industrial attachment report

N01310075A NATIONAL UNIVERSITY OF SCIENCE AND TECHNOLOGY DEPARTMENT OF APPLIED BIOLOGY AND BIOCHEMISTRY INDUSTRIAL ATTACHMENT REPORT Student Name: Adonis Msipa Student Number: N01310075A Page 1 Email Address: [email protected] Company: Beit bridge District Hospital Laboratory 1|Page ACKNOWLEDGEMENTS My utmost gratitude is directed to the Almighty for strengthening me throughout my industrial attachment period. I wish to express my indebted gratitude and special thanks to the whole Beit Bridge Hospital Laboratory team for their unwavering support and guidance throughout my workrelated learning period at their organization. Special mention goes to Mr. K. MAHALA, my incomparable supervisor and Mr. E Makondo the Lab Manager. My wonderful period was spent with a fantastic Lab staff who in spite of being extraordinarily busy with their duties, took time to hear, guide and keep me on the correct path during my internship learning. I also would like to acknowledge Mr. Mandibatsira’s efforts (Assistant Lab Manager) who always devoted his precious office time to come and evaluate my learning progress and achievements in the various departments that I was attached to. I would also like to thank all the Lab Technicians and lab assistants who guided me and motivated me in my daily procedures. Lastly, I would be remiss if I did not acknowledge a special extraordinary woman; my mother Miss Irene Msipa for being with me throughout this period; her motivational support, financial support and nurturing role was and Page 2 will always be greatly valuable. 2|Page ABSTRACT This report documents the practical and theoretical experience that was attained during the industrial attachment period at Beit Bridge District Hospital Laboratory. Beit Bridge District Hospital is ISO/IEC 17025 Accredited public Hospital laboratory in Zimbabwe. It boasts of five departments that are Bacteriology, Serology, Hematology, Blood bank and tuberculosis diagnosis. Departments attached to were Bacteriology, Serology, Hematology, Tuberculosis diagnosis and Blood bank. In Bacteriology tests conducted included Bacterial culture, identification and antimicrobial sensitivity tests. Hematology department conducts full blood counts, CD4 counts and Chemistry analysis (urea, electrolytes and liver function tests). Serology conducts serological tests on blood sera to detect antibodies of certain antigens that invade the body, these included rapid kits which use different immunochromatographic principles to detect the presence of the antibodies in whole blood, plasma or serum. The tuberculosis department involved the diagnosis of mycobacterium tuberculosis, the agent cause of the disease tuberculosis. The department used the Ziel Nielsen staining technique and the gene expert machine to identify the presence of the bacteria. The internship was greatly beneficial and was an eye-opener. It enabled me to have a vital feel of the real on-hands practical aspects in industry. It also improved personal attributes like good Page 3 communication skills, punctuality, organization, and appreciation of the need for teamwork. 3|Page TABLE OF CONTENTS TITLE PAGE CERTIFICATION...........................................................................................................................I ACKNOWLEDGEMENT ..............................................................................................................II ABSTRACT ..................................................................................................................................III INTRODUCTION.........................................................................................................................IV CHAPTER 1 1.0 1.1 SAFETY,CLEANING, DISINFECTION AND STERILIZATION.......................5-6 1.2 MEDIA AND REAGENT PREPARATION ……………………………………..7-8 1.3 SPECIMEN COLLECTION AND TRANSPORTATION ……………………9-10 CHAPTER TWO 2.0 THE LABORATORY SECTIONS AND VARIOUS TESTS PERFORMED…………… 2.1 HEMATOLOGY, IMMUNOHEMATOLOGY AND (BLOOD BANK) SECTION...11-15 2.2 SEROLOGY SECTION ………………………………………………….……………16-20 2.3 CLINICAL BIOCHEMISTRY SECTION …………………………………….………21-24 2.4 CLINICAL MICROSCOPY SECTION………………………………….…………….25-34 2.5 MICROBIOLOGY, CULTURE AND SENSITIVITY TESTS ……………………….35-52 CHAPTER THREE 3.0 SUMMARY, CHALLENGES ENCOUNTERED, RECOMMENDATION AND CONCLUSION………………………………………………………………..……………… SUMMARY……………………………………………………………………………...53 3.2 CHALLENGES ENCOUNTERED ………………………………………………….......53 3.3 CONCLUSION ………………………………………………………….……………......54 3.4 RECOMMENDATIONS …………………………………………………...………....54-55 3.5 REFERENCES ..............................................................................................................56-57 Page 4 3.1 4|Page CHAPTER ONE 1.1 LABORATORY SAFETY, CLEANING, DISNFECTION AND STERILIZATION 1.2 Laboratory safety and working conditions All students have to read and understand the information regarding laboratory safety and emergency procedures prior to the first laboratory session.  Personal protection which included protective equipment such as a lab coat, gloves, and closed shoes; are to be worn during all experiments. Hats and other articles which are not required during practical work were put into a separate room on the shelves.  Laboratory working area was neat and sterilized at all times.  Clean up and disposal procedures were adhered to at all times e.g. Needle disposal boxes were always available for all phlebotomists; broken glassware and sharps were kept separate from other waste and disposed of in the appropriate containers .  Hygiene practices were followed as well e.g. not pipetting by mouth, washing hands after using any substances that are hazardous to health, on leaving the laboratory and after visiting the toilet. 1.3 Cleaning, Disinfection, and Sterilization 1.3.1 Decontamination is a process which removes or destroys microorganisms to render an object safe for use. It includes cleaning, disinfection and sterilization. a) Manual Cleaning Cleaning is a process that removes foreign material (e.g. soil, organic material, micro-organisms) from an object. Cleaning of floor was done using cold water and 5% jik (particularly in work areas) because deposits of dust, soil and microbes on surfaces can transmit infection or Interfere with experimental results. Working benches and washing area were wiped using wet gauze with 0.1% Page 5 jik before and after each practical sessions for disinfection. 5|Page b) Disinfection This is a process that reduces the number of pathogenic microorganisms, but not necessarily bacterial spores, from inanimate objects or skin, to a level which is not harmful to health. alcohol (70%) was used to sterilize hands, objects and the skin area prior to venipuncture while 0.1% Jik was mainly used to wipe any spillage of samples either on the floor or working bench, and a cut wound by either a glass or syringe in the process of an experiment/washing of glassware, this is to minimize chances of infection. c) Sterilization Is a process that destroys all microorganisms, including bacterial spores. There were three types of sterilization; i. Dry heat sterilization using an oven, or an autoclave machine. The standard setting for a hot air oven was at least two hours at 160 °C (320 °F). A rapid method heat air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects. Dry heat was used on scalpels, spatula, slides and other heat-stable items that are adversely affected by steam. ii. Wet heat/steam sterilization by use of an Autoclave. 121°C for 20 min for unwrapped items such as used glass petri dishes and bio typing bottles prior to washing and 15 minutes for packaged items at 1.036 bar (15.03psi) above atmospheric pressure. This included; media, clean petri dishes and bio typing bottles prior to use. iii. Flaming; this was done to loops and straight-wires in Bacteriology lab when culturing stool or urine on media. 1.3.2 Washing of glassware Washing of glass ware was done almost on a daily basis in morning before proceeding with other activities. Used Glass slides, pipette and widal plates were dipped in 5% Jik for the whole night for adequate sterilization/disinfection then washed in the following day. Washing was done using 5% jik or dettol and a scrapper and cleaned using cold tap water. The glassware was then taken to Page 6 an Oven for drying/sterilization. Any broken glassware must be sterilized before being disposed. 6|Page 1.4 MEDIA AND REAGENT PREPARATION All media and reagents were assigned a preparation control number consisting the date the media or reagent was prepared and date Revised, and number of preparations made on the same date. For all media and reagents that are sterilized by autoclaving, a sterilization batch number was also assigned. The storage requirement for the media was recorded at the bottom of the page. If the material was to be used immediately and required no storage, then N/A was placed in that section. The ingredients and the chemicals used for preparing media and reagents were the product of any manufacturer as the comparative tests showed satisfactory results. Pre-mixed, dehydrated media was examined before use for indications of separation or deterioration. Each batch of medium was tested for sterility and growth promotion/inhibition characteristics, as appropriate following the QC procedures described by the manufacturer. 1.5 Media preparation Unless or otherwise indicated, up to 1 liter of a medium was sterilized by steam under pressure at 121 C (15-16 psi) for 15 minutes. Any departures from (i.e. preparation volumes, sterilization/heating requirements, formulations, etc.) required equivalency data to support the change(s) e.g. instructions from some manufacturers said to dissolve by gently heating while checking the media visually to determine if it is well dissolved to prevent overheating. This was an essential step in obtaining the correct pH for the final medium. Depending on the type and quantity of media needed, tubed media i.e. Biochemical media, Simmon Citrate agar and peptone water was either dispensed directly into tubes and sterilized by autoclaving or was autoclaved in bulk and then aseptically dispensed into presterilized tubes. Dilution tubes or sterile petri dishes were only dispensed after autoclaving. Examples include; X.L.D agar, Mueller Hinton (MH) agar, Nutrient Agar etc. Only distilled water (treated free from traces of dissolved metal, bactericidal, and inhibitory compounds) was used to prepare culture media, reagents, and dilution blanks. Inhibitor free water Page 7 is referred to as microbiologically suitable (MS) water. 7|Page 1.6 Results and Discussion Depending on the type and combination of nutrients, different categories of media were made. a. Ordinary culture media were routinely employed in a laboratory e.g. nutrient broth, nutrient agar, selenite broth and peptone water. b. Complex media are rich in nutrients, they contained water soluble extracts of plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and tryptone). Usually a sugar, often glucose was added to serve as the main carbon and energy source. Its purpose was to grow most heterotrophic organisms c. Defined media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e., the exact chemical composition of the medium is known. They contained a simple sugar as the carbon and energy source, an inorganic nitrogen source, various mineral salts and if necessary growth factors.It was used to grow specific heterotrophs and were often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays. d. Selective/differential media are media supplemented with growth-promoting or growthinhibiting additives. The additives are species- or organism-selective .Selective media (e.g. MacConkey agar (MAC), a Shigella strain such as S. flexneri) suppressed unwanted microbes, or encouraged desired microbes while differential media distinguished colonies of specific microbes from others.(E.g. MAC, contains lactose as a substrate and neutral red as an indicator, a lactose-non fermenting organism such as S. flexneri and a lactosefermenting organism such as E. coli and is typically used to prevent fungal growth in mixed cultures). e. Enrichment media is similar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating the rest of the bacterial population. An example of enrichment media is selenite broth used for primary isolation of enteric bacteria. Microorganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have adapted to the habitats most suitable Page 8 for their needs, in the laboratory, however, these requirements must be met by a culture medium. This is basically an aqueous solution to which all the necessary nutrients have been added. 8|Page 1.7 SPECIMEN COLLECTION AND TRANSPORTATION 1.7.1 Rationale for specimen collection Specimen collection was undertaken when laboratory investigation was required for the examination of a probable cause of disease for the patient. For example, a stool was requested (for the presence of transmissible parasites in stool e.g. amoeba), urine (for transmissible Urinary Tract Infections) and blood (to check for typhoid fever and blood borne pathogens) . 1.7.2     Specimen Safety considerations All specimens were treated as potentially biohazards. A white laboratory coat or tunic was to be consistently worn and fastened to the top. Gloves were always worn when handling specimen samples, and as an additional precaution, two pairs of gloves would be worn. All students and members of staff handling blood samples were advised to be vaccinated against Hepatitis B. 1.7.3   1.8 General guidelines for proper specimen collection and transport The procedures were explained to the individual and the reasons for taking the specimens.  Specimens were collected before administering antimicrobial agents.  specimens so as to prevent introduction of microorganisms during invasive procedures.  identification number, including date and place of collection with the initials.  specimens would yield false-negative results. Sterile equipment and asepsis were highly encouraged equipment to use when collecting The specimen must be labelled clearly on the container with the patient’s name and An adequate amount of specimen requires to be collected as inadequate amounts of All specimens collected are to be transported to the laboratory promptly. Urine specimens 10 to 50 mL of "clean catch-mid-stream" urine is required and is collected into a sterile urine tube Page 9 which was then tightly sealed. Urine sample was then dispatched to the laboratory as soon as possible or no more than four hours if kept at room temperature or up to 24 hours if kept at 4 9|Page degrees centigrade, because of the continuous growth of bacteria in vitro thus altering the actual concentration of organisms and misleading results. 1.8.1 Fecal Specimen 1 to 2 mg quantity is sufficient in a sterile polycot that is tightly sealed. If looking for trophozoite, a stool specimen s transported very rapidly to the laboratory to avoid disintegration of trophozoite. Stool samples should be examined within 30 minutes of collection of the specimen. 1.8.2 Blood specimen Blood (of about 50ml (2-3 tablespoons) was taken using a good aseptic technique.  The phlebotomist set the tourniquet around the upper arm of the subject, located the proper vein by inspecting and palpating and then sterilized the injection site using a med swab alcohol impregnated wipe. The skin was allowed to dry for least 30 seconds, the vein was anchored by placing the thumb about two centimeters below the vein and pulling gently to make the skin a little taut. After that, the needle, beveled upward, was pushed smoothly and quickly into the vein, to minimize the possibility of hemolysis as a result of vascular damage. Immediately after the insertion, the tourniquet was released to minimize the effect of hem concentration and blood was transferred into the Vacutainer purple cap 10 ml EDTA tube. The blood was carefully and gently mixed with the anti-coagulant by gently inverting the tube 5 to 6 times to allow the blood to gently coat the sides of the tube to dissolve powdered anti-coagulants. If there was any problem with blood flow during blood taking (e.g. collapsing vein), the procedure was discontinued and an attempt is made on the other arm. If that also failed, no further attempts were made and the blood collection for this particular participant was recorded as "failed". Anti-coagulant tubes contained specific amounts of the anti-coagulant Page 10 for specific amounts of blood. 10 | P a g e CHAPTER TWO 2.1 HEMATOLOGY, IMMUNOHEMATOLOGY AND (BLOOD BANK) SECTION In the hematology department, the analysis is carried out using the whole blood sample of patient for diagnosis of hematological diseases and abnormalities. Blood samples are collected in EDTA bottle tubes for analysis. Immunohematology Section Also known as the blood bank performs tests to provide blood and blood products to patients for transfusion purposes. The blood bank technologist relies on the phlebotomist to perform identification of the patient without error, since patients will die if given the wrong blood type. The analyses carried out in this section include: Full blood count, CD4+ count, ABO/D (Rh) typing, Blood grouping and Cross matching respectively. 2.1.1 FULL BLOOD COUNT (FBC) TEST  Introduction: The full blood count of a blood sample helps to know the total cells in the whole blood. It determines the total hematocrit (HCT), hemoglobin (HGB), red blood cell (RBC) count, white blood cell (WBC) count, platelet count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), neutrophils (NEUT), lymphocytes (LYMPH) and platelets (PLT)  Aim: To deduce the total counts of all blood components  Equipment/Apparatus: Hematology analyzer, whole blood sample in an EDTA bottle. Page 11 2.1.2 Procedure: Blood sample is collected into an EDTA bottle (purple Stoppered Tube) through venipuncture and is mixed with anticoagulant by inverting the bottle gently 8 times. The blood sample is then placed under the hematology analyzer sensitive probe. The probe button is pressed so that the probe 11 | P a g e can pick the sample into the machine for analysis. The result is then displayed on the screen of the machine and then printed out. 2.1.3. Conclusion: The machine is an efficient tool for the full blood count analysis. All parameters of the blood are read and displayed on the printed out paper. 2.1.4 CD4+ COUNTS 2.1.5 Introduction: CD4 T-cell laboratory testing through flow cytometry is considered an essential part of HIV care, since this parameter is used to stage disease and guide clinical management. Certain CD4 T cell thresholds are used as benchmarks to either initiate prophylaxis against opportunistic infections (OIs) and/or to begin antiretroviral therapy (ART). The CD4 cell count is also a relatively consistent indicator of treatment response. 2.1.6 Principle: Flow cytometry (cell measurement) is a process used to count, identify, and sort various types of cells. This technique is based on adding monoclonal antibodies (MAb) to a blood sample and running the fluid through a light source, usually a laser beam. If two antibodies are used, each is bound to a different fluorochrome (colored dye) and the machine can compare the numbers of cells with different proteins on their surfaces. For example, the ratio between CD4 cells and all white blood cells (leukocytes) can be measured using CD4 and CD45 antibodies, since all white blood cells have CD45 proteins on their surface. By placing detectors in different places in relation to the light source, different cell types of the same size can be distinguished because they emit light differently i.e. CD4 cells can be distinguished from similarly sized monocytes, which also carry CD4 proteins on their surface. Blood cells can be separated by size, shape, density, and epitope and the relative percentage of each cell subset of interest can be calculated. 2.1.7 Results: The results are calculated automatically by the electronic analyzers that come Page 12 with most of the machines used for CD4+ counts. The absolute T- Helper cell count is displayed in the first of four results windows of the analyzer (PIMA Machine).The operator has the option to print a result via an external PIMA printer. Other machines like the BD FACS machine give out results in the form of a printed out receipt that contains the results of the cell counts. The machines have a built in Quality Control Features to check the analyzer and reagent functionality. 12 | P a g e 2.1.8 BLOOD GROUPING AND GENOTYPING TEST  Introduction: Blood grouping of the A B O system is determined with Anti-A, Anti-B, and Anti-D sera, which form agglutination complex with antibodies found in the blood sample.  Aim: To determine the group and the rhesus of a patient’s blood  Equipment/Materials: Clean free grease tile, Pasteur pipette, Whole blood sample in an EDTA bottle, distilled water, applicator stick, test tube rack, clean white tile, cotton wool, applicator stick, cellulose filter paper, gloves and an incubator.  Reagents: Anti-A, Anti-B, Anti-D sera, Buffer for balancing, normal saline, Low ionic strength saline and Anti Human Globulin (AHG).  Procedure: For blood grouping: The blood sample was collected into an EDTA bottle through venipuncture. 10ul of blood was placed 3 spots on the tile with the aid of Pasteur pipette. The antisera A, B and D were placed carefully on each spots, ABO of the grouping system on the tile respectively and an applicator stick was used to thoroughly mix the drop of blood with the anti-sera one after the other without contamination. The tile was gently rocked from side to side for 2 minutes to allow Page 13 agglutination occurrence, then result was observed. 13 | P a g e Page 14  Result: The result for blood grouping was observed by studying the agglutination of red blood cells. 14 | P a g e  Conclusion: The result is to be observed according to the agglutination that occurred in each spots on the tile. Anti D determines the present of the rhesus ‘D’ factor in blood group. Factors that affect blood grouping are; wrong labeling of spot and confusion of anti-sera with spots, Contamination of test card or tiles with detergents, Expired anti-sera Page 15 Bio-medical significance; Blood transfusion, Blood compatibility, Antenatal screening. 15 | P a g e 2.2 SEROLOGY SECTION Tests done in this department are designed to detect the body's response to the presence of bacterial, viral, fungal, parasitic and other conditions which stimulate detectable antigen-antibody reactions in a test system to aid in the diagnosis of the patient. Most tests performed in this section are carried out under the principles of Immunoassay, some of them are; Cold agglutinins (CAG), Immunochromatographic assays. The specimen must be kept warm. Available tests in the laboratory included tests like, RPR test kits to diagnose syphilis, Pregnancy Testing, (PSA) prostate specific antigen testing, ABO Blood grouping and Rhesus testing. 2.2.1 HBsAg TEST FOR HEPATITIS, HBsAG is a rapid immunochromatographic test for the qualitative detection of Hepatitis B surface Antigen in human serum/plasma, it can be used for prenatal or transfusion screening, and during acute infection or chronic carriage of the Hepatitis B virus. Early detection of infection is essential for rapid initiation of adequate treatment. The presence of HBsAg can be detected within 10 minutes at the concerntration of 5ng/ml or higher, and 15 minutes at 1ng/ml. The test is intended for healthcare proffesional use. 2.2.1.1 PRINCIPLE The test uses a soloid phase “sandwich” immunoassays for the detection of HBsAg. The production, characterization and application of monoclonal antibodies for the detection of HBsAg. The Advanced Quality One step HBsAg test is colloidal gold enhanced immunoassy for the determination of HBV surface antigen HBsAgin human blood, plasma or serum. Goat anti – HbsAg antibody is immobilized in the test region on nitrocellulose membrane. During the assay, specimen is allowed to react with the colored conjugate (antibody – colloida gold conjugate); the mixture then migrates chromatographically on the membrane by the capillary action. An HBsAg positive specimen produces a distinct color band in the test region, formed by the specific antibodyHBsAg colored conjugate complex. Absence of this colored band in the test regionsuggests a negative result. A colored band always appears in the control region serving as procedural control Page 16 regardless of the test result. 16 | P a g e 2.2.3 PREGNANCY TEST The test was done using a pregnancy test strip (URINE). This is a rapid, one step for the detection of human chorionic gonadotropin (HCG) in urine. 2.2.3.1 PRINCIPLE The used atlas hCG one step pregnancy strip is a rapid chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin hCG in urine at the sensitivity 25ml/microliter to aid the early detection of hCG. The test utilizes a combination of antibodies and a monoclonal hCG antibody to selectively detect elevated levels of hCG. The assay is conducted by immersing the test strip in a urine specimen and observing the formation of colored lines. The specimen migrates via capillary action along the membrane to react with colored conjugate. Positive specimens react with the specific antibody- hCG colored conjugate to form a colored conjugate to form a colored line at the test line region of the membrane. Absence of this colored line suggests a negative result. To serve as a procedural control, a colored line will always appear Page 17 at the control line region if the test has been performed properly. 17 | P a g e 2.2.4 RPR TEST KITS FOR SYPHILIS DIAGNOSIS Syphilis diagnosis is achieved by the use of the SD BIO LINE Syphilis 3.0 test kit. This is a solid phase immunochromatographic assay for the qualitative detection of all isotypes (IgG, IgM, and IgA) against Treponemma pallidum. Treponemma pallidum is the causative agent of the venereal disease syphilis antibodies in human blood by immunoassay. 2.2.4.1 PRINCIPLE The SD BIO LINE syphilis 3.0 contains a membrane strip which is pre-coated with re combinant Treponemma pallidum antigens on test bound region. The recombinant Treponemma pallidum antigens on test bound region. The recombinant Treponemma pallidum antigens – colloid gold conjugate, patient sample and sample diluent moves along the membrane chromatographically to the test regions and forms a visible line as the antigen-antibody-antigen gold particle complex forms. Therefore, the formation of a visible line in the test region indicates a positive result for the detection of Treponemma pallidum specific antibodies (IgG, IgA, and IgM) when no treponemma pallidum specific antibodies are present in the sample, there won’t be any visible color band in the test region. 2.2.5 PROSTATE SPECIFIC ANTIGEN (TEST KIT) The advanced quality one step PSA test is a rapid immunochromatographic assay for the semi quantitative detection of prostate specific antigen (PSA) in serum, plasma or whole blood. The test is intended for healthcare professional use. 2.2.5.1 PRINCIPLE The one step PSA Test is a colloidal gold enhanced immunoassay for the determination of prostate specific antigen in human whole blood, serum or plasma. The nitrocellulose membrane was treated with mouse anti-human PSA McAb in the test region. During the assay, the serum specimen is allowed to react with the colored conjugate (antibody-colloidal conjugate); the mixture then migrates on the membrane chromatographically by the capillary action. The assay is designed to stronger than control band. The appearance of a test band that is weaker than the control band indicates the presence of PSA in the specimen at a level lower than 4ng/ml. Page 18 detect PSA at the cutoff level of 4ng/ml, the test band will appear with intensity that is equal to or 18 | P a g e 2.2.6 RDT MALARIA TEST KITS FIRST RESPONSE MALARIA Ag.HPRP2 CARD TEST (RAPID ONE STEP MALARIA Ag. P.falciparum {HRP2} TEST) 2.2.6.1 INTENDED USE The test kit is used for the qualitative determination of Malaria Histidine-Rich protein 2 (HRP2) in human blood as an aid in the diagnosis of malaria infection. 2.2.6.2 PRINCIPLE Immunochromatography relies on the migration of liquid across the surface of a nitrocellulose membrane. Immunochromatographic tests are based on the capture of parasite antigen from peripheral blood using monoclonal antibodies prepared against a malaria antigen target and conjugated to either a liposome containing selenium dye or gold particles in a mobile phase. A second or third capture monoclonal antibody applied to a strip of nitrocellulose acts as the immobile phase. The migration of the antigen-antibody complex in the mobile phase along the strip enables the labeled antigen to be captured by the monoclonal antibody of the immobile phase, thus producing a visible colored line. Incorporation of a labeled goat anti-mouse antibody capture ensures that the system is controlled for migration. Migration depends on several physical characteristics of the component reagents, primarily the porosity of the membrane controlling the flow rate and the components of the buffer solution used to transport the labeled antigen-antibody complex in the lysed blood sample. HRP-2 is a water-soluble protein produced by asexual stages and young gametocytes of P. falciparum. It is expressed on the RBC membrane surface, and because of its abundance in P. falciparum, it was the first antigen to be used to develop an RDT for its detection. pLDH and aldolase. pLDH, an enzyme found in the glycolytic pathway of the malaria parasite, is produced by sexual Page 19 and asexual stages of the parasite. Different isomers of pLDH for each of the four Plasmodium spp. infecting humans exist, and their detection constitutes a second approach to RDT development. Several other enzymes of the malaria parasite glycolytic pathway, notably aldolase, have been suggested as target antigens for RDT for species other than P. falciparum. 19 | P a g e 2.2.7 6 HIV SCREENING This is the diagnosis for Human Immunodeficiency Virus, an infectious agent that causes Acquired Immunodeficiency Syndrome (AIDS), a disease that leaves a person vulnerable to life threatening infection. HIV transmission occurs when a person is exposed to body fluids infected with virus, such as blood, semen, vaginal secretions and breast milk. To test for HIV, different rapid tests can be used. The Alere Determine HIV 1/2 can be used, First response for HIV 1/2 and the Chembio test kits can be used. The Alere determine HIV 1/ 2 is used as the screening test Kit, the first response as a confirmitory and the tier breaker which is the Chembio test kit. However the kits use a common principle which is the immunochromatographic principle. 2.2.7.1 PRINCIPLE Alere Determine HIV 1/2: A sample is added to the test kit at the sample pad. A buffer is applied to the sample. As the sample migrates through the conjugate pad, it reconstitutes and mixes with the sellenium colloid – antigen conjugate. The mixture continues to migrate through the solid phase to the immobilised recombinant antigens and synthetic peptides at the patient window site. If antibodies to HIV1 and or 2 are presentin the sample, the antibodies bind to the antigen at the patient window site. The HIV test result is negative when a line at the control window is seen. To be regarded positive of HIV, two bands of lines are observed , i.e at the control window and at the test window. The Alere determine HIV1/2 is a qualitative immunoassy for the detection of antibodies to HIV 1 Page 20 and 2 in human serum, plasma or whole blood. 20 | P a g e 2.3 CLINICAL BIOCHEMISTRY SECTION 2.3.1 INTRODUCTION This section deals with chemical analysis of serum or plasma in which many diseases of the major organs systems can be diagnosed such as heart attacks, hepatitis, renal failure, diabetes and Liver function. Blood samples may be collected into a Serum Separator Tube, Lithium Heparin or a plain tube. Tests that can be performed in this department include:  Blood Glucose;  Blood lipids (fat) Cholesterol level.  Electrolytes - sodium, potassium, CO2- (Bicarbonate), and chloride  Uric acid  Creatinine and Blood Urea  Liver function tests –AST (aspartate transferase), ALT (alanine transferase), alkaline phosphatase, and bilirubin. 2.3.2 MATERIALS AND EQUIPMENTS USED IN CLINICAL BIOCHEMISTRY SECTION Personal Protective Equipment (PPE), Blood collection materials Different tubes like; Lithium Heparin, Serum Separator and Fluoride Oxalate tubes, plain tubes, Chemical reagents and detergents, automated machines, centrifuge, Glucometer and Accu check. 2.3.3 BLOOD GLUCOSE 2.3.3.1 Test Overview: A blood glucose test measures the amount of a type of sugar, called glucose, in the blood. Glucose comes from carbohydrate foods. It is the main source of energy used by the body. Insulin is a hormone that helps the body cells use the glucose. Insulin is produced in the pancreas and released into the blood when the amount of glucose in the blood rises. Normally, the blood glucose levels increase slightly after one eats a diet containing carbohydrates. This Page 21 increase causes the rate in which the pancreas releases insulin so that blood glucose levels do not get too high. Blood glucose levels that remain high over time can damage a person’s eyes, kidneys, nerves, and blood vessels. 21 | P a g e There are several different types of blood glucose tests. i. Fasting blood sugar (FBS) measures blood glucose after a patient has not eaten for at least 8 hours and at most 14 hours. It is often the first test done to check for prediabetes and diabetes Materials/Reagents: Fluoride Oxalate and other blood collection equipment, Centrifuge, Insulin kit (NORUDIA® Insulin) Liquid reagent, automated machine, Glucometer or Accu Check. Procedure: The patient is ensured to have fasted for the required period of time before sample collection into a Fluoride Oxalate tube, and then the blood samples are centrifuged in order to obtain plasma. The plasma obtained is then decanted into a small cap which is then transported into the machine for analysis. The results are then deduced which is measured in mg/dl Normal Result: Normal result is less than or equal to 100mg/dl ii. 2-hours post-prandial blood sugar measures blood glucose exactly 2 hours after eating a meal. This is not a test used to diagnose diabetes. Procedure: After performing the same procedure for FBS sample collection, patients are then asked to return 2hrs as soon as they start eating, then another venepuncture is made and the same procedure is repeated. Result: Normal result is less than 140mg/dl for people age 50 and younger; less than 150mg/dl for age 50-60; less than 160mg/dl for age 60 and older. iii. Random blood sugar (RBS) also known as casual blood glucose test. RBS measures Blood glucose regardless of when was the meal ingested. Several random measurements may be taken throughout the day. Random testing is useful because glucose levels in healthy people do not vary widely throughout the day. Blood glucose levels that vary widely may mean a problem. Materials/Reagents: Same materials as FBS. Procedure: Glucometer or Accu Check are mostly used to perform this test. The Glucometer corresponding code strip is inserted and loaded, Patient’s thumb is disinfected using a70% alcohol pad and pricked with lancet. The blood is wiped off in order to avoid sampling error, pressure is applied below to enable blood flow again and the blood is placed on the strip. The result is then taken Page 22 Normal Results: 80-120mg/dl before meals and 100/140mg/dl after meals iv. Oral glucose tolerance test is used to diagnose prediabetes and diabetes. This test is a series of blood glucose measurements taken after you drink a sweet liquid that contains glucose. This test is commonly 22 | P a g e used to diagnose diabetes that occurs during pregnancy (gestational diabetes). This test is not commonly used to diagnose diabetes in a person who is not pregnant. Materials/Reagents: : Fluoride Oxalate and other blood collection equipment, Centrifuge, Insulin kit (NORUDIA® Insulin) Liquid reagent, automated machine, glucose solution Procedure: Patient is asked to take the required gram of sugar solution before sample collection. Samples are collected five times at 30min intervals and lastly collected once 1hr after the last sample collection of 30min intervals. The result is the deduced by the machines. Results: 75g of glucose; fasting 92mg/dl or more; 1hr 180mg/dl or more; 2hrs 153mg/dl or more 100g of glucose; more than or equal to 140mg/dl. 2.3.4 ELECTROLYTES TEST 2.3.4.1 Test Overview: Electrolytes are minerals found in the body tissues and blood in form of dissolved salts which help transfer nutrients into body cells and waste out of them. Electrolytes also maintain a healthy water balance and help stabilize the body’s acid/base (pH) level. The main electrolytes in the body are; Sodium and Potassium, others are; CO2(Bicarbonate), calcium and chloride ions. 2.3.5 URIC ACID TEST 2.3.5.1 Test Overview: This is a kidney or liver function test, which measures the amount of uric acid present in a blood sample. It is produced from the natural break down of body’s cells, from food consumed and then filtered out by the kidneys and passes out of the body in urine but if too much is being produced in the body, the kidney is unable to filter them normally, it becomes high and may cause solid crystals from within joint, which may lead to a painful condition called gout. These uric crystals can build up in joint and nearby tissues, thereby forming hard lumpy deposits called tophi. Results: Normal Ranges; In men 3.4 – 7.0mg/dl, in women 2.4 – 6.0mg/dl, in children 2.0 – 5.5 mg/dl Page 23 2.3.5.2 23 | P a g e 2.3.6 BLOOD UREA NITROGEN AND CREATININE TEST 2.3.6.1 Test Overview: This test is used to depict the function of the kidney. Blood Urea Nitrogen and Creatinine test can be used together to find the BUN-to-Creatinine ratio and when these substances are high in the blood it may lead to dehydration and heart failure. Blood urea nitrogen (BUN) measures the amount of Nitrogen in the blood that comes from the waste product urea. Urea is made when proteins are broken down in the body (Ornithine cycle). Urea is made in the liver and passed out of the body as urine. 2.3.6.2 Normal Results: Blood Urea Nitrogen (BUN); Adults: 10-20mg/dl, Children: 5-8mg/dl Blood Creatinine; Men: 0.6-1.2mg/dl, Women: 0.5-1.1mg/dl, Children; 0.4 to 1.0mg/dl 2.3.7 LIVER FUNCTION TESTS This is a group of blood tests that detects inflammation and damage to the liver and also check how well the liver works. Liver function test include ALT (Alanine amino trans- ferase) , AST (Aspartate aminotransferase) others are PT, INR, albumin, bilirubin ALP (Alkaline phosphatase). 2.3.8 BS-200E Chemistry Analyzer To conduct tests for clinical chemistry, the BS- 200E machine was used. This is floor-standing, discrete Page 24 and random access clinical chemistry analyzer offering constant 200 tests per hour. 24 | P a g e 2.4 CLINICAL MICROSCOPY SECTION 2.4.1 PREPARATION OF BLOOD FILM FOR MALARIA A blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide and then stained to allow the various blood cells to be examined microscopically. Blood films are usually examined to investigate hematological problems (disorders of the blood) and, occasionally, to look for parasites within the blood such as malaria and filarial.Two types of smears were prepared from the peripheral blood – one thin smear and the other thick smear. Blood films were made by placing a drop of blood on one end of a slide, and using a spreader slide to disperse the blood over the slide's length. The aim is to get a region, called a monolayer, where the cells are spaced far enough apart to be counted and differentiated. The monolayer is found in the "feathered edge" created by the spreader slide as it draws the blood forward. The slide was left to air dry, after which the blood was fixed to the slide by immersing it briefly in 70 % methanol (only for thin smear). Then slide was stained to distinguish the cells from each other using a Ramanowsky stain (Giemsa stain). 2.4.1.1 Thick smear: was made using Field stain , It was dried for 30 minutes and not fixed with methanol. This allows the red blood cells to be hemolyzed, leukocytes and any malaria parasites present are to be the only detectable elements. Thick smears were used to detect infection, and to estimate parasite concentration because they are examined using a larger sample of blood. 2.4.1.2 Thin smear: It was air dried for 10 minutes using a Fieldstain . After drying, it was fixed in 70% ethanol. This was done by either dipping the thin smear into ethanol for 5 seconds or by dabbing the thin smear with a ethanol-soaked cotton ball. Thin film examination was done for finding out the species of Plasmodium falciparum. Rinsing was done using neutral distilled water. 2.4.2 Interpretation of results Page 25 There are four species of malaria parasite namely P. falciparum (commonest), Plasmodium vivax (second common spp.) P.malariae (less common) and P.ovale (rare). In all stages of development, the parasite stains the same colour with Giemsa stain. These are: Chromatin which is part of the 25 | P a g e parasite nucleus is usually round in shape and stains a deep red; Cytoplasm occurs in a number of forms, from a ring shape to a totally irregular shape. It always stains blue, although the shade of the blue may vary between the parasite species.There are no RBCs in the stained thick blood film. The malaria parasites are seen though they appear smaller than in thin blood films. 2.4.3 DIAGNOSIS OF TUBERCOLOSIS 2.4.3.1 DIRECT SMEAR MICROSCOPY Direct smear microscopy (DSM) remains the most cost effective tool for diagnosing patients with infectious tuberculosis and for monitoring their progress on treatment. The World Health Organization strategy for Tuberculosis control, directly observed treatment short course (DOTS) relies on a network of laboratories that provide Acid Fast Bacilli (AFB) sputum microscopy. Sputum smear microscopy is, and is likely to remain for the foreseeable future, the only cost effective tool for diagnosing patients with infectious tuberculosis and to monitor their progress in treatment in low income and high tuberculosis prevalence countries. It is a simple, inexpensive, appropriate method, which is relatively easy to perform and interpret. It yields timely results with very high specificity of detection of tubercle bacilli and provides most of the essential laboratory epidemiological indicators needed for the evaluation of the National Tuberculosis Program (NTP) 2.4.4 ZIEHL NEELSEN (ZN) STAINING 2.4.4.1 PRINCIPLE When stained with concentrated Carbol fuchsin, tubercle bacilli will take up the red color of the stain. This is because the mycolic avid layers become permeable to carbol fuchsin on application Page 26 of heat. When decolorizing with acid alcohol, the bacilli will remain red (as the mycolic acid layers become impermeable). All the materials such as non-acid fast bacteria, cells and mucus, that are not color fast, will lose the color and take up the color of the counter stain. 26 | P a g e On examination under 100X objective lens, the bacilli will appear like red rods, with some slightly curved, more or less granular, isolated, in pairs or in group, standing out clearly against a blue or green background depending on which counter stain has been used. 2.4.4.2 REAGENTS a. Concerntrated Carbol Fuchsin (85% dye concerntration) b. 3% Acid – Alcohol Decolorizing Solution c. Methylene Blue or Malachite Green d. Tape Water 2.4.4.3 MATERIALS REQUIRED a. Alcohol soaked cotton swab or an alcohol/spirit lamp b. Staining racks c. Lighter or matches d. Timer e. Slide drying rack 2.4.4.4 METHOD Numbered smears are placed on the staining rack in batches of 12 (maximum) whilst ensuring that there is finger distance between slides. To the slides, as a quality control measure a known positive and negative smear are placed on every batch or whenever a freshly prepared stain is to be used. This quality control is done as a measure to ensure the staining capability of the solutions and of the staining procedures. It is also done to confirm that Acid Fast contaminants are not present in the stains. Prior to staining the carbol fuchsin is filtered directly onto the slide using a funnel containing filter paper. The slide is then gently heated beneath with the flame of alcohol soaked cotton swab or an alcohol lamp until steam rises from the stain. The slide must not be allowed to boil or dry otherwise it will scorch and re-crystalized substances will develop in the smear. The smear is then left for Page 27 five minutes and not allowed to dry. The smears are then heated for the second time as in the previous stage. They are then heat for the third and final time after another five minutes from the second heating. 27 | P a g e  Heat facilitates penetration of the dye through the mycolic layer of the cell wall of the bacilli. The slides are then tilted to drain off excess stain. Furthermore the slides are then rinsed off the staining solution with a gentle stream of cold tap water. It is convenient to use a beaker, flask, or squeeze bottle to pour the water onto the slides. During the process of rinsing, getting water streams directly on the smear must be avoided. Vigorous washing may cause the smear to lift. The slide is then tilted to drain off excess rinse water. The smear is then decolorized by covering the whole slide by 3% acid alcohol solution and leave it for a maximum of 3 minutes. If the carbol fuchsin stain is retained in the smear, it is under decolorized. The decolorization is repeated for another minute. The decolorizer is then washed off gently using stream of water. After that the slide is tilted to drain off the excess water. Using Methylene blue as a counter stain, the entire surface of the slide is covered with the methylene blue and left for 30 seconds. The counter stain is then drained off and the slide is rinsed with a gentle stream of water. Moreover the underside of the slide is then wiped with cotton wool or paper towel soaked in alcohol to remove soot and then the slides are placed on a slide drying rack to air dry. 2.4.5 PRECAUTIONS DURING SMEAR MICROSCOPY a. One must stick to the stipulated time to avoid under or over decolorization with acid alcohol b. Avoid making thick smears (smudges). This may interfere with proper decolorization and counter stains may hide the presence of acid – fast bacilli. Thick smears may also flake, resulting in loss of smear material and possible transfer of material to other slides. c. Avoid the use of stronger concerntrations of counter stain, (particularly, - Malachite green) as tis masks the presence of acid fast bacilli. d. Over counterstaining should also be avoided as this also masks the presence of acid fast Page 28 bacilli. 28 | P a g e 2.4.6 AURAMINE STAINING 2.4.6.1 PRINCIPLE The property of acid-fastness is based on the presence of mycolic acids in the mycobacterial cell wall. Primary stain (auramine) binds cell-wall mycolic acids. Intense decolorization (strong acids, alcohol) does not release primary stain from the cell wall and the mycobacteria retain the fluorescent bright yellow color of auramine. Potassium permanganate is used to quench fluorescence in the background; however, it provides little contrast for focusing and stains are therefore sometimes preferred, of which blue ink may be the best. Fluorescence microscopy allows smears to be examined more rapidly than is possible with the basic fuchsin procedures and is particularly indicated for high-volume laboratories. It may also be more sensitive for paucibacillary specimens, since it allows examination of more fields with less effort. However, it requires a stable power supply, greater expertise in reading and microscope adjustment, and a regular supply of the costly and short-lived bulbs. Cheaper systems using halogen lamps have less stringent requirements, but performance does not entirely match that of the standard mercury vapor lamps. 2.4.6.2 EQUIPMENT AND MATERIALS Alcohol sand jar (only if a loop is used, not needed with disposable sticks), Bunsen burner or spirit lamp, Diamond pencil or lead pencil (if frosted-end slides are available), Filter paper, appropriate for funnel size, Funnels, small, for filtering solutions in use, Forceps, Lens paper or soft tissue paper, Plastic bag for waste disposal, Bamboo or wooden sticks or wire loops, Fluorescence microscope with objectives of 20x or 25x, and 40x (ideally specific for fluorescence microscopy), and eyepieces of 10x , Slide staining rack, Slide boxes, New, clean slides (rinse in alcohol and dry if necessary), Timer, Staining reagents, Staining bottles, 250 ml beaker, Beaker for rinsing water, Sink and water supply, Disinfectant solution. 2.4.6.3 REAGENTS AND SOLUTIONS Page 29 Auramine staining solution, 0.1% Acid-alcohol decolorizing solution, 0.5% Counterstaining solution, Potassium permanganate 0.5%, or blue ink, 10%. 29 | P a g e 2.4.6.4 DETAILED INSTRUCTIONS Preparation of smears The working area is first disinfected using 0.1 Sodium hypo chloride. The slides are then labelled properly using the laboratory register serial number marked on the sputum container and then the slides are placed on each side on its corresponding container. Proceed to smearing, taking the labelled slides and opening containers one by one; doing the smearing behind the flame of a Bunsen burner or spirit lamp.  For a direct sputum smear, a small portion of purulent or mucopurulent material with the stick/loop and transfer it to the slide is selected.  If a smear is prepared after specimen decontamination, the concentrated material must be transferred to the slide with a sterilized loop to avoid splashing. Note: If a loop is used, it must be sterilized before use by heating until red-hot within the glass chimney of the Bunsen burner. After use, plunge the loop into the alcohol sand jar, moving it up and down to remove any remaining material, then heat it again until red-hot. The material is then spread carefully over an area equal to about 2–3 cm x 1–2 cm using repeated circular movements, without touching the edge of the slide. The smear must be as even as possible by continuing this process until no thick parts remain. The thickness of the smear should be such that a newspaper held under the slide can barely be read through the dried smear. Disinfect the working area after smear preparation and let the smears air-dry at room temperature; do not use heat to speed the drying. Where humidity is high, gentle warming will be needed on a slide warmer (or locally made box with glass top under which there is a 20-W light bulb). When dry, the slides are held in forceps and are fixed by passing three times slowly through the flame of a spirit lamp or quickly through that of a Bunsen burner, smear upwards; do not overheat or AFB staining will be poor. Smears must be always kept out of direct sunlight. Page 30 2.4.6.5 STAINING METHOD The slides are placed with the smear upwards, on the staining rack over a sink, about 1 cm apart. A new filter paper is then placed in a small funnel, keeping it over the first slide and is 30 | P a g e filled up with auramine staining solution. Let the solution filter through the paper, covering each slide completely. Do not heat and the smears are left for 20 minutes. Using forceps, each slide is tilted to drain off the stain solution. Rinse the slides well with distilled water or clean tap water from a beaker (not directly from the tap). The acid solution is poured over the smears, covering them completely, and allowed to act for 3 minutes. Again using forceps, each slide is tilted to drain off the acid-alcohol solution. Gently rinse each slide again with distilled water or clean tap water from a beaker (not directly from the tap). The smears are then flooded with potassium permanganate or blue ink solution for 1 minute. Time is critical because counterstaining for longer may quench the AFB fluorescence. Using forceps, tilt each slide to drain off the counterstain solution. Gently rinse each slide again with distilled water or clean tap water from a beaker (not directly from the tap). Using forceps, each slide is took from the rack and the water is drained off. Stand the slide on edge on the drying rack and allow to air-dry. 2.4.6.6 READING The stained smears must be kept in the dark (in a box or folder) and read as soon as possible – fluorescence fades quickly when exposed to light. Fluorescent lamp is switched on 5 minutes before use; leaving the lower ordinary lamp off. The nosepiece is rotated so that the 20x (or 25x) objective is in the light path. Select the filter set position suitable for auramine stain whilst checking that there is a strong blue light; if not, open shutters and/or the fluorescent light beam diaphragm. Load the positive control slide on the stage and move the stage to position the slide under the objective. Use the coarse adjustment first, and then the fine adjustment, to focus the objective. If this fails (i.e. in thin negative smears), turn the filter set to transmitted light, switch on the lower normal lamp and focus as with a light microscope. Then switch off the lower lamp and return to the required filter position. The field should now be in focus. Page 31 Note: Focusing and maintaining focus while moving the smears may prove quite difficult if the permanganate-quenched background is too dark. If the lamp works well (strong blue light seen from the side), background staining with blue ink is another alternative. 31 | P a g e Check that bright yellow fluorescent AFB are clearly seen. If not, the lamp is adjusted and/or the mirror position. Further check that the whole field is evenly lit. If not, center the diaphragm after partially closing it. The positive control is then changed for the first routine smear without changing focus or rotating the objective. The procedure is repeated with each smear to be examined. Using the 20x (or 25x) objective, the stained smear are scanned systematically from one side to the other and back again – at least one length must be scanned before reporting a negative. Acidfast bacilli appear bright yellow against the dark background material. Tubercle bacilli are quite variable in shape, from very short fragments to elongated types, and may be uniformly stained or with one or many gaps, or even granular. The typical appearance is of bacilli that are rather long and slender, slightly curved rods. They occur singly or in small groups, and rarely in large clumps. With good staining (always check a freshly stained positive control first), there may also be fluorescing (sometimes green) artefacts, which do not have the typical shape. Non-fluorescing bacillary shapes must also be considered as artefacts. The 40x objective is used for confirmation of AFB Slides are stored in a slide box in order of the numbers of the laboratory register; they will be needed for external quality assessment. Results should not be written on the slides. When finished, the power is turned off. When work needs to be interrupted for just a few minutes only, the light is blocked using the shutter or the switch to turn off the light source is used. After switching off a mercury lamp, wait at least 15 minutes before switching it on again. 2.4.6.7 RECORDING Because fluorochrome-stained smears are examined at magnifications of 200x to 400x, the number of AFB can roughly be divided by a factor 10 or 5, respectively (depending on the objective) to make them equivalent to fields seen on examination of fuchsin-stained smears at 1000x. 2.4.6.8 DOUBTFUL RESULTS If there is uncertainty about the presence of a bacillus because of the lower magnification, it is best objective. This is more efficient than re-staining by the Ziehl-Neelsen technique (sometimes Page 32 to inspect this carefully with the 40x objective or, if unavoidable, with a 100x oil-immersion 32 | P a g e recommended), which may result in bacilli being washed off or simply not found again. In such a case I had to seek advice from my supervisor. 2.4.7 THE GENE EXPERT MACHINE The Expert MTB/RIF assay is a new test that is revolutionizing tuberculosis (TB) control by contributing to the rapid diagnosis of TB disease and drug resistance. The test simultaneously detects Mycobacterium tuberculosis complex (MTBC) and resistance to rifampin (RIF) in less than 2 hours. In comparison, standard cultures can take 2 to 6 weeks for MTBC to grow and conventional drug resistance tests can add 3 more weeks. The information provided by the Expert MTB/RIF assay aids in selecting treatment regimens and reaching infection control decisions quickly. 2.4.7.1 PRINCIPLE The Xpert MTB/RIF assay is a nucleic acid amplification (NAA) test that uses a disposable cartridge with the GeneXpert Instrument System. A sputum sample is collected from the patient with suspected TB. The sputum is mixed with the reagent that is provided with the assay, and a cartridge containing this mixture is placed in the GeneXpert machine. All processing from this point on is fully automated. 2.4.7.2 Advantages of the Xpert MTB/RIF Assay Major advantages of the Xpert MTB/RIF assay are that Minimal technical training is required to run the test. The main advantages of the test are, for diagnosis, reliability when compared to sputum microscopy and the speed of getting the result when compared with culture. For diagnosis of TB, although sputum microscopy is both quick and cheap, it is often unreliable. It is particularly result usually takes weeks rather than the hours of the Gene Xpert test. Page 33 unreliable when people are HIV positive. Although culture gives a definitive diagnosis, to get the 33 | P a g e The main advantage in respect of identifying rifampicin resistance, is again the matter of speed. Normally to get any drug resistance result takes weeks rather than hours. Additionally, the Xpert MTB/RIF assay can quickly identify possible multidrug-resistant TB (MDR TB). MDR TB is TB that is resistant to both isoniazid (INH) and RIF, two of the most effective TB drugs. RIF resistance is a predictor of MDR TB because resistance to RIF, in most instances, co-exists with resistance to INH. Rapid diagnosis of RIF resistance potentially allows TB patients to start on effective treatment much sooner than waiting for results from other types of drug susceptibility testing. For patients who are found to NOT have TB disease, rapid results from the Xpert MTB/RIF assay may contribute to cost savings by avoiding unnecessary treatment and respiratory isolation in hospitals or other institutions. The test does however have disadvantages as it has advantages. 2.4.7.3 Disadvantages There are a number of disadvantages which include:      2.4.7.4 The shelf life of the cartridges is only 18 months; A very stable electricity supply is required; The instrument needs to be recalibrated annually; The cost of the test; The temperature ceiling is critical. Interpretation of Xpert MTB/RIF Assay Results As with other NAA tests, the Xpert MTB/RIF assay should be interpreted along with clinical, radiographic, and other laboratory findings. The Xpert MTB/RIF assay does not replace the need for smear microscopy for acid-fast bacilli, culture for mycobacteria, and growth-based drug susceptibility testing, in addition to genotyping for early discovery of outbreaks. Providers and laboratories are required to ensure that patient Page 34 specimens are available for recommended mycobacterial testing. 34 | P a g e 2.5 MICROBIOLOGY, CULTURE AND SENSITIVITY TESTS SECTION Microbiology involves the study of microbes. Although, microorganisms are generally beneficial and essential to life some are, however, pathogenic and cause infectious diseases. The diagnostic microbiology laboratory is engaged in the identification of infectious agents. These infectious agents are broadly classified as viruses, bacteria, mycostic agents and parasites (Protozoans and Helminthes) also this section analyses body fluids and tissues for the presence of pathogenic microorganisms primarily by means of culture and sensitivity (C&S). Results of the C&S tell the physician the type of organisms present as well as the particular antibiotic that would be most effective for treatment. 2.5.1 BASIC RULES FOR WORKING IN THE MICROBIOLOGY LABORATORY,  While working in the laboratory, it is important that one must adhere to the following basic rules;  Keep the work area clean especially before leaving the laboratory and disinfectant it thoroughly at the end of day.  Wash hands frequently with soap and water  Before leaving the laboratory, place the discarded glassware into the designated place.  Cultures are kept under incubation and should be inspected in the morning and findings must be carefully recorded.  Send the laboratory reports promptly. In case of emergency a special report is dispatched or communicated by telephone. 2.5.2 GENERAL REMARKS Page 35  All specimens for culture must be collected prior to the therapy. If the patient is on antibiotic, a microbiologist should be informed so that he/she can take measures.  Specimens should be collected in adequate amount from the infectious site. This usually instructed by the physician. 35 | P a g e  Always use the sterile bottle to transporting the specimen.  All specimen must be accompanied by a request slip with complete information, which include the patient name, age, sex, hospital number, source of specimen and clinical information is very important in order to choose the appropriate medium for the culture.  Details should be entered in the laboratory register before and after an examination has been done. All containers used for holding microbiological specimens must be sterilized before being used. Such as test tube, culture tube with and without cap, plates, container to collect sputum specimen, blood specimen for microbial culture, plane tubes for collection (CSF) of cerebrospinal fluid and other specimen container (universal bottle) for collection of urine specimen and stool specimen should always be sterile and if not, the specimen is rejected and added to the rejection list. 2.5.3 URINE ANALYSIS (URINALYSIS) Urinalysis (UA) simply means analysis of urine. The Urinalysis Test was mainly used to diagnose transmissible Urinary Tract Infections. For microbiological examination urine was collected as a "clean catch-mid-stream" specimen. Urine samples was dispatched to the laboratory as soon as possible or no more than four hours if kept at room temperature or up to 24 hours if kept at 4 degrees centigrade, because of the continuous growth of bacteria in vitro thus altering the actual concentration of organisms and misleading results. 2.5.3.1 Macroscopic Urinalysis Macroscopic analysis of the urine was done by inspecting the physical appearance of the urine. Normal urine is amber and clear. Macroscopic urinalysis noted the amount, color, clarity, and cloudiness of the urine as well as any other visible characteristics of the urine such as the presence Page 36 of glucose, blood clots etc. Detection of Urine for the presence of blood, protein and other analytes was usually undertaken with reagent strips, the results of which indicated that further laboratory investigation was required. 36 | P a g e 2.5.3.2 Interpretation of results The interpretation of the urinalysis by a dipstick was simply done by comparing the colors on the stick to the reference color changes that were readily available on the dipstick box. INTERPRETATION OF THE DIPSTICK IS SHOWN ON THE DIAGRAM BELOW DIAGRAM Page 37   SHOWING THE DIPSTICK INTERPRETATION Visible blood in the urine (gross hematuria) suggests a kidney stone or a bladder disease. Foamy urine indicates the presence of protein in the urine (proteinuria) due to certain kidney conditions that spill protein out of the kidney from circulating blood (nephrotic syndrome). 37 | P a g e  Blood in the urine (detected as hemoglobin in the urine).  Ketone in the urine (metabolic waste product {ketosis}).    Leukocyte esterase (suggests white blood cells in the urine). Nitrites (indicates evidence of any bacteria in the urine). Bilirubin, and urobilinogen in the urine (related to an elevated bilirubin level, denotes possible liver disease or red blood cell breakdown in the body). Certain medications however, changed the color of urine. The color change in each of the squares signified a specific abnormality found in the urine represented by that specific color. If there are no abnormalities in the urine, the squares maintained their original color. The changes in color took from a few seconds up to a couple of minutes to occur. Under normal circumstances urine is sterile. The lower part of the urethra and the genitalia are normally colonized by bacteria, many of which may also cause urinary tract infection. 2.5.3.3 Microscopic Urinalysis Microscopic urinalysis entailed placing a few milliliters of the collected urine sample onto a thin glass slide using a small pipette and analyzed under the microscope. The urine sample was analyzed to look for white blood cells, red blood cells, epithelial cells (cells that line the urethra or bladder), and bacteria in the urine. Under the microscope, an estimate of the number of these components was typically assessed and reported. The quantity of these cells provided additional clinical information. 2.5.3.4 Interpretation of results Epithelial cells are the cells lining many structures in the body, such as the urethra, bladder, ureters, vagina, or skin. The presence of epithelial cells in the urine represent contamination of the sample; however, these cells in urine are also associated with an inflammation or infection of the urethra or bladder. White blood cells (or leukocytes) in the urine is suspicious for a urinary tract infection (UTI). When Nitrites or leucocytes is detected in the urine (pus cells under a microscope), the urine is identification of bacteria. Page 38 cultured for identification of presence of bacterial infection as we will see under Culture and 38 | P a g e 2.5.4 STOOL TEST FOR OVA AND PARASITE (O&P) The stool ova & parasites (O & P) test involved examination of a stool (feces) sample for the presence of intestinal parasites associated with intestinal infections. A parasite is a worm that has a mature form, an immature form (larvae), and eggs (ova). A parasite is a protozoa with an adult form that lives in the intestines (trophozoite) and a round, encapsulated tranmissiable form (cyst). In the laboratory, the stool sample was examined for a variety of parasitic forms. Some parasites were large enough to be seen without a microscope. For others, microscope slides were prepared with either fresh unstained stool or stool dyed with special stains. These preparations were viewed with a microscope to detect the presence of parasites or their eggs. 2.5.4.1 Macroscopic examination The recovery of ova or parasite forms depends upon: Consistency of the stool (soft, loose or watery) cysts are found maximum in the formed stool while trophozoite are most abundant in watery stool; Presence of blood and mucus; Presence of round worms, thread worms or tapeworm proglottids; Colour and smell of the stool. 2.5.4.2 Microscopic examination (temporary wet mounts) A wet mount was prepared directly from faecal material or from the concentrated specimens. The basic types of wet mounts that were made from each sample included: a. Saline wet mount: It was used to detect worm eggs or larvae, protozoan trophozoite and cysts. In addition it revealed the presence of RBCs and WBCs. b. Iodine wet mount: It was used to stain glycogen and nuclei of the cysts; was examined for amoebic and flagella cysts. Normally, a drop of saline was placed on left half of the slide and one drop of 2% iodine solution on the right half. With an applicator stick a small portion of the specimen (equivalent to the size of a match head) was picked up and mixed with saline drop. Similarly, similar amount was picked up and mixed with a drop of iodine. The cover slip was placed separately on both and examined under the microscope. Ova, cysts, trophozoite and adult worms were identified as per their Page 39 characteristic features. 39 | P a g e 2.5.4.3 Interpretation of results Any abnormal result meant parasites or eggs were present in the stool. This was a sign of a parasitic infection. Such infections include:Amebiasis ,Giardiasis ,Strongyloidiasis and Taeniasis The most common and frequently detected parasites in the CMR lab are Giardia and Entamoeba histolytica (causes amebiasis). The distinct types of parasites differ with regard to their structures, life stages, and transmission forms. 2.5.4.4 Salient features of common trophozoite and cysts Cyst/Trophozoite Features Entamoeba histolytica trophozoite: 12-60 µ, asymmetric, purposeful directional motility, single Spherical nucleus. Entamoeba histolytica cyst: Spherical, 10-20 µ mature cyst has four nuclei with compact centrally located karyosome Giardia lamblia trophozoite: 9-21x5-15 µ, pear shaped with tapering ends, actively motile like falling leaf, 2 centrally placed nuclei, uniform granular cytoplasm. Giardia lamblia cyst: Oval, 8-12 µ long and 7-10 µ wide; clear space between cell wall and cytoplasm. Four median bodies are present. Entamoeba coli cyst: 10-35 µ, usually spherical, mature cyst contain 8 or rarely 16 nuclei, unevenly distributed in clumps. Some examples of other intestinal parasites that are detected with an O&P include Worms (helminths) such as Roundworms such as Ascaris, Strongyloides, Hookworms, and Tapeworms such as Hymenolepis nana, Taenia solium and Diphyllobthrium latum and Flatworms and flukes Page 40 such as Fasciolopsis buski. 40 | P a g e 2.5.5 CULTURE AND IDENTIFICATION OF BACTERIA 2.5.5.1 BACTERIAL CULTURE 2.5.5.2 Urine Culture The Qualitative isolation of urinary pathogens begun with primary culture on CLED (CysteineLactose-and Electrolyte- deficient) agar; however blood agar or MacConkey agar can also be used. MacConkey’s agar was utilized to further selectively isolate and differentiate those gram negative rods that ferment lactose (e.g. E.coli, Klebsiella, Enterobacter and Serratia). A quantitative culture was performed using a 1:1000 ml (0.001 ml) sterile, wire loop. The urine was gently swirled to ensure even distribution of any organisms before culturing. A wire loop was flamed and allowed to cool before use. The loop was dipped vertically into the mixed urine specimen and inoculated onto a CLED plate making a single line streak from the top of the plate to the bottom of the plate. Distribution of the 1:1000 ml urine dilution was done by cross-streaking the initial line of inoculation over the surface of the plate to obtain countable colonies. The plate was incubated overnight at 36 – 370C and examined the next day for countable colonies. The number of colonies present was multiplied by 1000. 1000 represents the reciprocal of your dilution factor “RDF” (i.e. from the standardized loop 1/1000). Multiplying the number counted “NC” by the (RDF) gives the number of organisms/ml of urine. CFU/ml = NC x RDF http://www.allinahealth.org/ahs/allinalabs.nsf/page/UAPlating052908.pdf/$FILE/UAPlating052908.pdf 2.5.5.3 Interpretation of Culture Results It took 24 to 48 hours to get results. Normal Results;"Normal growth" is a normal result. This means that there is no infection. Abnormal Results :A "positive" or abnormal test is when bacteria or yeast are found in the culture. Page 41 A positive or abnormal culture test meant there was growth of bacteria or fungi in the urine culture. In general, 100.000 or more bacteria p/ml (per milliliter) is indicative of an infection. Bacteria presence ranging from 100 to 100,000 is believed to be caused by contamination of a sample or 41 | P a g e could also be an infection. Bacterial count below 100 falls in the range of a negative culture result. It was noted though, that 100 or less bacteria per ml would also be caused by intake of antibiotics. The causative agents of UTI are Proteus mirabilis, S.aureus, E.coli, S. saprophyticus, Group B streptococci and Enterococci. Normal flora include Diphtheroids, Alpha-streptococci, Lactobacilli, Coagulase-negative staphylococci. E coli and other Enterobacteriaceae such as Enterococcus species. Between the two sexes, women ran a higher risk of developing urinary tract infections. The reason was largely biological, as the urethra in the female body is shorter and closer to the anus, making it easier for intestinal bacteria to sneak into the urethra. As for men, there have an added advantage in the fact that their prostate glands contain antibacterial elements which reduce the risk of infection.in case of a positive urine culture test, sensitivity were taken to determine the appropriate treatment. 2.5.5.4 EYE, EAR NOSE, WOUND AND PUS SWAB 2.5.5.4.1 Procedure: These samples are collected using a dry sterile cotton wool swab; they are then inoculated into Chocolate and Macconkey agar. Gram staining is then carried out on each specimen. Pathogens that are likely to be observed include; 2.5.5.4.2 Wound and Pus swab: Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfrigens 2.5.5.4.3 Eye swab: Haemophilus influenza, Pseudomonas aeruginosa, Betahaemolytic streptococcus etc. 2.5.5.4.4 Ear swab: Escherichia coli, Proteus spp etc. 2.5.5.5 VAGINAL, URETHRAL AND ENDOCERVICAL SWAB. 2.5.5.5.1 Test Overview: This is use to detect the causative organisms of female reproductive system Page 42 infections and their sensitivity to antibiotics. 2.5.5.5.2 Procedure: A swab stick was used to collect specimen from the affected area and then inoculated into sterile media which include Chocolate and MacConkey agar. These plates were incubated 42 | P a g e at 37c for 24hours and were examined for any pathogenic growth, if there is any growth then a sensitivity disc is placed on a pure culture of isolate. Gram staining is then carried out on each specimen, a wet preparation of the swab can be made by dropping normal saline into the swab container and the swab stick is rubbed on a slide covered with a slip and viewed under the microscope; pus cells, epithelial cells, yeast cells etc. can be viewed. 2.5.5.5.3 Result: Possible pathogens include; Neisseria gonorrhea, Trichomonas vaginals, Candida spp, Clostridium perfrigens etc. 2.5.6 Stool culture Stool culture ( fecal culture) was used to identify if bacteria or other germs were present in the food hadlers. A sample of the patient's feces was placed in a special medium that provided nutrients for certain organisms to grow and reproduce; the culture was done in a test tube (or on a flat round culture plate) which was kept at the proper temperature so that bacteria could grow. Stool culture took 48 hours or longer to complete, but some organisms may take several weeks to grow in a culture.The bacteria that grew in the culture were identified using a microscope and biochemical tests. 2.5.6.1 Interpretation of results Some bacteria that are normal inhabitants of the digestive tract are known as the enteric bacteria. Escherichia coli, Klebsiella, Enterobacter, and Pseudomonas are members of this group. The enteric bacteria usually do not cause infection in the digestive tract, and were reported as normal flora in a stool culture. Because the presence of normal flora helps to protect against pathogens, the absence of normal flora in a stool culture was also reported. When only normal flora were found the results were reported as "no enteric pathogens found." When normal flora were absent from the stool, a heavy growth of an organism not usually pathogenic was recovered and was reported in this case. Most intestinal pathogens are gram-negative bacilli. Infection-causing bacteria that aren't normally found in the digestive tract include Shigella, Salmonella, Campylobacter, enterotoxigenic E. cereus, and Escherichia coli. Page 43 Coli,Vibrio and Yersinia. Other bacteria that produce toxins are Staphylococcus aureus, Bacillus 43 | P a g e Although Escherichia coli is a normal bacteria found in the intestines, one toxic type of this bacteria can be acquired from eating contaminated meat, juice, or fruits. It produces a toxin that causes severe inflammation and bleeding of the colon. Routine cultures included MacConkey agar plate (differentiates those that can ferment lactose from those that cannot) and XLD (xylose lysine deoxycholate) plate, enhance the growth of Salmonella and Shigella by suppressing the growth of gram-positive organisms and gram-negative normal flora. They also differentiate lactose and sucrose fermenters such as E. coli from Salmonella and Shigella, which are not. An antibiotic sensitivity test was done after a bacteria were identified to show which antibiotics was to work the best in treating the infection. 2.5.7 IDENTIFICATION OF BACTERIA Bacterial colonies differ greatly in their morphologies, cellular morphologies and staining properties. These differences helped us in identifying different species of bacteria. Most of these tests were not extremely specific. Gram staining (Primary identification) allowed us to distinguish Gram positive from Gram negative organisms and rod-shaped organisms from coccusshaped organisms, but did not allow us to make a more specific identification. To aid in the more definitive identification of bacteria, a series of biochemical tests (Secondary identification) were used to differentiate closely related organisms. These various tests were designed to identify various metabolic properties of different bacterial species. Because clinical microbiologists often must identify bacteria quickly and accurately, a number of companies produce rapid identification systems. These systems contained an easy to read chart that allowed us to quickly identify an unknown isolate based on the color changes that occurred in the various tests. 2.5.7.1 GRAMSTAINING (PRIMARY BACTERIA IDENTIFICATION) Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell Page 44 walls. There were four basic steps of the Gram stain;applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture on a slide, Heat fixing killed some bacteria but was mostly used to fix the bacteria to the slide so that they didn't rinse out during the staining procedure; The addition of a mordant, which bounds to crystal violet and trapped it in the cell (Gram's iodine) 44 | P a g e Rapid decolorization with alcohol or acetone, and Counterstaining with safranin. Carbol fuchsin was sometimes substituted for safranin. The slide was allowed to air dry then observed under a microscope using (X100) power, oil immersion. 2.5.7.2 Interpretation of results If the bacteria was Gram positive, it retained the primary stain (crystal violet) and did not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria was Gram negative, it lost the primary stain and took the secondary stain, causing it to appear red when viewed under a microscope. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. 2.5.8 BIOCHEMICAL TESTS (SECONDARY BACTERIA IDENTIFICATION) These tests were grouped based on what we were looking for. They encompassed tests used to identify Gram Positive Bacteria and Tests used to identify Gram Negative Bacteria. There are carbohydrate, IMViC, enzymes, and respiration. The carbohydrate tests are phenol red and Kligler iron agar or KIA, Indole, and citrate make up the IMViC (IMViC is an acronym that stands for indole , methyl red, Voges-Proskauer, and citrate) tests. Enzymes test is urease, coagulase test while Respiration test is the catalase test. A Bunsen burner was used to sterilize the opening of any tubes that were opened while obtaining a bacteria sample. An inoculating loop was used to obtain samples from a tube or agar plate. The bench top was cleaned with bleach prior to starting the lab. All of the samples were placed into a Page 45 37° incubator for a period of time. 45 | P a g e 2.5.8.1  CATALASE,COAGULASE AND CAMP TESTS (Tests used to identify Gram Positive Bacteria) CATALASE TEST Tests for the presence of catalase in bacteria with the use of 3% H2O2 (hydrogen peroxide). This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas. To test for catalase, a sample of bacteria removed from agar plate using sterile wire loop was spread onto a microscope slide. A few drops of 3% H2O2 were added to the bacteria then mixed for observation. Since Catalase breaks H2O2 down into water and O2, the presence of oxygen was characterized by bubbles which indicate a (+) result. Most aerobic organisms displayed (+) results e.g. Staphyloccocus aureus. Some anaerobic organisms displayed (-) indicating that they do not produce catalase e.g. Clostridium, Lactobacillus,  and Streptococcus pyogenes. COAGULASE TEST This test is performed on Gram-positive, catalase positive species and is used to identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence factor of S. aureus. Coagulase is an enzyme that clots blood plasma. The formation of clot around an infection caused by this bacteria likely protects it from phagocytosis. This test differentiates Staphylococcus aureus from other coagulase negative Staphylococcus species Page 46 http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm 46 | P a g e 2.5.8.2 BIOTYPING (Tests used to identify Gram Negative Bacteria) They included, Simon’s Citrate, Urease test, Sulfur Indole Motility Test (SIM) and KIA (Kligler Iron Agar) test. Bio typing was done in a standard way; from KIA, SC, SIM and Urea respectively.  KIA (Kligler Iron Agar) test Is used to test for the fermentation of only glucose (yellow butt), fermentation of lactose and sucrose (all over yellow), CO2 formed (crack in agar), or ferrous ammonium sulfate produces (black precipitate). This media was used to separate lactose fermenting members of the family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose, like Shigella dysenteriae. The KIA agar had glucose with a 0.1% concentration and lactose and sucrose with a concentration of 1% present as well as phenol red (pH indicator) and nutrient agar. With a sterile needle, an isolated colony was picked from the TSA plate. The needle was stabbed to the bottom of the slant, and then streaked on the surface of the slant. The tube was incubated overnight at 37 degrees Celsius to pink and the results were recorded. If the medium became acidic, then the phenol red turned yellow. If the medium became alkaline, then the phenol red turned purple. If an organism would only ferment glucose, then the medium initially turned yellow. Because there was so little glucose in the medium, however, the bacteria quickly exhausted the glucose supply and begun to oxidize amino acids for energy. The oxidation of amino acids produced ammonia as a by-product. The ammonia caused an increase in pH and a return to a red or purple color on the surface of the slant. Therefore, organisms only fermented glucose produced a slant with a red surface and yellow butt. If the organism being tested would ferment lactose and sucrose, then the entire tube turned, and remained yellow. The production of gas was detected in KIA slants by the presence of bubbles within the agar. Furthermore, if the produced gas was H2S, it reacted with the ferrous sulfate to produce glucose positive, lactose negative, sulfur reducing enteric. Page 47 ferrous sulfide, a black precipitate. Proteus mirabilis (pictured here, second from right) is 47 | P a g e http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm  Simon’s Citrate This is a defined medium used to test for the ability of organism to utilize citrate as a carbon source. It is often used to differentiate between members of Enterobacteriaceae. Contents of Simmon citrate include; sodium citrate as the carbon source, mono ammonium phosphate as the nitrogen source and bromthymol blue indicator Bacteria with citrate lyase/ citrase break down citrate to form pyruvate. Pyruvate is further reduced in fermentation. If CO2 was produced, it reacted with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turned the pH indicator (bromthymol blue) from green to blue. This was a positive result (the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative (green). http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm  Sulfur Indole Motility Media (SIM) This is a differential medium used to test the ability of an organism to reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to Page 48 differentiate members of Enterobacteriaceae. SIM tubes were inoculated with a single stab to the bottom of the tube containing 1mL of indole broth and then incubated at 37 degrees C for 2 hours. 48 | P a g e If an organism was motile then the growth radiated from the stab mark and made the entire tube appear turbid. Sulfur was reduced to H2S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide was produced, a black color formed in the medium. Proteus mirabilis is positive for H2S production. The organism pictured on the far left is positive for hydrogen sulfide production. Bacteria that have the enzyme tryptophanase, converted the amino acid, tryptophan to indole. Indole reacted with added Kovac’s reagent to form indole dye which was red in color (indole +). Escherichia coli was indole positive. The organism pictured second from left is E. coli and is indole positive. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we worked with were motile. http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm  Urease test This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It was used to distinguish the genus Proteus from other enteric bacteria. A urea broth was used. The hydrolysis of urea formed the weak base, ammonia, as one of its products. This weak base raised the pH of the media above 8.4 and the pH indicator, phenol red, turned from yellow to pink. Pink broth indicated the breakdown of urea and Proteus mirabilis was a rapid hydrolyzer of urea (center tube) below. The tube on the far right was inoculated Page 49 with a urease negative organism and the tube on the far left was uninoculated. 49 | P a g e http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm The unknowns were identified using a chart that was based on the results from the tests. While doing the tests there were some reactions that did not occur as quickly as they should. Some of the tests provided immediate results while others were to be incubated for a period of time. For example the Voges-Proskauer test took a while to complete after the two solutions were added. Identification of unknown bacteria meant that an antibiotic was to be made to treat an illness. This was carried out in Sensitivity tests. 2.5.9 ANTIMICROBIAL SUSCEPTIBILITY TESTING /SENSITIVITY TEST 2.5.9.1 Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics. Antibiotic susceptibility testing (AST) was carried out to determine which antibiotic was most successful in treating a bacterial infection in vivo. 2.5.9.2 Sensitivity analysis Sensitivity analysis determined the effectiveness of antibiotics against microorganisms such as bacteria that were isolated from cultures.Sensitivity analysis was performed along with Clean catch urine culture and stool culture . The Kirby-Bauer method /disk diffusion method was used for determining antimicrobial resistance as it was convenient, efficient and less costly Compared to other methods ( Stokes method, E-test (also based on antibiotic diffusion), Agar and Broth dilution Page 50 methods for Minimum Inhibitory Concentration determination). Any commercially available discs with the proper diameter and potency were used (Antibiotic discs). Stocks of antibiotic discs were kept at -200C. A small working supply of discs was kept in the refrigerator for up to one month. 50 | P a g e On removal from the refrigerator, the containers were left at room temperature for about one hour to allow the temperature to equilibrate. This procedure reduced the amount of condensation that occured when warm air reached the cold container. A growth medium, usually Mueller-Hinton agar, was first evenly seeded throughout the plate with the isolate of interest that had been diluted at a standard concentration (approximately 1 to 2 x 108 colony forming units per ml). Commercially prepared disks, each of which were preimpregnated with a standard concentration of a particular antibiotic, were then evenly dispensed and lightly pressed onto the agar surface. The test antibiotic immediately begun to diffuse outward from the disks, creating a gradient of antibiotic concentration in the agar such that the highest concentration was found close to the disk with decreasing concentrations further away from the disk. After an overnight incubation, the bacterial growth around each disc was observed. If the test isolate was susceptible to a particular antibiotic, a clear area of “no growth” was observed around that particular disk. 2.5.9.3 Interpretation of results http://en.wikipedia.org/wiki/Antibiotic_sensitivity biology.ed.ac.uk Colonies of microorganisms were combined with different antibiotics to see how well each stopped each colony from growing. The zone around an antibiotic disk that has no growth is referred to as the zone of inhibition since Page 51 antibiotic 51 | P a g e this approximates the minimum antibiotic concentration sufficient to prevent growth of the test isolate. The result of the susceptibility test was the classification of the microorganism in three categories of susceptibility as Susceptible, Intermediate susceptibility and Resistant. Zone diameters, to the nearest whole mm for various antimicrobial agents with disc content specified for each one for interpretation as susceptible, intermediate and resistant are usually provided, this zone was then measured in mm and compared to a standard interpretation chart . The effectiveness of individual antibiotics varies with the location of the infection, the ability of the antibiotic to reach the site of infection, and the ability of the bacteria to resist or inactivate the antibiotic. Some antibiotics actually kill the bacteria (bactericidal), whereas others merely prevent the bacteria from multiplying (bacteriostatic) so that the host's immune system can overcome them. Page 52 For example, gram (+) bacteria are sensitive to penicillin. 52 | P a g e CHAPTER THREE 3.1 SUMMARY 3.1.1 SUMMARY OF OTHER ATTACHMENT ACTIVITIES During my period at the Beit Bridge District Hospital as a student, I also had the opportunity of cataloguing some information materials for the laboratory and I also did some activities at the reception such as: attending to nurse aides, confirming and examining their request forms, entering their details into the register, detailing them concerning the test they were bringing the specimens for and directing them how and to where the tests were to be carried out. I was also given the task of being an assistant quality manager of the laboratory. It was a good tenure as I got to know and understand some fundamental aspects of quality control and management of the laboratory. We did some statistical analysis of data in the lab and l gained a lot of experience in the management of standard operation procedures in the laboratory. I was heavily taught the safety conducts in the laboratory. All in all it was a great working experience overall. 3.2 CHALLENGES ENCOUNTERED The main problem encountered was financial support for survival in the industrial site. At first it was difficult to pay rent as l was not getting any incentive from the institute at the same time I was trying to clear out my balance at the University. It was very tough, however at the later stages the hospital had to offer me accommodation at the nurses, somehow the burden was halved. It was quite challenging for me that l had to cater for everything using my own resources without any assistance from my institution and l had to feed myself every time to get to the organisation every working day. I was not given any remuneration or allowance, other problems encountered during the training was some Venda cultures which was fantastic. Page 53 attending to different people with different personalities at the reception. However l got to learn 53 | P a g e 3.3 CONCLUSION The whole internship period has been a success. I gained experience on various techniques and was exposed to various medical laboratory practices/analysis .This includes specimen collection, transportation and processing for food handlers certification as well as antimicrobial susceptibility testing, reading, interpretation and reporting of the results It gave me the opportunity of a hands-on experience in industrial laboratory work, more befittingly in an ISO/IEC 17025: 2005 accredited laboratory/institution. The student benefited much in learning new methods not taught at University, in using sophisticated equipment not available at the university and on an exposure to a more professional and hierarchical work environment. Modern equipment like the Flourescence microscope, BD FACS machine, Mindray machines, Partec machine , centrifuges, and sterilizers were some of the equipment that the student got the opportunity to operate in running tests. The student got the chance to appreciate how quality assurance is implemented through ISO accreditation, internal and external audits, and training. Personally, I gained and developed good communication skills, better organization, punctuality and a team-work oriented mindset. The student wishes to acknowledge the Beitbridge District Hospital Laboratory management on allowing student internship programmes despite the high unemployment rate in the country that has forced many companies to suspend such programmes. 3.4 RECOMMENDATIONS My first recommendation is to my institution (NUST). I am recommending an inter-company rotation schedule for students on attachment whereby a student will not only be attached at a single company but will rotate on a number of companies that are in different fields. This will improve students’ exposure to various industrial fields. For example one student could rotate from Parirenyatwa (Medical Institution), Nestle (Food Manufacturing), SIRDC (Research Institution) and Irvine’s Zimbabwe (Agricultural Company-Poultry). The rotation schedule would be drafted Page 54 by the school authority in association with the respective companies I recommend that all institutions or bodies involve in Student Industrial Working Experience Scheme, should provide places for industrial attachment for Student Industrial Training Fund and also pay some 54 | P a g e allowances to students and the company should provide more safety equipment to prevent further environmental and health hazards. I also recommend for the enlargement and installation of a safety hood in the bacteriology department for purposes of handling of specimens and cultures. The design of the cabinet should be such that air is drawn through the cabinet to a filter and then to an outlet funnel (by means of an extractor fan). The flow of air has the purpose of carrying infected particles and aerosols away from the environment of the operator to the outside atmosphere where they are at once diluted and destroyed by the ultraviolet light in the sun’s rays. This will help in creating a bacterial and fungalfree atmosphere. Also in the bacteriology department there is the need of constantly having an air Page 55 freshener since the odor of some fecal and urine specimens can be so unpleasant. 55 | P a g e REFERENCES 1. Lubert Strayer (1998) Biochemistry, W.H Freeman and Company, New York. 2. "Bacteria." Biology Online. 22 July 2008. 2 Dec. 2008 <http:// www.biologyonline.org/dictionary/bacteria>. 3. Madigan, Michael, John Martinko, Paul Dunlap, and David Clark. Brock Biology of Microorganisms. 12th ed. Boston: Benjamin-Cummings Company, 2008. 414-15. 4. Potter, Dr. Beth. "Biochemical Tests." Microbiology 202. Penn State Erie, 30 Oct. 2008 – 11 Nov. 2008. 5. Clendeman, T. E., G. W. Long, and J. K. Baird. 1995. QBC® and Giemsa stained thick blood films: diagnostic performance of laboratory technologists. Trans. R. Soc. Trop. Med. Hyg. 86:378. [PubMed] 6. Dunn, C. R., M. J. Banfield, J. J. Barker, C. W. Hinham, K. M. Moreton, D. T. Balik, R. L. Brady, and J. J. Holbrook. 1996. The structure of lactate dehydrogenase from Plasmodium falciparum reveals a new target for anti-malarial design. Nat. Struct. Biol. 3:912–915. [PubMed] 7. Iqbal, J., P. R. Hira, A. Sher, and A. A. Al-Enezi. 2001. Diagnosis of imported malaria by Plasmodium lactate dehydrogenase (pLDH) and histidine-rich protein 2 (PfHRP-2)based immune capture assays. Am. J. Trop. Med. Hyg. 64:20–23. [PubMed] 8. 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Braunstein GD, J Rasor, H Danzer, D Adler, ME Wade “Serum Human Chorionic Gonadotropin levels throughout normal pregnancy “. Am. J. Obstet. Gynecol. 1976; Page 57 126(6):678-681 57 | P a g e