GENERAL INSTRUCTIONS

Before starting the microbiology practicals, read the following instructions and

follow them accordingly.

I. It is essential to maintain cleanliness while working in a laboratory. Keep the

apparatus, microscope and the working table clean and orderly. Student should wear a

clean apron in the lab.

II. Eating, drinking, and using mobile phones not allowed in lab.

III. Wash hands with soap at the start and end of the lab.

IV. Take care not to spill any culture on the desk.In case of accident clean with a

disinfectant.

V. All non-infectious solid waste like cotton, paper should be placed in the waste basket.

VI. The culture loop should be sterilized vertically in flame before and after making a

smear.

VII. Glassware such as test tube, pipettes, and lask should be carefully plugged with non-

absorbent cotton.

VIII. Use clean and dry slides and coverslips. Just before making a smear pass slide through

flame few times.

 THE MICROSCOPE

Acquaint yourself with mechanical construction and names of various points on the
manipulation and ease of instrument.

The use of the microscope

I. Remove the microscope from cabinet by grasping the microscope arm firmly with the right
hand and the base with the left hand, carry it close to the body and place it gently on laboratory
table.
II. Place the microscope with its arm facing the user approximately 15 cm from the edge of the
laboratory table.
III. The stage should be kept in a horizontal position. This prevents immersion oil or other
liquid from running off slide.
II. Bring the draw tube to the std. length for which the objective is corrected. The exterior lens
of the eyepiece should always be carefully cleaned before use by whipping with lens paper.
Being exposed, this lens easily accumulates dust from the air.
IV. Objective lens should be always kept clean. They are best cleaned by wiping lens paper.
Under no conditions should objectives be removed from the resolving nose piece unless it's
necessary to clean back lens for this purpose follow the same procedure as under.
V. Dried cedar wood immersion oil may be removed by rubbing the objective lens with a piece
of lens paper previously moistened with a drop of xylol. If paraffin oil is used instead of cedar
wood oil, use of solvent is not necessary.
VI. Set up the microscope so that the light is properly reflected through the plate or mirror.
VII. If the microscope is equipped with a sub-stage condenser, always use the plane mirror. If
used without a condenser, either surface maybe employed. the concave mirror yielding more
intense illumination.
VIII. First focus the slide under low power objective (10X) then under high power objective
(40X/45X) and oil immersion objective (100X) if necessary.
IX. In focusing low power objective, first place slide upon the stage of the microscope under
the spring dips. Swing low power objective under body tube and using the coarse adjustment
until the image is distinct.
X. Focusing the high-power objective, first swing the lens under the body tube then while
looking between the objective and the slide, slowly lower lens with coarse adjustment until the
front of the objective almost touches the slide focus upward by means of the fine adjustment
until images is sharp focus.
XI. In focusing oil immersion objective, first place a drop of oil over the object on the slide.
Lower objective very carefully with the coarse adjustment until contact is made with glass.
Don't lower the objective too rapidly; otherwise, there will be danger of cracking the glass
slide. Then focus upward with fine adjustment until images are a sharp focus.
XII. If the fine adjustment fails to function its lens has probably been summed up too far. Screw
it down about half length of threads before focusing on object.
XIII. The sub-stage diaphragm serves several purposes. It may be used to control the intensity
of illumination, but it's not recommended for this purpose. Neutral filters should be used to
reduce the intensity of light. Too much light will cause an uncomfortable glare, whereas too
little will cause undue exertion; both should be avoided. The main functions of the diaphragm
are to increase contrast and to improve definition. Different diaphragm openings are required
for different types of work within the same slide preparation.
XIV. Never close one eye when using a microscope. This causes eyestrain; make a habit of
keeping both eyes open. This may seem difficult but will become a habit soon.

The care of Microscope:

I. Keep it free from dust- This rule should be observed at all times
II. When handling microscope, grasp it by handle arm.
III. Do not tip over microscope.
IV. Do not use alcohol on lacquered parts of instrument as it acts as solvent lacquered prevents
oxidation of metal and should not be removed. Although modern finishes are relatively alcohol
resistant some of them are nevertheless more soluble in alcohol than in xylol.
V. Keep coarse adjustment free from dust if rack and pin ion need lubricating, use small amount
of non-corrosive petroleum jelly.
VI. Do not use oil for lubricating a sliding bearing.
VII. Don't carelessly twin the coarse adjustment screw to permit violet contact of the front lens
of objective with glass slide. This may twin objective lens.
VII. Make it a habit to clean immersion lens after using the microscope. Use lens paper for
wiping the lens.
AIM : To study the tools used in microbiology laboratory

1) Inoculation loop: It is a simple tool used by microbiologists to retrieve an inoculum from a

culture of microorganisms. It is used to transfer the culture on to the plates or tubes into the
culture media. The loop of the wire might be platinum, tungsten or nichrome. It is sterilized in
a flame before and after use.

2) Inoculation needle: It is a laboratory equipment used to transfer and inoculate living

microorganisms. It is usually a nichrome or platinum wire attached to a metallic handle The
base of the needle is dulled, resulting in a blunt end.

3) Autoclave: This instrument is used for moist heat sterilization. When water boils, steam is
generated. It works on the principle that on increasing the pressure, the temperature also
increases according to Charle’s law. Hence, pressure inside a closed vessel increases the
temperature (boiling point) of water. Ssaturated steam is generated which has a greater
penetrating power. The steam condenses upon coming in contact with a cool surface, hence
provides moist conditions. All microbes are destroyed by denaturation and coagulation of
proteins. The conditions for moist heat sterilization are 121°C temperature, pressure 15 psi and
time 15-20 minutes.

4) Hot air oven: It is based on the principle of sterilization using dry heat or hot air. It is less
effective than moist heat. Microbes are destroyed by protein denaturation and oxidation due to
slow burning. For dry heat sterilization, oven should be operated at 160°C to 180°C for 2
hours.

5) Incubator: It is used to provide favourable conditions for growth of microorganisms in a

culture. It is similar to the oven in construction, it consists of an insulated cabinet fitted with a
heating element. The temperature is controlled by a thermostat. It has double doors, inner one
is glass to view the contents without admitting outside air.

6) Refrigerator: It is used for storing media and stock cultures at 4°C.

6) Quebec colony counter: The colonies produced on a petri plate are counted by this. They
are of 2 types, a manual colony counter which is a small cabinet having a circular grid in the
center of a provision to illuminate the grid. Petri Plate is placed on the circular grid of the
colony counter. Colonies are counted using a large magnifying glass. In electrically operated
counters there is a pencil shaped electrode and a LCD display.

7) Laminar air flow: It is a cabinet in a carefully inclosed bench designed to prevent

contamination of any particle sensitive materials. Air is drawn through a high efficiency
particulate air (HEPA) filter and blown towards the user. It is made of stainless steel with no
gaps or joints. They exist in both horizontal and vertical configurations with a variety of airflow
patterns.The air is claimed to be 99.97% free from microbial contamination. This is based upon
removal of dioctylphthalate particles of size 0.3μm and larger. Quality control procedures must
be adopted to evaluate and monitor the quality of laminar air flow. Air quality is evaluated
using settling plate technique, particle counters and microbial air samplers.
AIM :- To prepare culture media for growth of bacteria.

REQUIREMENTS

● Apparatus: 1000 ml beakers , glass rod, spatula , conical flasks

● Chemicals: Beef extract ,peptone, NaCl, Agar Agar, distilled water

1. Composition of Nutrient Broth

Beef Extract-3g
Peptone - 5g
NaCl-5g
Distilled water - 1000ml

Introduction: For any bacterium to be propagated for any purpose it is necessary to provide
the appropriate biochemical and biophysical environment. The biochemical (nutritional)
environment is made available as a culture medium. Media are the substances over which
micro-organisms are cultured or grown to harvest in a larger amount. Depending upon the
special needs of particular bacteria, a large variety and types of culture media have been
developed with different purposes and uses. Culture media are employed in the isolation and
maintenance of pure cultures of bacteria and are also used for identification of bacteria
according to their biochemical and physiological properties.
Nutrient broth and agar is a general-purpose/basal medium that supports the growth of a wide
range of non-fastidious organisms (microbes that can grow and thrive without specific
nutritional or environmental conditions). It is commonly used for cultivation and maintenance
of microbial cultures for scientific study or identification.
However, the nutrient agar/broth media can be enriched with the addition of blood or other
biological fluids (usually 10%) such as serum, to make the medium suitable for the cultivation
of associated fastidious organisms.
The relatively simple formulation of nutrient broth/agar is ideal for the cultivation of
microorganisms that are not specific in their nutritional requirements. The medium consists of
beef extract (or yeast extract), peptone, sodium chloride and agar (in case of nutrient agar).
The beef extract/yeast extract contains water-soluble substances, including carbohydrates,
water-soluble vitamins, organic nitrogen compounds, and salts.
Peptone (enzymatic digest of animal proteins) is the primary source of organic nitrogen,
particularly amino acids and long-chained peptides for the growing bacteria.
Sodium chloride maintains the osmotic equilibrium between the microorganisms and the
medium, agar acts as a solidifying agent and has no nutritive value.
Agar, a polysaccharide obtained from sea weeds is used as a solidification agent.
Procedure: Weigh the ingredients of nutrient broth and dissolve in distilled water. Warm water
can be used for dissolving the ingredients. Adjust pH to 7.2 with pH paper or bromothymol
blue indicator. Make the volume to 1 litre with distilled water. Pour approximately 10 ml into
each test tube. Plug the test tubes with non-absorbent cotton and sterilise at 15 psi and 121°C
for 15 min in an autoclave.

2. Nutrient Agar: To nutrient broth add 1.5 % Agar. Boil the media with a constant stirring
until all agar has dissolved. For making slant and stab, pour about 10 ml of nutrient agar in
each test tube and plug with cotton. Sterilise at 15 psi and 121°C For 15 mins. After sterilisation
keep the slant tubes in an inclined position till agar solidifies..For agar stab tubes keep tubes in
a vertical position. For making agar plates, pour nutrient agar in sterile dishes while it is hot.
After solidification of nutrient agar, slant and stab tubes can be inoculated with desired species
of microorganism. Nutrient agar plates can be streaked using desired microorganisms.
AIM – Techniques of inoculation of nutrient broth, stab, slant and streak plate.

REQUIREMENTS-
• Apparatus – Test tubes, inoculation loop, Petri plates
• Cultures- any broth culture
• Sterile nutrient broth, slant, stab and Petri-plates having sterile nutrient agar

PROCEDURE-
Disinfect the working area by cleaning table surface with disinfectant and carry out the work
between two burners (Aseptic area)
A. Inoculation of Nutrient Broth

1. Take tube in your left hand & inoculating loop in your right hand.
2. Heat inoculating loop in flame until nichrome wire is hot and then allow it to cool for 45
secs.
3. Remove the closure one at a time from the tubes or simultaneously considering
minimum time available for inoculum (to avoid microbial contamination and heat at
neck of the tube with burner.)
4. Insert the sterile inoculating loop into stock culture and remove small amount of
culture.
5. This inoculum is then transferred to the uninoculated broth.
6. Reflame the neck of the tubes and close with respective caps.
7. Flame the inoculating loop after inoculation.
8. Put the culture tube in the rack meant for it & loop at its proper place.
9. At the end put all inoculated tubes in the incubator set at 37°C and observe growth as
turbidity after 24-48 hours.

B. Inoculation of Stab Culture

1. Prepare a sterile nutrient agar media.
2. Inoculate the culture of given microorganism in stab by sterile needle.
4. Incubate the culture tube at 37°C for 24-48 hours in an incubator and observe the growth of
microorganism.

C. Inoculation of Slant Culture

1. Prepare a sterile nutrient agar slant as described in the previous experiment.
2. Inoculate the culture of given microorganism in slant tube by sterile inoculation loop
3. Incubate the culture tube at 37°C for 24-48 hours in an incubator and observe the growth of
microorganisms.
4. Slants and Stabs can be stored as stock cultures for 2-3 months when kept at 4 °C in a
refrigerator.

D. Streak plate method

1. Prepare sterile nutrient agar Petri-plates as described in the previous experiment.
2. Take loop full of culture & streak in a manner as shown in the figures using different patterns
for streaking.
3. Incubate the Petri-plates, upside down at 37°C for 24-48 hours in an incubator and observe
the growth of microorganisms.
AIM - To study the bacterial morphology by Negative staining.

REQUIREMENTS –
❖ Culture of Klebsiella pneumoniae
❖ Stain: Nigrosin Black or Congo red
❖ Apparatus: Glass slides, filter paper, Bunsen burner, inoculating loop
❖ Equipment: Microscope.
❖
PRINCIPLE-
Negative (indirect) staining is a technique by which bacterial cells are not stained but are made
visible against dark background. The acidic Nigrosin black, Congo red, eosin, Rose Bengal
stains are thus used. Acidic stains are negatively charged; therefore, they don’t bind to
negatively charged bacterial cell surface. On the other hand, they form a deposit around the
cell resulting in the appearance of colorless bacterial cell against the dark background. Negative
staining technique has an advantage even the direct or positive staining methods for the study
of morphology of cells. This is because heat fixation is not required for cells that do not receive
vigorous physical or chemical treatments, therefore the natural size and shape of the micro-
organisms can be seen by this method. It is possible to observe bacteria that are difficult to
stain e.g. Spirillium
PROCEDURE -
(a) Using sterile technique, place a loopful of the bacterial culture on the slide with help of
inoculation loop.
(b) Immediately add a drop of Nigrosin black stain.
(c) Mix and spread out in a thin film (thick films will crack and peel off) using a second
slide held at an angle of 45 degrees.
(d) Air dry and observe the slide under oil immersion objective.
PRECAUTION –
1. Do not heat fix the slide,
2. Too thick layering of the staining solution will not allow light to pass through the object
and stain will crack on drying.

RESULT:
AIM- To study the bacterial morphology by capsule staining technique.

REQUIREMENTS-
● Culture: 48 hours nutrient broth culture of Klebsiella pneumoniae.
● Stains and Reagents:
Solution A : Congo Red (1% in distilled water)
Solution B: Maneval's Reagent
Composition of Maneval’s Reagent:
5% Phenol solution (30 mL),
20 % Acetic acid (100 ml),
30%. Ferric chloride solution (4 ml)
1%. Acid Fuschin solution (2ml)

● Apparatus : Staining tray, Bunsen burner ,innoculating loop. glass slides.

● Equipment: Microscope.
.
PRINCIPLE- A capsule is a gelatinous outer layer that is secreted by the bacterial cell and
adheres to the cell wall. Capsulated bacteria are generally virulent and capable of producing
disease as capsule gives protection against phagocytosis by macrophages in host body.
Capsules can be made of polysaccharides, polypeptide, or glycoprotein.
Capsules are non ionic in nature and so they do not get stained easily. By using negative
staining method, demonstration of capsule becomes easy. Congo red (Stain A) stains acts an
acidic stain and stains the background red. When we add Maneval’s reagent (Stain B), acid
fuschin stains the cell pink. Due to acetic acid in Maneval’s reagent, pH is shifted to the acidic
side, due to which congo red stain turns blue and thus background turns from red to blue. Thus
a clear contrast is created created between pink cell, colourless capsule and blue background.
PROCEDURE
1) With the help of inoculation loop, aseptically take a drop of bacterial culture on a clean
grease free slide. Do not heat fix the slide.
2) Place a drop of solution A on the slide and mix with a drop of the given culture.
3) Spread this evenly in the form of a smear and allow it to air dry.
4) Place a drop of solution B on one end of the slide and prepare thin film with the help of
another slide keeping it at 45-degree angle.
5) Allow it to air dry.
6) Observe under oil immersion lens.
AIM -To study the bacterial motility by hanging drop technique.

REQUIREMENTS -
Cultures: Broth cultures of Escherichia coli and Saccharomyces cerevisiae
Chemicals: Vaseline or petroleum jelly
Apparatus: Cavity (depression) slides, Inoculation loop, burner, cover slip
Equipment: Microscope

PRINCIPLE -
Living microorganisms can be studied to determine the natural size and shape of cells, cellular
arrangement motility, reactions to various chemicals and response to environmental factors. Wet
mount and hanging drop techniques are routinely used for the study of bacterial motility. This
technique has the following advantages.
1. It is less time consuming and easy to study the motility of the bacterial
2. Capacity of aeration is increased as the hanging drop is surrounded by an air space.
3. This technique is also used for the observation of size, shape of microorganisms in living
state.
Disadvantages are -
1. The hanging drop may evaporate in some time.
2. In this technique, three different refractive indices are involved in the light path. Hence good
microscopy is not possible with this preparation.
Flagella are responsible for motility of bacteria and are thus called the organ of locomotion. The
number and arrangement of flagella are characteristics of each bacterium. Flagella may be
arranged on the bacterial body as monotrichous, lophotrichous , amphitrichous or peritrichous. The
flagella has three basic parts: filament, hook and basal body.

PROCEDURE-
1) Clean a depression slide and a cover slip and place it on the table.
2) Clean a coverslip and apply petroleum jelly to each of the four corners of the coverslip.
3) Using fine needle, make a thin layer of petroleum jelly on the periphery of the cavity of the
slide.
4) Place a loopful of bacterial suspension to be examined aseptically in the centre of the cover slip
5) Invert the cavity slide over the coverslip containing the culture drop in such a manner that the
drop comes in centre of the cavity.
6) Allow coverslip to adhere to the jelly and gently turn the slide so that the drop of culture hangs
in the cavity.
7) Place the slide on microscope stage and carefully focus the edge of the drop so that it appears
cross the centre of the field.
8) Locate the droplet with the aid of low power objective and then observe under a high
pressure lens.
9) Observe the behaviour of microorganisms in the inner side of the drop edge and record size,
shape and motility.
10) Check whether the motility is true motility (microorganism changes its position and moves
from one place to another) or Brownian movement (vibrating or dancing movement of bacteria in
suspension)
AIM: To study the cell wall of the given microorganism by staining technique.

REQUIREMENTS
Cultures: 24 Hour nutrient broth culture of Bacillus subtilis and Saccharomyces cerevisiae
Stains: Crystal violet (0.5%), Congo red (0.5%)
Reagents: Tannic acid (5%)
Apparatus: Glass slides, staining tray, inoculating loop, Bunsen burner.
Equipment: Microscope

PRINCIPLE:
Cell wall is a rigid structure which gives a definite shape to the cell, situated between the outermost
slime or capsule and cytoplasmic membrane. It is involved in growth and cell division of bacteria.
Cell wall contains diaminopimelic acid (DAP), muramic acid and teichoic acid. These substances
are joined together to give rise to a complex polymeric structure known as peptidoglycan which
provides rigidity to the cell.
The cell wall has low affinity for the stain and therefore, it is not stained with most of the
staining techniques. In cell wall staining technique mordant is applied which increases the affinity
of cell wall for stain. It also increases apparent thickness of cell wall due to deposition of fine
precipitates.
Cell wall and cytoplasm are acidic in nature where cell wall is more acidic than cytoplasm. So,
when we apply the basic stain Crystal Violet, it stains the cytoplasm as well as the cell wall. Congo
red is a selective decolorizing agent it selectively decolorizes the less acidic portion that is
cytoplasm. Hence, cell wall remains stained by Crystal violet and cytoplasm appears colorless.

PROCEDURE:
Ringen et al method-
1) Prepare a smear of the given culture, air dry and heat fix.
2) Flood the smear with 5% aqueous solution of tannic acid for 30 minutes.
3) Rinse the slide with water. Do not dry.
4) Stain the smear with crystal violet for 2-3 minutes.
4) Wash the smear with water.
5) Treat the smear with 5% Congo red solution for 2-3 minutes.
6) Wash with water and allow it to dry.
7) Examine the slide under an oil immersion objective.
AIM - To study the morphology of the given bacterial culture by Gram’s staining (differential
staining).
REQUIREMENTS –
1. Cultures: Nutrient broth (24 hours) culture of Escherichia coli, Bacillus subtilis,
Saccharomyces cerevisiae , Mixed culture
2. Stains: Crystal violet, Safranin
3. Reagents: Gram's Iodine, Decolorizer (Alcohol and acetone in 1:1 ratio)
4. Apparatus: Staining tray, Bunsen burner, inoculating loop, glass slides
5. Equipment Microscope.
PRINCIPLE –
Gram staining is one of the most important and widely used differential staining, which was
discovered by Christian Gram in 1884. This technique divides bacteria into two groups-
a. Gram negative: Those which loose the primary stain on application of decolorizer and take the
colour of a counter- stain like safranin or basic fuchsin. Red/Pink in appearance.
b. Gram positive: Those which retain primary stain like crystal violet and appear deep violet in
colour.
The gram staining uses four different reagents:
1. Primary stain (crystal violet): The crystal violet stain is used first and stains all cells deep violet
in colour.
2. Mordant (Gram's Iodine): A mordant may be defined as any substance that forms an insoluble
compound with stain and serves to fix the colour to bacterial cell. It should be noted that mordant
is not a stain. It leads to form crystal violet Iodine - magnesium ribonucleate complex in Gram-
positive bacteria. This complex is not formed in Gram-negative bacteria, as magnesium
ribonucleate is absent in the cell wall. Hence, only CV- I complex is formed in Gram-negative
bacteria.
3. Decolorizing agent (Acetone-alcohol in 1:1 ratio): Cell wall of Gram-positive cell wall contains
less lipid. On application of decolorizing agent like alcohol or acetone, shrinkage of cell wall takes
place due to dehydration and decreases the permeability for CV-l-Mg ribonucleate complex. Thus,
the complex is retained in the cell and cell is stained deep violet in colour. On the other hand, the
treatment of decolorizing agent extracts lipid from cell wall of Gram-negative cell and there
is increase in permeability property of cell wall. Due to this, CV- I complex extracted and cells get
decolorized.
4. Counterstain (Safranin). This is the final reagent to stain red, those cells that have been
previously decolorized. Since only Gram-negative cells are decolorized, they may now absorb the
counterstain. Gram positive cells retain the violet colour of the primary stain.

PROCEDURE-
1. Take a clean, grease-free slide and prepare a smear from a 24 hour nutrient broth culture.
2. Air dry and heat fix it.
3. Cover the smear with crystal violet stain for 1 minute.
4. Wash the smear with water and cover it with Gram iodine (mordant) for 1 minute
5. Wash the smear with water and decolorize with acetone alcohol very carefully till the washing
does not contain violet colour.
6. Rinse the smear with water and counterstain with safranin for 1 minute.
7. Wash smear with water and let it air dry.
10. Examine under oil immersion objective and record your results.
AIM: To perform the test for sterility of the given samples.

Introduction: Test for sterility is intended for detecting the presence of viable/spore forms of
microorganisms in all parenteral preparations and on all pharmaceutical preparations which need
to be sterile. A number of products need to be sterile like ophthalmic preparations, infections,
infusions, implants, syringes, bandages, needles, surgical instruments etc and must be checked for
contamination of microorganisms. The test must be carried out under aseptic conditions to avoid
accidental contamination of the product during the test. The working area in which the test is
performed should be routinely monitored by sampling air and surface of the working area.

Principle: The test for sterility is based on the principle that when microorganisms are placed in a
medium which provides nutrient material and water and incubated at a suitable temperature, the
organism will grow, and the growth is indicated by turbidity in an originally clear medium.

Random sampling :

Th samples in a batch are selected for sterility testing by random sampling as per guidelines given
in pharmacopeia.

The table below indicates the number of items recommended to be tested in the batch as per I.P.
Culture Media:

Culture media must be capable of promoting vigorous growth of a small number of contaminating
aerobic and anaerobic organisms.

1. Fluid thioglycolate media: It is used with clear fluid products.

2. Alternative thioglycolate media: It is used for turbid/viscid products and for devices having
tube with small lumina.
3. Soyabean-Casein digest media: It is used for growth of fungi.

The pH of media is adjusted to 7.2 and sterilized by autoclaving.

The table below gives the composition of the media used in sterility testing.
Sterility of the Media: To be tested by incubating the small portions of the autoclaved media at
30-35 0C (FTM/Alternate FTM) and 20-25 0C (Soyabean Casein digest media) for 48 hours.

Growth Promotion Test: Test each autoclaved load of media for its growth promoting quality by
separately inoculating duplicate test containers of each medium with about 100 viable
microorganisms of each of the strains listed in Table below. The test media is satisfactory if clear
evidence of growth appears in all inoculated media containers within 7 days.

Controls: Two types of controls are required to be incorporated in all sterility testing experiments

1. Negative control: To ensure sterility of medium

2. Positive control: To determine suitability of the medium used for testing
Methods for Sterility Testing:

1. Method A: Membrane Filtration

2. Method B: Direct Inoculation

1) Method A:Membrane Filtration: It is preferred where the substance under examination

is an oil, an ointment that can be put in a solution, a non-bacteriostatic solid not readily
soluble in culture media, a soluble powder or a liquid that possesses inherent
bacteriostatic/fungistatic properties. For liquid products where volume of container is more
than 100 ml, only Method A should be used.
Membrane generally used is for sterility testing has porosity of 0.45 µm, diameter about to
47 mm, flow rate 55-75 ml of water/minute at a pressure of 70mm of Hg. The complete
unit should be sterile and entire operation should be carried out aseptically.

Dilution fluids: Generally, two types of dilution fluids are used:

1. Fluid A: Dissolve 1 gm of peptic digest of animal tissue (such as bacteriological peptone) in

one liter of water. Filter and adjust the pH to 7.1 ± 0.2. Dispense fluid into the flasks (100 ml)
and sterilize at 121 oC for 20 minutes.

2. Fluid B: If the test sample contains lecithin or oil, use fluid A, for each liter of which has been
added 1 ml of polysorbate 80. Adjust pH to 7.1 ± 0.2 and sterilize at 121 oC for 20 minutes using
an autoclave.

Quantities of sample to be used:

(i) For parenteral preparations: Whenever possible use the whole contents of the container but
not less than the quantities prescribed in Table.1, if necessary, dilute to about 100 ml with a suitable
diluent (e.g. fluid A).

(ii) For ophthalmic and other non-injectable preparations: Take an amount within the range
prescribed in column I of Table.2, if necessary, use the contents of more than one container and
mix thoroughly. For each medium use the amount specified in column II of Table.2, taken from
the mixed sample.
Table 1: Quantities of injectable preparations used for sterility testing
 Table 2: Quantities of ophthalmic and other non-injectable preparations for sterility
testing

Method of test:

(a) For aqueous solutions: Transfer aseptically the quantities of the preparation being examined
prescribed in the two media onto one membrane. Draw the liquid rapidly through the filter with
the aid of a vacuum. The membrane is removed aseptically and cut into two, one part is then
immersed in 100 ml of soyabean casein digest medium and incubated at 20 to 25 oC for not less
than 14 days. Similarly, the other part is immersed in 100 ml of fluid thioglycollate medium and
incubated at 30 to 35 oC for not less than 14 days.

(b) For liquids immiscible with aqueous vehicles and suspensions: Carry out the test described,
for aqueous solutions but add a sufficient quantity of fluid ‘A’ to the pooled sample to achieve
rapid filtration.

(c) For oils and oil solutions: Filter oils or oily solutions of sufficiently low viscosity without
dilution through a dry membrane. Dilute viscous oils are necessary with a suitable diluent.

(d) For ointments and creams: Dilute ointments in a fatty base and emulsions of the water-in-oil
type to give a fluid concentration of 1% w/v, by heating, if necessary, to not more than 40 oC with
a suitable sterile diluent such as isopropyl myristate. If ointments and oils are insoluble in isopropyl
myristate then use Method B.

(e) For soluble solids: Dissolve not less than the quantity of the substance being examined as
prescribed in Tables 1 and 2, in a suitable sterile solvent such as fluid ‘A’.

(f) For solids for injection other than antibiotics: Constitute the test articles as directed on the
label and carry out the test as described for aqueous solutions or oils and oily solutions.
(g) For antibiotic solids, bulks, and blends: Aseptically remove a sufficient quantity of solids
from the mix to obtain a composite sample, equivalent to about 6 g of solids, and transfer to a
sterile flask. Dissolve in about 200 ml of fluid A, and mix.

(h) For antibiotics in packages of 5 g or less: From each of 20 containers, aseptically transfer
about 300 mg of solids into a sterile flask, dissolve in about 200 ml of fluid A, and mix.

(f) For sterile devices: Aseptically passes a sufficient volume of fluid ‘B’ through the 20 devices
to recover not less than 100 ml from each device. Collect the fluids in sterile containers and filter
the entire volume through a membrane filter. In the case of sterile, empty syringes, draw sterile
diluent into the barrel through the sterile needle, if attached or through a sterile needle attached for
the test, and express the contents into a sterile polling vessel. For catheters where the inside lumen
and outside surface are required to be sterile, either cut them into pieces such that the medium is
in contact with the entire lumen or fill the lumen with medium and then immerse the intact unit.

2) Method B: Direct Inoculation

Transfer the prescribed quantity of preparation directly into the culture medium so that the
volume of the medium under examination is not more than 10% of the volume of the medium,
unless otherwise specified. When it is necessary to use large volume of the product, it may be
preferable to use a concentrated culture medium prepared in such a way that it takes account of
subsequent dilution. The table below gives the quantity of the test sample and media required
for direct inoculation.
 If the test solution is bacteriostatic/fungistatic, use a suitable sterile neutralizing agent as
given in the table below.

Methods of test:
(a) For aqueous solutions and suspensions: Remove the liquid from the test containers with a
sterile pipette or syringe. Transfer the quantity of the preparation under examination (Table.1)
directly into the culture medium so that the volume of the preparation under examination is not
more than 10% of the volume of the medium unless otherwise prescribed. When the quantity in a
single container is insufficient to carry out the tests, the combined contents of two or more
containers are to be used to inoculate the media. The inoculated medium is incubated for 14 days
at 30 to 35 oC in the case of fluid thioglycollate medium and at 20 to 25 oC in the case of soybean-
casein digest medium. If the preparation under examination has antimicrobial activity, carry out
the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient
quantity of culture medium.

(b) For oils and oily solutions: Use media to which have been added 0.1%, w/v of (4-tert-
octylphenoxy) polyethoxyethanol or 1% w/v of polysorbate 80 or other suitable emulsifying
agents, in an appropriate concentration and proceed with the test.

(c) For ointments and creams: Prepare by diluting to ten-fold by emulsifying with the chosen
emulsifying agent in a suitable sterile diluent such as fluid ‘A’. Transfer the diluted product to a
medium not containing an emulsifying agent.

(d) For solids: Transfer the required quantity of the material to the medium.
(e) For surgical dressings and related articles: Aseptically remove two or more portions of 100
to 500 mg each from the innermost part of the sample or package under examination. From
individually packaged, single-use materials, aseptically remove the entire article. Immerse the
portions or articles in each medium and proceed with the test.

(f) For sterile devices: Immerse the device in 1000 ml of culture medium. If immersion is
impracticable, flush the lumen of 20 units with the fluid thioglycollate medium and soybean-casein
digest medium separately and recover 15 ml of each medium. Incubate with not less than 100 ml
of each medium. For catheters where the inside lumen and outside surface are required to be sterile,
either cut them into pieces such that the medium is in contact with the entire lumen or fill the
lumen with medium and then immerse the intact unit.

INTERPRETRATION OF RESULTS:
At intervals during the incubation period and at its conclusion, examine the media for
macroscopic evidence of microbial growth. If no evidence of microbial growth is found,
preparation under examination complies with test for sterility. If evidence of microbial growth is
found, then the preparation under examination does not comply with test for sterility. Do not repeat
the test unless it can be clearly shown the tests for sterility were invalid for causes unrelated to
preparation under examination.
The test may be considered invalid only when one/more of following conditions are fulfilled.
a) Microbial growth is found in negative control
b) Data on microbial monitoring of sterility testing facility shows a fault
c) A review of testing procedure used for test in question reveals a fault
d) After identifying micro-organisms isolated from the container showing microbial
growth, the growth may be described without any doubts to fault with respect to material
and/or technique used in conducting the test procedure.
If the test is declared to be invalid, repeat with same number of units as original test. If no
evidence of microbial growth is found in repeat test, preparation under examination
complies with test for sterility. If the microbial growth is found in repeat test, preparation
under examination complies with test for sterility. If the microbial growth is found in
repeat test and confirmed microscopically, the preparation under examination does not
comply with the test for sterility.