A TECHN! STUDENT INDUSTRIAL WORK “HEME (SIWES) AL REPORT ON EXPERIE! UNDERTAKEN AT STANDARD MEDICAL DIAGNOSTIC LABORATORY, EDE, OSUN STATE. SUBMITTED TO THE SIWES COORDINATOR DEPARTMENT OF MICROBIOLOGY FACULTY OF SCIENCE OBAFEMI AWOLOWO UNIVERSITY IL! BY JIMOH ABDULLAH] ADEKILEKUN MCB/2006/119 COURSE CODE: MCB 399 IN PARTIAL FULFILLMENT OF THE AWARD OF A BACHELOR OF SCIENCE DEG ([Link]) IN MICROBIOLOGY OBAFEMI AWOLOWO UNIVERSITY ILE-IFE. MAY, 2010DEPARTMENT OF MICROBIOLOGY, OBAFEMI AWOLOWO UNIVERSITY ILE-AFE, OSUN-STATE. 16™ MAY, 2010, THE COORDINATOR, STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME, DEPARTMENT OF MICROBIOLOGY OBAFEMI AWOLOWO UNIVERSITY ILE-IFE OSUN- STATE. DEAR SIR. LETTER OF TRANSMITTAL In partial fulfillment of the requirement for the award of a Bachelor of Science degree {[Link]} in microbiology I. Jimoh Abdullahi Adekilekun hereby submit a copy of the report of the industrial training undergone at STANDARD MEDICAL DIAGONOSTIC LABORATORY, EDE ,OSUN STATE. ‘Yours Faithfully Jimoh Abdullahi AdekilekunDEDICATED TO. The ALMIGHTY ALLAH for his grace upon my life and also my mom, brother, sister, my friends and big daddy for being mine.ACKNOWLEDGEMENT I would want to genuinely appreciate my mom for her persistence on my behalf, patience, love and financial support. 1 also would want appreciate my brother, sister and my friends for being just the best. Sincere thanks to big daddy for his love and support. A big thank you to the organizers of this SIWES program, it was indeed an educating program. I would also like to thank the scientists at Standard Medical Diagnostics Laboratory for their patience in answering our questions and also for giving necessary explanations when due and my classmates and friends with whom I underwent this SIWES program, Thanks to Almighty Allah for making all this possible, I am very gratefulTABLE OF CONTENTS. Title Page Letter of Transmittal ‘i Dedication. ii Acknowledgement ii Table of Content iv Chapter 1 1.1 Brief history of SIWES and Objectives of SIWES. 1.2 Structural organization of STANDARD MEDICAL DIAGONOSTIC LABORATORY, EDE, OSUN STATE 3 Chapter 2 2.1 General Laboratory Equipments. 34 2.2 Care and Safety in the Laboratory $6 Chapter 3 3.1 Microbiology laboratory.......ssscsessssseossessecsessesnssnsstee 5.1 Chemical Pathology Chapter 6 6.1 Experience gained and problems encountered during the period of the SIWES program. 37 6.2 Recommendation 6.3 Conclusion 64 Appendix. 6.5 References.HAPTER ONE Ll STRUCTURAL ORGANISATION OF STANDARD MEDICAL DIAGNOSTIC LABORATORY, EDE, OSUN STATE. Biomedical Services + Reception Laboratories Health Records ‘Chemical pathology Microbiology Hematology 61.2 BRIEF HISTORY OF [Link]. SIWES was established in 1973 by the Industrial Training Fund (ITF) as one of her programmes. It was designed to give Nigerian students studying occupationally- related courses in higher institutions the experience that would supplement their theoretical leaning in order to solve the problem of lack of adequate practical skills preparatory for employment in industries by Nigerian graduates of tertiary institutions The Scheme exposes students to industry based skills necessary for a smooth transition from the classroom to the world of work. It affords students of tertiary institutions the opportunity of being familiarized and exposed to the needed experience in handling machinery and equipment which are usually not available in the educational institutions, Participation in SIWES has become a necessary pre-condition for the award of Diploma and Degree certificates in specific disciplines in most institutions of higher learning in the country, in accordance with the education policy of government. Usually there are three modules: The first module is for two months and this is taken by all 200- level Engineering and Food Technology students in University. This module of industrial Training is designed to expose the students to engineering and technology operations at the shop floor level. The second module is for three months. This is for the 300-level students of Engineering, Food Technology. Geography, Biochemistry, Nursing, Pharmacy, Geology, Physics, and Library Science. The third ‘module is however for six months and itis taken by 400-Level students of Engineering, Food Technology, Botany, Microbiology, Industrial Chemistry, Computer Science, Zoology, Agriculture and Physiotherapy. SIWES is operated by the ITF, the coordinating agencies (NUC, NCCE, NBTE), employers of labor and the institutions concemed (universities and polytechnics). Funded by the Federal Government of Nigeria. Beneficiaries-Undergraduates students of the following: Agriculture, Engineering.Technology, Environmental, Science, Education, Medical Science and Pure and Applied Sciences. Duration - Four months for polytechnics and Colleges of Education , and six ‘months for the Universities. A SURVEY OF THE INSTITUTIONS PARTICIPATING IN SIWES 9 Universities Polytechnics s of Education This survey was carried out year 2008 13 OBJECTIVI SIWES is a program organized for students of higher institutions to acquire practical knowledge of their various discipline in a real standard establishment different from the kind of experience or knowledge gained within the four walls of the classroom or school laboratoryCHAPTER TWO 2,1 GENERAL LABORATORY EQUIPMENTS ‘THE LIGHT MICROSCOPE ‘The microscope employs a hollow, extremely intense cone of light concentrated on the specimen. The field of view of the objective lens lies in the hollow, dark portion of the ccone and picks up only scattered light from the object. The clear portions of the specimen appear as a dark background, and the minute objects under study glow brightly against the dark field. This form of illumination is useful for transparent, unstained biological ‘material and for minute objects that cannot be seen in normal illumination under the microscope, AUTOCLAVE The autoclave is effective equipment used for steam sterilization at pressures above the atmospheric pressure. Thus. it is possible to steam at higher temperature then the boiling point, which a lot of microorganisms cannot withstand. Autoclaving is the most effective ‘method for sterilizing culture media. When sterilizing culture media with autoclave, we do so at 1.05Kg per square centimeter for 15 minutes to eliminate contaminations. REFRIGERATOR This is used to preserve samples, reagents etc, which are used for daily analysis and cannot be exhausted at once. The refrigerator helps provide optimum environment for materials to be preserved. INCUBATOR ‘The incubator is mainly used to incubate culture media as microbes have different optimum temperatures for growth and reproduction. The temperature of an incubator can be set to the preferred temperatures WATER BATH This is required to incubate bottle of culture media, liquids in flasks or other large Containers, and when incubating samples in the test tube racks. WEIGHING BALANCE This is a delicate instrument used for weighing essential, reagent, stains and culture ‘media that requires adequate weighingSTRAIGHT WIRE It is made up of a thick metallic lower part and a straight thin upper metallic part usually made up of platinum. This straight wire is used for stab culture and for picking discrete colonies. Usually sterilized before, during and after usage. This is achieved by flaming on Bunsen bumer red hot and allowed to coo! a bit before use. WIRE LOOP Made up of a thick metallic lower part and a straight thin upper metallic part curved into a small circle usually made up of platinum. Wire loop is used generally for inoculating samples and picking colonies sterilized by flaming red hot before, during and after use. It is always better to use the sides of the loop rather than the apex during inoculation, MYCOLOGY NEEDLE It is made up of a thick metallic lower part and a short straight thin upper metallic part usually made up of platinum. Used for needle mount preparations of fungi and fungi inoculation. It is usually sterilized by flaming. GLASS SLIDES Used for preparation of slides for microscopy. Sterilization is by flooding with alcohol and flaming off excess alcohol. COVER SLIPS This is use for covering wet smears of preparations. It is sterilized by flooding with alcohol and flaming off excess alcohol PETRI DISH Used for the preparation of culture media. It is usually bought sterilized. The disposable type cannot used a second time while the glass ware type can be reused be usually sterilized by autoclaving. FORCEPS A pair of forceps is a metallic object used for handing hot object or contaminated materials. It is sterilized by flaming red hot. ‘Others include: Other Laboratory equipments include includes sterilized slide, Giemsa Stain, needle, syringe, ethanol, sterilized bottle, agar (MacConkey or Chocolate), Gram positive, Gram 0negative sensitivity kit , cotton wool, EDTA, microscope, oil immersion, ethanol, sterilized slides, swab sticks, cotton wool, spirit, giemsa stain, lancets, surgical blades, oil immersion, pipette, light microscope, hot plate (dryer), centrifuge, hand gloves, microhaematocrit centrifuge, capillary tubes for measuring PCV, sealant, microhaematocrit reader, anaerobic jar, test tubes, bottles, water bath, weighing balance, microscope. pipette, beakers, bio safety cabinet, cotton2.2 SAFE WORKING PRACTICES IN AM EDICAL LABORATORY The following are some of the important points which apply when working with infectious materials: Never mouth-pipette. Use safe measuring and dispensing devices. Do not eat, drink, smoke, store food, or apply cosmetics in the working area of the laboratory Use an aseptic technique when handling specimens and cultures, Always wash your hands after handling an infectious material in the laboratory, when leaving the laboratory and before attending to patients. Cover any open wound with a water proof dressing. Wear appropriate protective clothing when working in the laboratory. Ensure it is decontaminated and laundered correctly ‘Wear protective gloves and when indicated a face mask, for all procedures involving direct contact with infectious materials. When wearing gloves, the hhands should be washed with the gloves on, particularly before doing ant clerical work. Centrifuge safely to avoid creating aerosols. Know what to do should a breakage occur when centrifuging. ‘Avoid practices which could result in needle stick injury. Do not use chipped or cracked glassware and always deal with a breakage immediately and safely Avoid spillages by using racks to hold containers, work neatly and keep the bench surface free of any unnecessary materials. Decontaminate working surfaces at the end of each day's work and following any spillage of any infectious fluid. Report to the laboratory officer in charge, any spillage or other accident involving exposure to infectious material Know how to decontaminate specimens and other infectious materials. Use and control an autoclave correctly. Dispose laboratory waste safelyCHAPTER THREE ACTUAL ACTIVITIES CARRIED OUT IN THE UNE {In Standard Medical Diagnostics Laboratory, where | underwent my SIWES program, we have the following sections: Reception, Chemical Pathology, Hematology, Microbiology laboratory 3.1 AT THE RECEPTION The receptionist on seat, collects samples from patients waiting to be transferred to the laboratory, put bills on the patients cards depending on the kind of tests to be done, register the patients cards and then also register results before they are given out to Patient, they also give out universal, anticoagulant bottles to patient and give them necessary instructions on how to collect into the bottles that is being given to them. Some of the laboratory materials are stored in the reception. Listed below are a few steps to follow when dispatching microbiological specimens: 1. Keep a register of all specimens dispatched. Record the name, number, and ward ‘or health centre of the patient, ype of specimen, investigation required, date of dispatch, and the method of sending the specimen. When the report is received ‘back from the microbiology laboratory, record the date of the receipt in the register. Check the specimen container is free from cracks, and the cap is leak-proof. Use sufficient packaging material to protect a specimen especially when the container is a glass tube. When the specimen is fluid use sufficient absorbent ‘material to absorb it should a leakage or breakage occur. 4. Mark all specimens that may contain highly infectious organisms. 3.2 MICROBIOLOGY LABORATORY In this laboratory the following tests are carried out Malaria parasite test ‘+ Urine Analysis, MCS * Malaria Parasite Test 13 ae'* Stool microscopy Semen Analysis '* Blood Microfilaria 3.21 URINE ANALYSIS, MCS URINE BENCH Pathogens that could be found Bacteria © Gram positive: Staphylococcus, Haemolytic Streptococci ‘© Gram negative: Escherichia coli, Proteus species, Pseudomonas, Aeruginosa, Klebsiella strains, Salmonella typhi, Neisseria gonorrheae The following activities are carried out on the urine bench: Urine macroscopy ie. Appearance which includes the color, turbidity etc. The microscopy to check out for possible parasite. Then culture of urine samples. URINE MACROSCOPY AND MICROSCOPY ‘Some other urine parasites include Wiucheraria brancofti. Onchocerca ete. Collection of urine Urine is collected in clean universal bottles. The mid part ofthe first early morning sample is preferred. MACROSCOPIC EXAMINATION Appearance: the normal urine color should be either amber or yellow. Other colors could be red brown black or white. ‘Turbidity: it could be slightly turbid, turbid or clear. MICROSCOPIC EXAMINATION Note: You culture before you spin the urine samples in the centrifuge to avoid contamination the samples, The urine samples are poured inside test tubes and labeled with the laboratory number of the patient. It is then arranged inside the centrifuge and allowed to spin for 10minutes, so as to separate the urine into layers. The supernatant part of the spinned urine is then disposed off into a container containing «disinfectant and then the sediment is placed on the glass slide. The sediment of urine 4sample on the slide is covered with a coverslip and then examined under the microscope. The following could be seen under the microscope: bacteria cells, epithelial cells, cellular casts, red blood cells and white blood cells. HOW TO CULTURE URINE Culture on Cysteine Lactose Electrolyte Deficiency Agar ([Link].D) and MacConkey agar (both are differential agar) which differentiate between lactose and non-lactose fermenting organisms. C.L.E.D does not allow swarming of Proteus. A sterile wire loop is used to culture urine. PROCEDURE- Dip a wire loop inside the universal tube containing the urine (open the cork of the tube with the side of your palm and keep holding the cover while you dip the wire loop into the urine) and then inoculate your plate. The inoculation is done by introducing urine into the plate and making a smear, from the inoculums a primary and secondary streaking is made. To make the primary streaking, spread from the inoculums at angle 90 and the secondary streaking is done by spreading from the primary streaking, Then incubate overnight -that is putting inoculated plate in the incubator at 35°C for 18-24 hours. The plate can then be read the next day. On CLED, the lactose and non-lactose fermenting organisms are checked and then confirmed on MacConkey agar. For lactose fermenting organisms, the colonial appearance is recorded and then the gram staining is done. If tis gram negative, the organism present could either be Klebsiella or Escherichia coli. Biochemical test can then be done by inoculating citrate, urea and Peptone water. The peptone water is used for sensitivity test on nutrient agar (DST); the plate is then incubated at 37°C and also the urea and citrate for 12-18 hours (overnight), Klebsiella is evident if citrate is positive or urea is positive. Citrate is positive when it is blue in color whereas urea is negative when itis yellow and positive when itis red. Citrate is negative and urea is negative when Escherichia coli is evident FOR NON-LACTOSE FERMENTERS, oxidase test is done. Positive oxidase test shows evidence of Pseudomonas species in urine while negative oxidase test shows Wemevidence of Salmonella, Shigella, Proteus, Vibrio cholerae. Biochemical testis then done for these organisms. 3.22 MALARIA PARASITE TEST ‘Some parasites that could be detected in the blood are: Plasmodia, Trypanosomes, Leishmania, filarial worms. SPECIMEN. A one meter in blood diameter of the blood film of the patient Specific identification of parasites requires a permanent stain. For permanent staining, two types of blood films can be prepared. Thick films allow a larger volume of blood to be examined, thus making it easier to detect light infections with fewer parasites, while species identification is difficult. Thin films are necessary to see the morphological characteristics of the parasites and to identify them. PROCEDURES PRECAUTION: It is necessary for one to be very careful while collecting and preparing blood samples. A number of parasitological, bacterial and viral diseases can be transmitted through blood. Blood film should be prepared preferably within one hour of collection The time of collection should be mentioned on the specimen as well as on the result sheet and also the laboratory number for correlation. It is preferable to prepare blood films with fresh blood without anticoagulant. If itis not possible, blood pant coagulated with EDTA (10mg/Sml) should be used, Step! An absolutely clean, grease-free slide, well-washed slide cleaned with 70% ethanol is recommended (at Standard Medical Diagnostics Laboratory new slides are used). The slide is labeled with the patients laboratory number. Step 2 ‘Swab the top of the patient’s third finger or thumb with cotton wool soaked with ethylated spirit to disinfect and clean the possible micro organisms present on the surface ‘of the skin. Step 3 Prick the point cleaned with a sterilized lancet and discard immediately. Step 4 16Apply pressure on the lower side of the top with your own hand so the blood would be able to come out in few trickles as a drop or two will be placed on either sides of the slide since the laboratory number labeled on the slide will be in the middle. Step 5 ‘You prepare a thick blood film for the malaria parasite test. To make a thick blood film, place two or three small drops of fresh blood without anticoagulant on a clean slide with a sterilized end of another slide. Mix the drops in a circular motion over an area about two centimeters in diameter, (continue mixing for about thirty seconds to prevent formation of fibrin strands that may obscure the parasites after staining, if anti- coagulated blood is used, itis not necessary to continue mixing for thirty seconds). Step 6 Allow the film to dry in air at room temperature on a dryer (hotplate) to fix the film. Step7 After drying, the slide is placed directly into an aqueous stain called Giemsa stain to ‘make the thick blood film to Iyses the red blood cells and to remove hemoglobin so that the parasites can be easily detected GIEMSA STAINING TECHNIQUE. Giemsa stain is a Romanaosky that requires dilution in buffered water or buffered saline before use, Giemsa stain (stock solution) Giemsa stain powder 0.6g Methanol, absolute (acetone-free) SOml Glycerol 50m! Dissolve the Giemsa stain in methanol in a brown bottle containing a few glass beads. ‘Add glycerol, mix and place the bottle in a water bath at 50-60 degree centigrade for two hours to dissolve the stain. Shake gently at half-hour intervals. The stain should stand at room temperature for three weeks and should be filtered before use. If kept air-tight, the stain is stable for several months Preparing a working solution of Giemsa stain For thick films the commercial stock solution is diluted with the ratio 1:50 with a neutral oF slightly alkaline buffer (7.0 to 7.2) € .g phosphate or tap water if the pH is satisfactory. aTECHNIQUE USED The labeled slide with the blood film on either end of itis directly stained in diluted Giemsa stain for like 15 minutes. It is then brought out and rinsed properly with tap water, gently flushing the stain off the slide with water. Dip the slide briefly in the buffer or rinse under gently running tap water. © Wipe the under surface of the slide to remove excess stain. © Allow it to airsdry in a vertical position, © View under the light microscope with the oil immersion lens, ¢ A drop of oil is placed on each dried, stained and fixed blood film and then viewed for malarial parasites. ‘TROPHOZOITE OF MALARIAL PARASITE AS VIEWED UNDER THE LIGHT MICROSCOPE, ‘Trophozoite is the growing form of the parasite in the peripheral blood of man after the invasion of the red blood cells by merozites. When the mature schizonts rupture the merozites penetrate the red blood cells and develop into trophozoites. Immature trophozoites are concave disc appearing as ring forms in stained preparation. It consists of, L.A rod-shaped nucleus (chromatin dot) stained red. 2. A peripheral rim of cytoplasm that stains blue and 3. An unstained clear area or vacuole in the centre that pushes the chromatin dot to the periphery of the cytoplasm. 4. Three stages of the asexual life cycle occur in man, namely the trophozoites, schizont and the gametocytes. The parasites reside in the peripheral red blood cells. Each species is identified on two basic parameters 1. Appearance of the infected red blood cells. 2. Appearance of the parasite Results: Malaria Parasite ‘Chromatin of parasite: Dark red 18Cytoplasm of parasite: Blue ‘Schufiier's clots: Red Maurer's dots: Red-mauve 3.23 BLOOD MICROFILARIA TEST In this test, blood is gotten from the patient's vein A rubber tube rope is tied on the upper part of the patients arm. A vein is located between the middle fold of the arm and the upper section of the arm. When the vein is located, the spot where it is found is swabbed with a cotton wool soaked in spirit, A sterilized needle is used to prick the vein and blood is drawn and immediately transferred into the small anticoagulant bottle gotten from the reception, itis corked tightly Immediately you are through drawing the blood you loosen the rubber rope on the arm to reduce pressure and the blood stops coming out You clean the spot on the person's arm after all these with a clean sterilized cotton wool * Note the blood gotten can be used for either microfilaria test or malaria parasite test TEST Blood Microfilaria can be detected in the direct wet mount of fresh blood by their characteristics, shape and motility For identification of microfilaria worm in stained blood films, the following characteristics are looked for: @ (ii) (ii) Presence of absence of a sheath. Presence or absence of nuclei at the tip of the tail Size of microfilaria worm Size of cephalic space in sheathed microfilaria worm.3.24 STOOL MICROSCOPY ‘Three protozoan parasites which may be found in human stool are: - Rhizopodea (amoebae) e.g. Entamosbs histolytica = Zoomastigophora (flagellates) e.g. Giarelia Intesinalis - Ciliatea (ciliates) e.g. Balantidium coli EXAMININATION OF FAECES. It is viewed under light microscope at x10 and x40 First you view macroscopically for the following: Form, color, Smell, consistency. presence of blood, and mucus, nematode, tapeworm, and segments. ‘When viewed under the microscope in normal saline ‘* You place a drop of normal saline on a thin slide with the pipette in the normal saline bottle. © Pick a tiny bit of the stool sample and make a smear in the normal saline with it. ‘© View under the light microscope for cellular exudates such as helminthes egg. protozoa cyst, and actual larva of nematode worms. ‘+ When viewed under the microscope in iodine it is the same process as listed in the first two steps above, just use iodine in this case and not normal saline When viewed under the light microscope, stained protozoa cysts are more visible. Other things that could be seen under the microscope are: fat globules, undigested starch, vegetable cells, and air bubbles. Cysts can be concentrated by the formal ether technique or by a simple floatation in concentrated zine sulphate 3.25 SEMEN ANALYSIS Semen Analysis with Microscopy This involves the analysis of semen by culturing and performing sensitivity test. Part A (i) Physical examination © Volume: Imi, 2ml and above © Viscosity: Watery or Normal 20‘© Appearance: creamy, whitish or Creamy — whitish (ii) Microscopy © Motility © High power © Normal © Abnormal N.B. The best sperm count is about 90x10° total counts but normal count is 45 x 10°, bbut when the total count is 25 x 10° the diagnosis could be infertility (i) After the examination in part A, sterilize inoculating loop by flaming, culture the semen sample on MacConkey and Chocolate agar. NOTE® to always culture on chocolate agar you cut the agar in the middle and throwing of this cut part into the waste to prevent organisms from swarming in the agar. (ii) Incubate for 24 hours (iii) Examine the colony if there is growth, gram stain, set up biochemical tests (iv) Inoculate peptone water for flooding of DST (sensitivit Antibiotic disc). HOW TO CARRY OUT SENSITIVITY TEST: Flood the nutrient agar with inoculated peptone water, place the antibiotic disc on the flooded plate and incubate overnight for 12-18 hours. At the end of the stipulated time test using the right any antibiotic surrounded by a region where no microorganism grew can proof useful against the microorganism discovered present. 4.7 Procedures for gram stain (i) Crystal violet solution (ii) Todine solution (functions as a mordant) (iii) Acetone (decolorizes) iv) Safranin (counter stain) Procedure 1. Prepare a heat fixed smear from a 18-24 hour old culture 2. Stain with crystal violet solution for 1 ~ 2 minutes and rinse off the solution. 3. Rinse off with iodine solution for 1 minute Nee oo4. Rinse off the iodine solution and wash the slide with acetone until the crystal violet dye no longer runs from the slide and this will last only S-15 seconds. S. Rinse under gentle ~ running tap, and counter stain with safranin for 30 seconds. 6. Wash with water, blot dry and examine under microscope. ‘Observation 1 Gram-positive cell appear purple, or crystal violet iodine complex 2 Gram negative cells are red or pink Note* Cells could be either bacilli or cocci