water

b. Gram's iodine

iodine 1g
potassium iodide 2g
distilled water 300 ml

c. 70% alcohol

Ethyl alcohol 70 ml
Distilled water 30 ml

e. Safranine 0.5%

Safranine stain 0.5 g

Distilled water 100 ml

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(Table VII.7)
Gram-negative organisms Gram-positive organisms
Escherichia coli Staphylococcus aureus
Klebsiella species Staphylococcus epidermidis
Enterobacter species Alpha-haemolytic streptococci (Viridans)
Proteus species Streptococcus pneumoniae
Salmonella typhi Streptococcus faecalis (group D)
Salmonella species Streptococcus pyogenes (group A)
Pseudomonas aeruginosa Streptococcus agalactia (group B)
Neisseria meningitides Listeria monocytogenes
Hemophilus influenza Clostridium perfringens
Bacteroides fragilis (anaerobe) Peptococcus species (anaerobes)
Brucella species Peptostreptococcus species
Pseudomonas pseudomallei Candida albicans and other yeast-like
fungi (e.g. Cryptococcus)

XII. Ziehl-Neelsen Stain

1. Procedure

a. Prepare smear from sputum or from culture and let it for air-drying
b. Fix by passing through the flame.
c. Cover the slide with carbol Fuchsin, heat and wait for 5 minutes (don't boil but look
for steam vapour).
d. Wash with Distilled Water.
e. Decolourise with acid-alcohol, cover for 1-2 minutes and check for complete
decolorization.
f. Wash with tap water
g. Counter stain with methylene blue for 30 seconds.
h. Wash with tap water.

2. Reagents

a. Ziehl Neelsen Stain:

Solution A:
Basic Fuchsin 1.5 g
95% Ethanol 50 ml

Solution B:
Phenol 25 g
Distilled water 500 ml

Working Solution: Mix 10 ml from Solution A with 90 ml from Solution B.

Warning: This solution is highly corrosive and poisonous.

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 b. Acid Alcohol

HCL concentrated 3 ml
Ethyl alcohol 95% 97 ml

c. Aqueous Methylene Blue

Methylene blue, Aqueous 0.3 g

Distilled water 100 ml

XIII. Direct Microscopic Examination for Fungi:

Traditionally, the potassium hydroxide preparation has been the recommended method for
direct microscopic examination of clinical specimens. Between 10-20% of the specimens,
which show fungi by this method, are negative on culture.

1. Procedure:

The fungi may be demonstrated in direct smears by placing some of the material (hair,
skin scraping, nail snipping or pus, etc.) in a drop of 10% KOH and warming the
preparation slightly. Smears are then covered and the examination (for mycelia and
spores) is made after 5-15 minutes. The KOH dissolves the tissue cells and clears the
fungi cells. Study is usually made under low power.

2. Sample collection:

Samples collected from lesions may be obtained by scraping the skin or nails with a
scalped blade or microscope glass slide, and infected hairs are removed by plucking
them with forceps.

Skin scrapings:

a. Cleanse the affected area with an alcohol swab.

b. Scrape or remove crusts close to the margin with a sterile scalpel.

Nails:

a. Cleanse the nail with an alcohol swab.

b. Take snipping of the infected part of the nail with sterile scissors or a scalpel.

Hairs:

a. Remove fluorescent or broken, dull hairs with sterile forceps for examination.

Storage: Room temperature.

Transportation: No special requirements.

233
234
PART VIII – REFERENCE VALUES

235
236
 REFERENCE VALUES

(Table VIII.1)
Test Conventional Units Factor SI-Units
Haematology
M: (4.5-5.5)x106/mm3 (4.5-5.5)x1012/L
Erythrocyte (RBCs) Count 106
F : (4.2-5.2)x106/mm3 (4.2-5.2)x1012/L

MCV (76-96) µm3 1 (76-96) fL

MCH (27-32) pg 1 (27-32) pg

MCHC (30-36)% 0.01 (0.30-0.36) Conc. fraction

Leucocytes (WBCs) Count (4.0-10)x103/mm3 106 (4.0-10)x109/L

Platelets (150-400)x103/mm3 106 (150-400)x109/L

Reticulocytes (0.2-2.0) % 0.01 (0.002-0.02) No. fraction

M: (13.5-18)g/dl (135-180)g/L
Haemoglobin 10
F : (12.0-16)g/dl (120-160)g/L
M: (40-54)% (0.4-0.54) Vol. fraction
Haematocrit 0.01
F : (37-47)% (0.37-0.47) Vol. fraction
M: (1-10)mm/hr, (1-10)mm/hr,
ESR 1
F : (3-15) mm/hr (3-15)mm/hr

Clotting Time (5-12) minutes 1 (5-12) minutes

Bleeding Time (1-5) minutes 1 (1-5) minutes

Biochemistry
FPG (70-110) mg/dl 0.05551 (3.9-6.1) mmol/L

Urea (15-50) mg/dl 0.1665 (2.5-8.3) mmol/L

Creatinine (0.6-1.3) mg/dl 88.4 (53-115) µmol/L

M: (3.7-7.8) mg/dl (226-468) µmol/L

Uric Acid 59.48
F : (2.7-7.3)mg/dl (140-430) µmol/L

Cholesterol (150-250) mg/dl 0.0258 (3.9-6.5) mmol/L

HDL-Cholesterol (35-60) mg/dl 0.0258 (0.9-1.6) mmol/L

LDL-Cholesterol < 150 mg/dl 0.0258 < 3.9 mmol/L

M: (40-160) mg/dl (0.45-1.8) mmol/L
Triglycerides 0.01143
F : (40-140)mg/dl (0.45-1.6) mmol/L
Total Bilirubin : Adult <1.1 mg/dl 17.1 <18.8 µmol/L
: Neonatal < 12 mg/dl 17.1 <205 µmol/L

Direct Bilirubin < 0.25 mg/dl 17.1 < 4.3 µmol/L

Total protein (6-8) g/dl 10 (60-80) g/L
Albumin (3.8-5.1)g/dl 10 (36-51)g/L

237
 Test Conventional Units Factor SI-Units
SGOT / AST < 37 U/L 1 < 37 U/L

SGPT / ALT < 40 U/L 1 < 40 U/L

Alkaline phosphatase Men : 80-306 U/L 1 80-306 U/L

Women : 64-306 U/L 64-306 U/L
Children : upto 644 U/L upto 644 U/L

238