Anti-d Blood Group

CHAPTER ONE
INTRODUCTION
The industrial attachment is an important part of the course structure of the university. This is the time students appreciate the theoretical work done in the lecture halls when seen practically. This attachment introduces the student to the outside world in terms of working experience and also prepares them for the future.
The department of applied biology therefore introduced the industrial attachment program in its learning curriculum to give students a chance to combine theoretical knowledge with practical skills in the working environment. It requires a student to go for industrial attachment in his chosen field of profession for a period of eight weeks. At the end of the attachment program, the student is expected to write a report that incorporates work experiences gained linking it to the theoretical knowledge acquired in school. OBJECTIVES OF SUPERVISED INDUSTRIAL ATTACHMENT To provide the student an opportunity to apply his or her knowledge in real situation thereby bridging the gap between university work and actual practice. To expose the student to work methods not taught in school and to provide access to equipment not available in the university environment. To assess the interest of the student in the occupation he or she plans to take. To expose students to the structures, operations and integration of different departments in laboratory such the microbiology, biochemistry, parasitology and hematology. To expose the student to the behavior of organizations both in the internal and external environment and encourage then to be productive and hardworking whilst developing chemists for the future.

A BRIEF HISTORY OF THE HOSPITAL
HISTORY OF THE MILITARY HOSPITAL
The Military Hospital is a general hospital situated about 4km from the Accra International Airport on the main Airport - Central road (Independence Avenue Road). It is a walking distance from the Golden Tulip Hotel, a popular and modern 4- Star Hotel and located directly opposite the Army Officers Mess. During World War II, it became operationally and logistically necessary for Britain to take over the defense and security of the West African sub – region. The responsibility was given to the war office with Lt Gen Giffard as the General Officer commanding the West Africa region. The General established his operational Headquarters in the Gold Coast.
As a result of the war, there was an urgent need for Casualty Clearing Stations (CCS) and general Military Hospitals within the colonies to attend to the medical needs of the numerous service personnel deployed in combat in several theatres. General Giffard requested the war office in the UK for the necessary logistics and administrative support for setting up a Military Hospital in the Gold Coast. The Public Work Department was commissioned by the colonial officers to construct the buildings and by 4th July, 1941 the Base Hospital in Accra was completed to begin operation as the 37th general Hospital within the British Empire.
Soon after the war in 1946, the size of the hospital was re- designated Military Hospital to the Gold Coast to serve as one of the support service providers within the Armed Forces.
Over the years, the hospital has grown to become the second largest medical facility in Ghana’s capital, Accra and the third largest in the country after Korle – Bu (Accra) and Komfo-Anokye (Kumasi). As a Military institution and for operational flexibility, technical efficiency and administrative ease, the hospital has been well structured to carry out its roles and task. It is therefore organized into working units or subunits- Divisions and Departments on the basis of medical, paramedical and administrative lines, with their own heads. The subunits operate with a Commanding Officer (CO) at the apex. The CO is responsible to higher Headquarters for the smooth running of the hospital.
The pathology as one of the divisions is made up of the Chemical pathology and Immunology, Haematology, Microbiology, Parasitological, Histopathology, Blood Bank / Transfusion, Science / Serology and Morbid Anatomy Departments. It provides medical laboratory services to clinicians to help them monitor treatment and confirm the success or otherwise of such treatments.
The 37 Military Hospital provides Medicare for troops, all civilian employees of the Ministry Of Defense (M0D) and their families and as well as the general public. It also serves as the United Nations Level IV medical facility in the West Africa sub regions. The hospital indeed lives to its motto “HEALTH FIRST” and all those who have passed through it can attest to it.
CHAPTER TWO
THE DEPARTMENT AT THE LABORATORY OF 37 MILITARY HOSPITAL
1. PARASITOLOGY DEPARTMENT
2. HAEMATOLOGY DEPARTMENT
3. SEROLOGY
4. BLOOD BANK
5. CHEMISTRY DEPARTMENT
6. MICROBIOLOGY DEPARTMENT
7. HISTOPATHOLOGY
8. MORBID ANATOMY DEPARTMENT
SAFETY RULES AND REGULATIONS UNDERTAKEN IN THE LABORATORY After taking a skin snip, disinfect the instruments. Thus needles and surgical blades.

Laboratory coats must be worn at all times in the laboratory. This will protect your clothing from contamination and discoloration.

Eating, drinking is forbidden at all times in the laboratory.

Gloves must be worn at all times when working on samples.

When a sample accidentally spill, disinfect the place immediately.

After using disposable equipment and samples, they should be disposed properly.

Clean the laboratory benches with disinfectant before and after work.

All samples should be properly labeled. This will prevent improper use of material.

After working in the laboratory, observe good hygiene by washing your hands with soap.

Collect samples (stool and urine) in clean specimen bottles.
ACTIVITIES UNDERTAKEN IN THE PARASITOLOGY LABORATORY Routine Stool Examination (RST) Routine Urine Examination (RUE)
ROUTINE STOOL EXAMINATION
Objective
The routine examination of stool is used to detect the presence of intestinal parasites including ova, larva, trophozoites and cyst of Helminthes and protozoa respectively.

Test Procedure Emulsify about 1g of faecal material in physiological saline bringing it to suspension using an applicator stick Label a clean glass slide using a grease pencil Place a drop of stool suspension on each end of the viewing portion of the slide Cover gently with a cover slip Place the preparation under the microscope, lower the condenser to obtain a low light intensity Observe for trophozoites of intestinal protozoa, ova, larva, Helminthes, red blood cells, crystals, white blood cells, Ascaris, Hook worms using the *40 objective
Reporting Test Results
Macroscopy: Describe stool specimen as to consistency (formed, semi formed, loose, watery etc. and presence of blood and mucous)
Microscopy: Note the name of any parasite present or “No ova, larva, or protozoa seen”.
ROUTINE URINE EXAMINATION
Objective
The objective is to screen for the detection of diabetes, metabolic abnormalities, liver diseases, biliary and hepatic obstructions, haemolitic diseases, diseases in the region of the kidney and urinary tract as well as parasites and other formed elements.

Test Procedure Label a 5ml or 10ml centrifuge tube using a grease pencil Mix urine and pour into the centrifuge tube until it is about half full of the specimen Observe the appearance and colour of the urine specimen and record it Dip the reagent strip into the urine for approximately 1 second Remove 30 to 60 seconds later and record the biochemical reaction of the reagent strip to the colour chart Ignore any colour change after 120 seconds (2 minutes). Spin the urine at 3000rpm for 3 to 5 minutes Decant the supernatant and re-suspend the deposit in the residual fluid Label a glass slide and place a drop of urine deposit onto it Apply a cover slip gently on it .Place it under a microscope, lower the condenser to obtain a low light intensity Examine the preparation preferably using the 40X objective Observe for pus cells (WBC), RBC’s, epithelial cells, cast (granular, blood and leucocytes); crystals, ova of Schistosoma haematobium, trophozoites of Trichomonas vaginalis and fungi (yeast-like cell)
Reporting Test Results
Macroscopic:
Colour: Normal freshly passed urine is amber (pale to dark yellow in colour. The yellow is due the pigment urochrome, urobilin and porphrings.
Appearance: Normal passed urine is clear. Abnormality may be due to: Urinary tract infections in which the urine appears cloudy because it contains pus cells and bacteria Urinary schistosomiasis in which the urine often appears red and cloudy because it contains blood (haematoria) Black water fever and other conditions causing intravascular haemolysis in which the urine appears brown and cloudy because it contains free haemoglobin (haemoglobinuria) Jaundice in the urine may appear yellow-brown or green-brown because it bile pigments or increased amount of urobilin (oxidized urobilinogen) Bancroftian filariasis in which the urine may appear milky-white because it contain chyle
PH: Normal reaction of freshly passed urine is slightly acidic, around 6.0
Protein: Positive results indicate the urine might contain urinary schistosomiasis, urinary tract infections, and nephritic syndrome, renal disease such as pyelonephritis and glomerulonephritis and renal tuberculosis. It may also be found in pregnant women and sometimes healthy young individuals.
Glucose: Found in the urine of diabetic patients and occasionally in healthy individuals. Ketone: Found in the urine of untreated diabetics or persons suffering from starvation
Bilirubin: Found in the urine of persons with hepatocellular jaundice or cholestatic (obstructive) jaundice
Urobilinogen: Found in the urine of persons with conditions of abnormal haemolysis
Blood: Found in the urine in urinary schistosomiasis, bacterial infections, acute glomerulonephritis (inflammation of the glomeruli of the kidney), sickle cell disease, leptospirosis, infective endocarditis, kidney stones and Specific gravity (S.G): The normal specific gravity of urine varies between 1.002-1.025, depending on the state of hydration of the person and time of the day. High specific gravity is found in urine because of the following: glucose, protein, or other particles.
Nitrite: Causes of nitrite in urine are because bacteria that produce the enzyme nitrate reductase are able to reduce nitrate to nitrite. Nitrate reducing bacteria are Escherichia coli, proteus species, Citrobacter species, Aerobacter species and Salmonella species A negative test cannot rule out a urinary tract infection because the causing organism may be one that does not reduce nitrate to nitrite, e.g. Enterococcus faecalis, Staphylococcus albus, Staphylococcus saproptyticus, Pseudomonas species or Candida species.
NB: The following are some abnormal urine colours, straw, coke, light amber, deep amber etc.
Haemorrhagic conditions
OCCULT BLOOD TEST (IN STOOL)
Objective
Occult blood test is done to detect the presence of occult (hidden) blood in stool samples. This test is not carried out during menstruation or diarrhea
Test Procedure Open the front part of the hemoplus test card Take a small sample of stool using the spatula, smear the red circles completely with the stool sample Open the back part of the hemoplus card and drop two drops of hemoplus developing reagent on each of the circles Close the hemoplus test card with its flap The result is read after some seconds according to the manufactures instructions
Reporting Test Result
Positive results: A blue-black couloration of the sample occurs.
Negative results: No change in coularation of the sample
ACTIVITIES UNDERTAKEN IN HAEMATOLOGY LABORATORY
The main activities include; Blood Film (BF) for Malaria parasites Sickling test FBC (Full Blood Count) ESR (Erythrocyte sedimentation Rate)
BLOOD FILM FOR MALARIA PARASITES
Objective
To detect the presence or the absence of malaria parasites

Principle
Stained thick films are used to diagnose malaria and other diseases caused by blood parasites
Test Procedure Place a large drop of well- mixed blood in the center of a clean glass slide Using the corner of a second slide, carefully spread the drop of the blood over an area of 10-12mm To determine the correct thickness of the film; place a piece of newspaper on the slide , the film must just be visible Dry the film at room temperature Prepare 1 in 10 dilution of the stock Giemsa stain Cover the entire slide with diluted Giemsa and stain for at least 10 minutes Wash gently in tap water and leave slide to dry in an upright position at room temperature Examine under *100 objective for the presence or absence of malaria parasites
Reporting Test Result
+ = 1-10 parasites per 100 thick fields
++ = 11-100 parasites per 100 thick film fields
+++ =1-10 parasites per single thick film fields
++++ =more than 10 parasites per single thick film field

To prepare Giemsa stock solution
Giemsa powder: 3.8g Absolute methanol: 250ml Glycerol (pure): 250ml
Weigh and dissolve the powder in the alcohol in a dry conical flask. Stir using a glass rod until the powder has completely dissolved. Add the glycerol. Filter and store in a reagent bottle and label.
Working solution;
To prepare 5% Giemsa solution:
Giemsa stock 5ml Phosphate buffer 7.2 or tap water 95ml
Mix the solutions in a measuring cylinder.
SICKLING TEST
Objective
Detection of Haemoglobin Sickling in human red blood cells

Principle
Under low oxygen tension, erythrocytes containing haemoglobin Sickling assume the characteristic sickle –shape (Crescent Shape). This is enhanced by the addition of a reducing agent e.g. 2% Sodium Metabisulphite solution.
Test procedure Combine an equal volume of blood and sickling fluid Mix very well on the slide Apply coverslip gently to avoid air bubbles Allow to stand for at least 30 minutes Examine under the microscope for sickled red blood cells
Reporting Test Result
Report as sickling Positive or Negative
To prepare 2% Sodium Metabisulphite Solution: Sodium Metabisulphite 0.1 g Distilled water 5ml
Weigh the salt and dissolve in 5ml distilled water in a test tube. Transfer into an appropriate bottle and label.

FBC (FULL BLOOD COUNT)
The count of white blood cells (WBC),red blood cells (RBC), estimation of haemoglobin concentration(HB),platelets(PLT),HCT,MCV,MCH,MCHC,Lymphocytes(LYM),Neutrophils(NEUT) among other blood components in a whole blood is known as full blood count (FBC).
Test procedure The blood samples are placed on a roller for 10-15 minutes to ensure well mixed anti-coagulated blood They are then fixed under the SYSMEX BD HAEMATOLOGY ANALYSER (an automated machine) to be analyzed and the results are printed out automatically through a computerized printer.
ESR (Erythrocyte sedimentation Rate)
Objective/Principle ESR measures the sedimentation of erythrocytes in their native. The numerical value in millimeters is obtained by measuring the distance between the lowest point of the surface meniscus to the upper limit of the erythrocyte sedimentation in a column of anti-coagulated and diluted blood that has stood in the selected tube for 60 minutes.
Test Procedure The ratio of blood to anticoagulant is 4 : 1 Fill a clean dry standard tube with the blood and adjust the level to the “O” mark Place the tube in a strictly vertical position on the bench, not exposed to direct sunlight and free from vibration and draughts. After exactly one hour , read the distance from the bottom of the surface meniscus to the top of the column of sedimentating red cells (where the full density is apparent) in millimeters and record as the ESR value
Reporting Test Result
The test is reported mmfall/hr
Reference range: Men up to 10mm/hr Women up to 14mm/hr
Erythrocyte Sedimentation Rate is a non-specific indicator of inflammation and tissue damage.
To prepare sodium citrate anticoagulant: (100ml) Tri-Sodium citrate 3.8 g Distilled water 100ml

Weigh the sodium citrate and transfer it to a proof bottle. Add 100ml of water and mix to dissolve the chemical. Label the bottle and store preferably at 2-8 degrees Celsius.
ACTIVITIES UNDERTAKEN IN THE CHEMISTRY DEPARTMENT The following are various tests undertake in the chemistry department LFT [Liver Function Test] BUE creatinine[Blood Urea Erythrocyte Creatinine] FBS[Fasting Blood Sugar] Lipid profile Glycosylated hemoglobin Glucose-6-phosphate dehydrogenase(G6PD)
Test procedure Collect all blood samples and balance them in the centrifuge to separate the blood into their various components, thus plasma (serum) from the white (leucocytes) and red (erythrocytes) blood cells. Pour the serum of the blood samples into the cups of the chemical analyser, these cups are fixed in cup adapters on the trays of the chemical analyser. Fix the trays into the chemical analyser and allow to rotate

There are four trays in all, and each tray can contain only 10 cups, so as the trays rotate in the chemical analyser, the serum is collected by a pipette in the biochemical analyser which then undergoes several processes to produce specific results needed for diagnoses through a connected printer.
Objectives for carrying out these tests
BUE+CREATININE - Kidney function FBS -Glucose metabolism
LFT (Liver function test) - Liver enzymes and protein
GLUCOSE 6 PHOSPHATE DEHYDROGENASE (G6PD)
Principle
G6PD is involved in the generation of phosphorylated nicotinamide-adenine dinuclotide (NADPH) in the pentose phosphate pathway (hexose monophosphate shunt). NADPH is needed to provide glutathione (GSH) which maintains haemoglobin and other red cell proteins in a reduced active form. Exposure to an oxidant drug increases the need for NADPH and GSH. G6PD deficiencies prevent this need from being met, resulting in the oxidation of haemoglobin into methahemoglobin. This precipitate to form Heinz bodies which attach to the red cell membrane. Cells containing Heinz bodies are easily damaged and lysed extravascularly.
Test procedure
Set up 3 test tubes as follows:
Test Positive control Negative control EDTA Blood 1ml 1ml1ml
Na Nitrite Dextrose 0.05ml 0.05ml -
Methylene Blue 0.05ml - 0.05ml
Reporting result: Defect or No defect
To prepare Reagent:
Glucose 5.0g Dissolve in 100ml
Sodium Nitrite 1.25g distilled water
Methylene Blue 0.15g Dissolve in 100ml
PROCEDURE TO DETERMINE BLOOD GLUCOSE LEVEL.
URINE SUGAR TEST
The urine glucose test is used to monitor a person with diabetes. When the blood glucose level rises above 160mm/dl, the glucose will be detected in the urine; consequently glycosuria may be the first detector that diabetes or another hyperglycemia condition is present. PROCEDURE Early morning urine is collected in a small container The urine strip is dip in the urine for about 5 seconds The rate of color change formed is compared with the color chart and the results are interoperated REPORTING OF RESULTS
If no color change occurs it is negative otherwise positive. Depending on the color changed of the strip; a positive results are recorded as +, ++, +++, ++++
PRINCIPLES
This method employs a specific reaction of the enzyme glucose oxidase which catalyzes the conversion of glucose to gluconic acid and hydrogen peroxide second enzyme, peroxidase, promotes the reaction of hydrogen peroxide generated with chromogen to form a brown oxidized compound. The intercity of the color depends of the amount of sugar present in the urine

GLYCATED HEMOGLOBIN (HbA1C) TESTS
Summary and explanation:
Hemoglobin A1C is formed by the non-enzymatic glycation of the β-chain of hemoglobin AO. The level of hemoglobin A1C is proportional to the level of glucose in the blood over a period of glucose approximately two months. Thus, hemoglobin A1C is accepted as an indicator of the mean daily blood glucose concentration over the preceding two months. Studies have shown that the clinical values obtained through regular measurement of hemoglobin A1C leads to changes in diabetes treatment and improvement of metabolic control as indicated by a lowering of hemoglobin A1C values.
LIVER FUNCTION TESTS (LFTs)
The diagnosis of liver disease is often obvious from the clinical presentation and comprehensive diagnostic work-up might not be necessary. However, patients with mild jaundice, which may be hematological in origin, and anicteric patients who have disease of the liver (e.g. malignancy, alcoholism, etc.) will usually require further investigations.
Evaluations of these patients should first focus on the routine LFTs and if necessary, followed up with specific LFTs. Routine LFTs done in the Military Hospitals pathology division (biochemistry laboratory) include: Total protein, Albumin, Bilirubin, Alanine, Aminotransferase, Alkaline phosphates, Gamma-glutamyltransferase. The LFTs are done with the automated chemistry analyzer (ELLIPSE/BT 3000) manufactured by Assel company in Germany.

TESTS
TOTAL PROTEINS
CLINCAL SIGNIFICANCE
In human plasma, albumin accounts for 50%-60% of total proteins; the remaining fraction usually contains globulins. Most plasmatic proteins are synthesized by the liver, except immunoglobulin. Increase of the plasmatic volume (salt retention syndrome, intoxication with water…), or its reduction (dehydration related to vomiting, diarrhea…), induce respectively relative hyperproteinaemia.
Abnormal total protein rates only occur in the event of disorder affecting the concentration of albumin and immunoglobulin, thus, severe protein insufficiency (malabsorption, maldigestion, dietary insufficiency), renal and hepatic diseases result in hyperproteinaemia. If total protein concentration is lower than 40g/L, oedema can be observed. Hyperproteinaemia can be seen in the case of hyperimmunoglobulinemia (multiple myeloma infection).
ACTIVITIES UNDERTAKEN IN THE BLOOD BANK
They include Blood grouping Hepatitis B and C[HBsAg and HCV respectively] Retro screening for HIV/AIDS VDRL test

BLOOD GROUPING
Objective/ principle
The ABO and rhesus blood grouping is done to ensure that blood donors and patients are grouped correctly since incompatible blood transfusion may result in the death of the recipient. An individual’s ABO blood group depends on the A, B or O gene located on the chromosome inherited from each patient
Reagents Needed
Anti-A reagent (Anti-A) Anti-B reagent (Anti-B) Anti-D reagent (Anti-D)
Test procedure Put three drops of patient’s blood separate from each other on a white tile or slide Put a drop of Anti-A by first drop of blood, Anti-B by the second drop of blood and Anti-D by the third drop of blood 25 Mix each of the content to a uniform mixture and place it on an orbital shaker to mix for about 3 minutes Take the tile off the orbital shaker and observe for agglutination of red cells with the antiserum
Reporting Test Results
Positives
Blood group A positive (A Rh “D” positive): Agglutination with Anti-A and Anti-D Blood group B positive (B Rh “D” positive ): Agglutination with Anti-B and Anti-D Blood group AB positive(AB Rh “D” positive): Agglutination with Anti-A, Anti-B and Anti-D Blood group O positive (O Rh “D” positive ): Agglutination with only Anti-D
Negatives
Blood group A negative (A Rh “D” negative): Agglutination with Anti-A, but no agglutination with Anti-D Blood group B negative ( B Rh “D” negative): Agglutination with Anti-B, but no agglutination with Anti-D Blood group AB negative (AB Rh “D” negative): Agglutination with Anti-A and Anti-B, but no agglutination with Anti-D Blood group O negative (O Rh “D” negative): No agglutination with Anti-A, Anti-B and Anti-D
HEPATITIS B SURFACE ANTIGEN (HBsAg) AND HCV SCREEN
Objective/ principle
A chromatographic immunoassay for the qualitative determination of Hepatitis B surface antigen and Hepatitis C virus in serum or plasma
Test procedure Remove the dipstick from its wrapping pouch by tearing the pouch along the split Holding the strip vertically, carefully dip it into the specimen (10-15 sec). Do not immerse the strip past the maximum line Interpret the results within 15 minutes
Reporting Test Results
Positive Result: The presence of two purple colour bands within the result area regardless of which band appears first
Negative Result: The presence of only one purple colour band within the result area
RETROSCREENING/ RAPID TEST FOR HIV-I/II ASSAY
Principle
The test involves the trapping of antibodies to HIV 1 and 2 by a capture reagent which is absorbed on a porous membrane. It is a sexually transmitted disease which is usually carried out to know the antigen-antibody reaction. A kit is usually used for this test.
Test procedure Remove cover from the test kit Write the sample number on the device Using the smaller pipette, discharge a drop of blood unto the kit Add a drop of buffer solution unto the sample and allow to react Read results within 10 minutes
Interpretation of results Negative: One small distinct red band (control band) Positive for HIV 1 and or HIV2: Two small distinct red bands
Invalid: No band shows
VDRL Objective / principle
Venereal Disease Research Laboratory (VDRL) test is used to detect the presence of regain (circulating antibodies directed against tissue components) in serum or plasma which is indicative of Syphilis. Syphilis is a sexually transmitted disease caused by spirochete, Treponema pallidum. Infection causes the production of antilipoidal antibodies. This type of antibody is produced as a result of the tissue damage that occurs in syphilis and is directed against tissue components.
Test procedure Centrifuge blood sample at 3000rpm for 5 minutes Remove cover from the VDRL test strip Drop the strip 90 degrees into the serum of the blood sample to a required level After some seconds remove strip and allow it to react for about 2-3 minutes
Reporting Test Results Positive: Appearance of two bands, meaning the antigens coated on the strip reacted with the antibodies in the serum Negative: Appearance of one band, thus control band
ACTIVITIES UNDERKEN IN THE MICROBIOLOGY DEPARTMENT
These include the following Sputum for acid fast bacilli[AFB] Culture media and sensitivity test Semen analysis High vagina swab[HVS] Coagulase and catalase test Skin snip
SKIN BIOPSY (SNIP) FOR MICROFILARIA
Objective/Principle
Physiological saline causes the microfilaria present in the skin tissue to come out and can be observed. The disease is also known as river blindness because invasion of the eye leads to loss of vision
Test Procedure Obtain a skin snip by cutting a piece of skin from the buttocks and back of the shoulder Place the skin snips on the glass slide and add a drop or two of physiological saline Apply a cover glass carefully and leave the wet mount to stand for at least 30 minutes Examine the mount after the stipulated time period scanning with the *10 and *40 objectives for detailed morphology .Look for the microfilaria of onchocerca volvulus and Mansonela streptococci
Onchocerca volvulus
Onchocercavolvuls causes river blindness because of microfilaria damage to the eye LEISHMANIA PARASITE
Objective
To determine leishmania parasites
Principle
Leishmania parasites occur as Amastigotes and are thus demonstrated microspically in blood or other tissue smears
Sampling Conditions
Anticoagulated blood, peripheral blood, bone marrow aspirates, lymph node fluid, spleen or liver aspirate, occasionally nasal secretion and skin are used
Test Procedure Prepare smear from aspirate or blood Air dry the smear and fix in methanol for 2 minutes Stain the smear in 1:20 Giemsa for 30 minutes Examine the smear microscopically using the oil immersion (*100) objective Look for Amastigotes which are small, round to oval shape with a nucleus and kinetoplast
NB: The Leishmania parasite causes trypanosomiasis in human
SPUTUM FOR ACID FAST BACILLI (AFB)
Objective
To identify acid-fast bacilli in sputum in order to diagnose pulmonary tuberculosis (TB)

Principle
The principle is based on the ability of AFB to hold the red dye of CarbolFuschin using the ZiehlNeelson technique
Test procedure Label the clean glass slides Make a smear to cover 2/3 the length of the slide Dry and heat fix by passing through flame 3 times Flood the slide with strong CarbolFuschin Heat gently with a flame until steam rises (avoid boiling) approximately 3 minutes Repeat the steaming three or four times in the course of 5-10 minutes. The film must not be allowed to dry , add fresh stain if necessary Wash thoroughly in running water Decolorize with 20-25% H2SO4 until no stain comes off Wash well in running water Counter stain with hydro soluble methylene blue for 15 to 30 seconds Wash with water and air dry Wipe the back of the slide clean, do not blot Examine under the microscope using the oil immersion lens. Acid and alcohol fast bacilli should show up as bright red/ pink rods where as other organisms are blue.
Reporting Test Result No AFB / 100 fields Scanty 1-9 AFB per 100 fields 1+ - 10-99 per 100 fields 2+ - 1-10 per 10 fields 3+- greater than 10 per field
Report as;
ZN AFB present (+, ++, +++)
ZN No AFB seen. CULTURE MEDIA
A culture media is a media containing nutrients such as ions, moisture and correct pH for pathogens growth for microscopy and identification CULTURE PREPARATION
Blood agar: 8.5g
Water: 15 ml Test procedure Prepare the agar medium as directed by the manufacturer Sterilize by autoclaving at 121 degrees Celsius for 15 minutes and transfer to water broth of 50 degrees Celsius\ When the medium cools to 50-55 degrees Celsius, mix well and dispense aseptically in sterile petri dishes Give date and number to the medium in the petri dishes Store at 2-8 degrees Celsius at room temperature INOCULATION OF CULTURE MEDIA IN PETRI DISHES
The inoculation of culture media in petri dishes is referred to as looping out or plating out .before inoculating a plate of culture medium, the surface of the medium must be dried otherwise a single colony will not be formed Using a sterile loop, inoculate a small area of the plate and flame loop until sterile SENSITIVITY TEST Sensitivity test is an application of antibiotic drugs to see which of them will be sensitive to the micro organism. This can be done by using Disc diffusion technique A dilution technique Disc diffusion technique
A disc of blotting paper is impregnated with known volume and appropriate concentration of antimicrobial, and this is placed on a plate of susceptibility. Testing agar uniformly inoculated with the test organism. The microbe diffuses from the disc the medium and the growth of the test organism is related to the susceptibility of the organism.
Interpretation of zones
Resistant – Organism will not respond to treatment with the drugs
Intermediate-Infections respond to treatment in layer dose
Susceptible- Infection responds to treatment with drugs in normal dose
HIGH VAGINAL SWAB (HVS) ROUTINE EXAMINATION
Objective
To identify Candida species and Trichomonas vaginalis
Test procedure
Direct Saline Preparation Prepare a smear or gram stain Put a few drops of saline to cover the cotton bud of the swab Mix thoroughly the saline with the sample on the cotton bud Label the grease free glass slide using a marker Make a wet mount from the contents in the tube I.e. put about 2 drops of the contents on the slide and apply a cover slip Examine the wet mount under the microscope scanning with the 10* objective and the 40* objective for detailed morphology Look for yeast-like cells (Candida sp) and motile trophozoites of Trichomonas vaginalis, pus cells and epithelial cells
Reporting Test Result
Yeast-like cells (Candida sp.) and/ or Trichomonas vaginalis present
Number of puss cells and epithelial cells per high field
Gram stain: report due cells and / or gram-negative intracellular diplococci (GNID) present or not seen.

COAGULASE AND CATALASE TEST
Objective /principle
To identify and isolate cultured microorganisms, most especially organisms in the genus Staphylococcus COAGULASE Test
Test procedure Pipette 9 drops of peptone water and add to 1 drop of blood plasma Inoculate the cultured organism Examine for the precipitation or clotting of the blood plasma
Reporting Test Result
Coagulase positive: Precipitation / clotting of blood plasma e.g. Staphylococcus aureus and Staphylococcus epidermidis
Coagulase negative: No precipitation / clotting of blood plasma. CATALASE Test
Test procedure Inoculate the organism in hydrogen peroxide oxide Observe for oxygen bubbling
Reporting Test results
Catalase positive: Presence of oxygen bubbling. E.g. Staphylococcus epidermidis
Catalase negative: Absence of oxygen bubbling. E.g. S. epidermidis
NB: Coagulase and Catalase tests are used to identify and isolate for the presence or the absence of Staphylococcus aureus, Staphylococcus epidermidis etc.
Other microbiological tests include Semen Analysis, inoculation and culturing of various

CHAPTER THREE
EXPERIENCE/KNOWLEDGE ACQUIRED
Some experience/knowledge acquired is as follows I learnt how to culture urine samples, blood samples, stool samples and other samples. Every sample has a required medium on which to culture. Some samples may require more than one medium. For example fresh blood samples are first stored in a brain-heart infusion broth and later cultured on blood agar, MacConkey agar and chocolate agar. Also urine samples are cultured on Cystine Lactose Electrolyte-Deficient (CLED) agar.
The chocolate agar is normally used for the growth anaerobic microorganisms such Neisseria gonorrhea. I also learnt that all stored media in the refrigerator must be dried first in the incubator at a temperature of 35 ̊C – 37 ̊C for about 15 minutes before using them for culture. The working area must be disinfected to prevent contamination. When culturing one must not talk. The wire loop must be flamed before and after use with a bunsen burner to keep it sterile. Also, I learnt that the use of normal saline for the stool emulsification is mainly to keep the morphology of any parasite such as hookworm present in the stool. Slides prepared with urine and stool samples for the microscopy must not dry before viewing under the microscope. In routine urine examination the results for the biochemistry aspect must be read on time. Example, glucose is read 30 seconds after the test strip is dipped into the urine sample. The container containing the urine test strip must not be left open.

CHAPTER FOUR CONCLUSION In conclusion, my practical attachment at 37 Military Hospital, Accra has been an immense success in my academic life as well as my social life; it has given me the experience in work ethics and has also broadened my knowledge on the application of microbiology and parasitology in the hospital and laboratory setting. It is therefore necessary to give proper attention to this compulsory industrial attachment to give students more knowledge and experience in and after school. RECOMMENDATION
I recommend that the school should have a link with various hospitals, research institutes, industrial sectors to alleviate the challenges of attachment location and students should be given more practical lessons on campus to enhance effective understanding whenever they go on industrial attachments. I also recommend that, the period of attachment should be extended to enable us acquire more experience in the work ethics and can as well be able to apply and learn more microbiologically related issues.

APPENDIX
EDTA - Ethylenediamine tetra-acetic acid
LTF – Liver Function Test
VDRL – Veneral Diseases Research Laboratory
R/E – Routine Examination
CLED - Cystine lactose electrolyte-deficient
SS - Salmonella Shigella
XLD - Xylose lysine deoxycholate
KIA - Kligler ion agar
TSI - Triple Sugar Ion
BA - Blood Agar
CA – Chocolate Agar
MAC – MacConkey
Z-N - Zeihl-Neelsen
MRSA - Methicillin Resistant Staphylococcus aureus
LDC - Lysine decarboxylase
WBC – White Blood Cell
RBC – Red Blood Cell

BIBLIOGRAPHY
Monica Cheesbrough (2006). District Laboratory Practice in Tropical Countries, Second Edition, Part 2. New York: Cambridge University Press.

Bibliography: Monica Cheesbrough (2006). District Laboratory Practice in Tropical Countries, Second Edition, Part 2. New York: Cambridge University Press.