In a peripheral laboratory, microscopic examination can provide rapid and economical presumptive diagnosis which may have significant bearing upon control and prevention strategies.

4.1 Cleaning and storage of microscope slides

4.1.1 Cleaning of new slides

Soak the slides in a vessel containing soap water solution for a few hours. Place the slides either in running tap water or several changes of clean water for few hours. The slides should be wiped dry using a dry, clean, lint-free cloth. Always handle the cleaned slides by the edges to avoid finger marks.

4.1.2 Cleaning of used slides

Soak the slides for at least 60 minutes in 1-2% hypo-chlorite solution. Wash in hot soap water scrubbing both the sides with the brush, taking particular care to wash only a few slides at a time to prevent scratching. Clean the slides individually with gauze or cotton wool. Transfer the slides to a fresh detergent solution. Wash in running tap water or several changes of clean water. Wipe dry with a clean lint free cotton cloth.

4.1.3 Storage of Slides

Initially, after washing and cleaning, the slides should be kept in a dry place or a warm air cabinet. Thereafter slides should be stored in packages of l0 which should be wrapped in thick paper and secured with adhesive tape or rubber bands.

4.2 Microscopy for pyogenic meningitis

Pyogenic meningitis is an acute bacterial infection of the meninges, commonly caused in epidemic form by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae.

For the laboratory confirmation of the diagnosis, the following clinical specimen should be collected.

4.2.1 Cerebrospinal Fluid (CSF)

CSF following lumbar puncture should be collected in 3 separate clean sterile containers (Bijou bottles) for following investigations:

General guidelines for collecting CSF

4.2.2 Examination of CSF

Macroscopic:

Look for the presence of turbidity, blood or coagulum.

Cytology

Cytologic examination to be done only when there is no coagulum in the CSF.

Material Required:

Neubauer's counting chamber, WBC diluting fluid, WBC pipette, Compound microscope.

The cell count should be done by the usual procedure of WBC count using a Neubauer's chamber and count the number of leukocytes per cmm of fluid.

The normal CSF should be absolutely clear, free of any coagulum and should not contain more than 0-8 lymphocytic cells/cmm.

In pyogenic meningitis, appearance of CSF is turbid and contains more than 8-10 leukocytes/cmm, the cells being predominantly polymorphs in nature.

Microbiological examination:

Microscopy

Microscopic examination is required to directly visualise the causative organism in the CSF.

Requirements Clean slides Coverslips Table top centrifuge Centrifuge tubes Pasteur pipettes Clean glass vials Reagents of Gram's staining. Rubber teats Discarding jar Neubauer counting chamber WBC pipette. Procedure: Transfer about 1-2 ml of CSF in a sterile centrifuge tube. Centrifuge at 3000 rpm for 5 minutes. Keep the supernatant fluid for Latex Agglutination test. From the deposit, make smears on 3 clean glass slides and air dry. In case of a clotted CSF, transfer 3 small pieces of clot on three different glass slides. Tease the clots using needles or wooden sticks or the edge of the slide and after spreading make the smears. Air dry. In case of scanty CSF, several drops of CSF should be placed at one particular spot on the slide, each being allowed to dry before the next is added. Air dry and heat fix the smear. Stain the smears by Gram's staining method as given subsequently. Examine under oil immersion lens. Observations Presence of Gram negative bean shaped diplococci, both intracellular and extracellular suggests the presence of Neisseria meningitidis (Meningococcus). Other organisms which can be seen are Streptococcus pneumoniae (Pneumococcus), which appear as gram-positive diplococci, Haemophilus influenzae which appear as gram negative thin filaments rods.

4.3 Diagnosis of pulmonary tuberculosis by sputum examination:

Tuberculosis is a disease of great public health importance caused by Mycobacterium tuberculosis and some other species of Mycobacteria.

The diagnosis of pulmonary tuberculosis can be established by demonstrating the bacillus in the sputum of the patient by microscopy.

4.3.1 Sputum collection

Collect the sample preferably early in the morning. For optimum results, 3 consecutive days samples should be tested. In case sputum is scanty, a 24 hour collection may be examined. A nebulized and heated hypertonic saline may be used to induce sputum production in patients unable to bring out the sputum. Sputum should be collected in a sterile wide mouthed container with a tight lid.

The sample should be delivered to the laboratory with minimum delay.

Specimen that cannot be delivered or processed immediately should be refrigerated at 4-8oC for a maximum of 3-4 days.

Materials required for sputum microscopy:

Properly collected sputum specimen Wooden sticks Clean glass slides Spirit lamp/Bunsen burner Petri dish Inoculation hood Face masks Reagents for Zeihl-Neelsen staining Glass rods, plastic clay.

Procedure:

Preparation of the smear

In an inoculation hood or in an isolated room, wearing a face mask, transfer a portion of the sputum to a petri dish. Using a wooden stick, tease out a small portion of caseous, purulent or bloody material and transfer it to a clean slide. Using the same wooden stick or an inoculating wire loop, spread this material uniformly over a large area, covering at least two thirds of the slide. Air dry the slides and flame them immediately and stain according to the Ziehl-Neelsen staining method as given below:

4.3.2 Ziehl Neelsen Staining (Acid fast staining)

Requirements

Staining Procedure:

Observations:

4.4 Diagnosis of plague

Plague is an ancient scourge of mankind. It is a bacterial disease caused by Yersinia pestis. It is enzootic in rodents. In man, plague occurs mainly in three forms, bubonic, pneumonic and septicaemic.

The presumptive diagnosis of Plague can be established by microscopic examination.

Sample collection:

4.4.1 Sputum Collection in Pneumonic Plague:

Collect the sputum sample in a sterile wide mouth screw capped container. Label the specimen Transport the specimen to laboratory at 2-8oC. In the Laboratory: Make three smears out of the same portion of exudate/sputum taking precautions not to form aerosols. Air dry the smear. Stain smears either by methylene blue/Gram staining/Wayson's stain.

4.4.2 Gram Stain

This is a routine laboratory procedure used for examining specimens suspected to contain bacteriologic agents. Direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis. Gram stain results, the shape of cell (cocci, bacilli), the type of cell arrangement (single, chained, clustered) visualized under light microscopy, can provide a quick assessment of what the etiologic agent may be.

Principle

The Gram stain forms the cornerstone of microscopic bacteriology. It was described by Hans Christian Gram over l00 years ago. Crystal violet (gentian violet) is the primary stain that will bind to the peptidoglycan present in the cell walls of some bacterial cells. Iodine is added as a mordant to fix the dye. If the cell wall does not contain peptidoglycan then crystal violet is easily washed off with acid or alcohol (decolorizer). A secondary dye, safranin (counterstain), is added after the decolorization step. If the primary dye did not bind, the cells will easily adsorb safranin. Thus gram-positive cells are purple, while gram-negative cells are pink/red.

Requirements

Procedure:

Or

Decolorize with 100% acetone. First, tip off the iodine and hold the slide at a steep slope. Then pour acetone over the slide from its upper end , so as to cover its whole surface. Decolorization is very rapid and is usually complete in 2-3 seconds. After this period of contact, wash thoroughly with water under a running tap.

When to use this procedure and what you expect to see:

Y. pestis appear as fat, short, gram-negative coccobacilli about 1ì by 0.5ì. Gram stains are typically done on cultures/subcultures, bubo aspirates, spleen, liver and sputum smears.

Critical value/Action to be taken:

When gram stained material reveal small coccoid gram-negative bacilli, material should be further processed for culture isolation and identification. No notification is needed at this time.

Interpretation:

Y. pestis appears as a fat short, gram negative coccobacilli about 1 ì by 0.5 ì.

4.4.3 Wayson stain for visualizing Yersinia pestis:

Wayson stain is a polychromatic differential stain used as a presumptive test for the presence of Yersinia and Pasteurella spp.

Principle

Basic fuschin and methylene blue in the Wayson stain bind to bacterial cells which appear under light microscopy as bipolar, closed safety pin-shaped cells. The differential polychromatic morphology can be visualized with many different types of organisms; therefore, Wayson stain alone is not diagnostic for Y.pestis.

Critical values/Action to be taken:

When stained material has a characteristic "safety pin" morphology, it is Wayson stain positive. Further processing of specimen for culture isolation and identfiication must follow. No notification is needed unless submittor specifically requests notification.

If Wayson bi-polar organisms known to have "safety pin" morphology cannot be visualized after staining, check reagents and check for possible technical problems. Repeat stain until characteristic morphological results are obtained with control cultures.

Materials needed for this test:

Wayson stain:

Interpretation:

Consistent, striking bipolar "safety pin" morphology of small, fat bacilli are characteristic of the Yersinia and Pasteurella spp. Other bacteria may exhibit bipolar appearance as well, especially if the specimen is taken from areas with a wide variety of normal flora (nasal, pharyngeal, and fecal). Hence, report " Organisms morphologically resembling Y. Pestis seen; culture report follows".

"All Y.pestis are Wayson positive, but all Wayson positive strains are not Y.pestis".

Quality control measures:

Test each lot of Wayson stain using known Yersinia/Pasteurella spp. (positive control) and with Escherichia coli or other enteric bacteria as negative controls. When examining tissue smears, controls slide prepared with plague bacilli infected and uninfected tissue smears should also be examined.

4.4.4 Methylene blue staining

Material required

Procedure

Observation:

4.5 Malaria

Malaria is a parasitic disease caused by Plasmodium species. In India, the disease is commonly caused by P.vivax and P.falciparum. The laboratory diagnosis is based on demonstration of different stages of the parasite in the peripheral blood film of the patient.

4.5.1 Collection of sample

Peripheral blood smear:Time for taking blood:

4.5.2 Preparation of blood smear

Both thick and thin films should be made on the same slide. Blood sample should be collected from the tip of the ring finger of the left hand. However in small children, sample should be collected either from the heal or the tip of the big toe of the foot taking all aseptic precautions using a sterile needle or a lancet . Apply gentle pressure to the finger and collect a single small drop of blood on to the mdidle of the slide. This is for the thin film. Apply further pressure to express more blood and collect 2 or 3 large drops on the slide about 1 cm from the drop intended for the thin film. Wipe the remaining blood away from the finger with cotton wool.

Thin film: Using another clean slide as a 'spreader' and with the slide with the blood drops resting on a flat firm surface, touch the small drop with the speader and allow the blood to run along its edge. Firmly push the spreader alongwith the slide away from the largest drop keeping the spreader at an angle at 45oC. Make sure the spreader is in even contact with the surface of the slide all the time the blood is being spread.

Thick film: Always handle slides by the edges or by a corner to make the thick film as follows:

Using the corner of the spreader, quickly join the larger drops of blood and spread them to make an even thick film. The blood should not be excessively stirred but can be spread in a circular or rectangular form with 3 - 6 movements.

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

Wrap the dry slide in clean paper and despatch with the patient's record form tothe laboratory as soon as possible.

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

4.5.3 Staining of blood smears:

GEIMSA STAIN

Materials and Reagents :

l. Geimsa stain powder/ready Giemsa stain solution.

2. Alcohol

3. Methanol

4. Marking pen

5. Staining jars

6. Boric acid Borax buffer - pH 7.2.

Preparation:

Take 50 ml of solution I and adjust the pH to 7.2 using appropriate volume of solution II. Then make up the volume to 200 ml with distilled water.

Staining technique:

4.5.4 J.S.B. Stain

Materials and reagents required:

Preparation:

J.S.B. II

Dissolve 2 gm eosin yellow in 1 lit. of distilled water and store in the dark for 4 weeks before use.

J.S.B.I

Staining technique:

4.5.5 Observation

Examine thin film first. If no parasite is found then only examine thick film. If parasites are seen in the thick film but the identity is not clear, the thin film should be reexamined more thoroughly so as to determine nature of infection.

Thin film examination:

Observation on thick smear:

Appearance in thick film

4.6 Examination of blood for microfilaria

Filariasis is a disease of the lymphatics caused mainly by the nematode Wuchereria bancrofti and rarely by Brugia malayi.

Laboratory diagnosis:

This is based on the demonstration of the larval stages of the parasite in the peripheral blood of the cases/carriers.

4.6.1 Collection of blood:

The blood should be preferably collected between l0 p.m. and 2 a.m. specially in areas where microfilaria shows nocturnal periodicity.

4.6.2 Examination of unstained preparation:

Take 2-3 drops of blood on a clean glass slide. Put a coverslip on it. The rim is then smeared with vaseline to prevent drying up of the blood. Examine the slides under low power microscope immediately or within 24 hours of collection of blood. Wriggling microfilaria present in the blood can be seen.

4.6.3 Examination of stained smear:

Thick film:

Thin film:

Observation:

Microfilaria of Wuchereria bancrofti are seen.

Microfilaria of Brugia malai.

4.7 Examination of throat Swabs for diphtheria.

Diphtheria is a disease caused by a rod shaped gram positive bacteria corynebacterium diphtheria. Laboratory diagnosis (Presumptive) is based on demonstration of rod shaped bacteria arranged in a Chinese letter pattern (Cuneiform arrangement) and showing presence of metachromatic gravels in the throat swab smear from the suspected case of diphtheria.

However, confirmation of the diagnosis needs cultural examination.

4.7.1 Collection of throat Swab

Collect the throat swab from a suspected case of diphtheria by vigorously subbing the beck of the throat, both tonsils and any areas of inflammation and membranes formation using a sterile cotton tipped swab stick : case should be taken to avoid touching the tongue or lips with the swab Always, obtain two samples, one for smear preparation and another to be sent for culture

4.7.2 Preparation of smear

Take a clean, graceful slide, label it using a diamond pencil on the right end corner of the slide. Prepare a smear in the centre of the slide using the collected throat swab by gently rubbing the swab in a rotatory fashion, covering at least middle 2/3 rd of the slide. let the smear air day and carry out Albert`s's staining

4.7.3 Albert's starting

A. Albert's I:

Dissolve the dyes in alcohol and add to the water and acetic acid. Allow to stand for one day and then filter.

B. Albert's iodine* (Albert's II)


Iodine Potassium iodide Distilled Water

6 mg. 9 mg. 900 ml.




Procedure

(i) make smear, dry in air and fix by heat

(ii) Cover slide with Albert's stain I and leave for 4-6 minutes.

(iii) Wash in water and blot dry.

(iv) Cover the slide with Albert's stain II and allow to act for 1-2 minutes

(v) Wash, blot dry, and examine under oil immersion of microscope. C. diphtheriae appear as bacilli with dark green protoplasm and blue-black granules. Other bacteria will stain light green.

* Note - If Albert's iodine is not available then Gram's iodine may be used.

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5.1 LATEX AGGLUTINATION TEST FOR MENINGITIS

The ideal immunological test, which is also a rapid test and easy to perform in a district laboratory, is the latex agglutination (LA) Test. test is done to detect the bacterial antigen (capsular polysaccharide) in CSF samples collected from patients.

The available commercial kits are designed to provide diagnosis for meningitis caused by:-

The general procedure for performance of the test is given below, however, the laboratory personnel are advised to go through the instructions provided by the kit manufacturer, carefully, and strictly adhere to the same.

5.1.1 Equipment required: (but not supplied with the kit)

Pasteur pipettes (sterile) Rubber teats Container with disinfectants (for discard)

5.1.2 Procedure

Systematically heat all CSF specimens for 5 minutes at 80-l00oC. Centrifuge the CSF samples at 2000 rpm for l0 minutes, preserve the supernatant for further use. Shake each latex suspension well. In the corresponding fields of the slide, dispense one drop of each of the latex suspension followed by one drop of the CSF supernatant. Mix with a stirring stick; use separate stick for each combination of CSF and latex suspension. Rotate the slide, and read within 2 minutes. 5.1.3 Controls Periodically check- a) that none of the four latex reagents agglutinate in presence of 0.15 mol/L NaCl solution. b) that each of the four latex reagents do agglutinate with respective positive control.s. 5.1.4 Reading Negative reaction: The CSF latex suspension mixture remains a "milky suspension" (disregard any granules that may occur with S.pneumoniae). Positive reaction: Distinct rapid agglutination (clumping) occuring within 2 minutes (normally 30 seconds). 5.1.5 Interpretation Aglutination with one of the latex reagents indicates presence of the corresponding antigen in the CSF sample. Advantages of LA test Most sensitive method available Rapid Good field applicability Can diagnose the disease even in antibiotic treated patients. No special equipment/instrument required. Disadvantages of LA test: Commercial kits not produced in India; to be imported. Expensive The test does not yield any bacterial isolate; other parameters cannot be tested. 5.2 DIAGNOSIS OF HEPATITIS B VIRAL INFECTION: Diagnosis of Hepatitis B viral infection is very important, not only in case of viral hepatitis and liver cirrhosis patients, but also in the screening of donor blood samples, to ensure safe blood transfusion and to control or check the spread of hepatitis B infection through unsafe blood transfusion.This is achieved by detection/demonstration of "Hepatitis-B surface Antigen" (HBsAg; the 'Australia Antigen') in the patient/donor blood samples. A simple latex agglutination test for rapid detection of HBsAg, which is very much feasible in the district laboratories, is described below: LATEX AGGLUTINATION TEST FOR RAPID DETECTION OF HBsAg (AUSTRALIA ANTIGEN) 5.2.1 PRINCIPLE A distinct agglutination occurs, when serum sample containing HBsAg is mixed with latex particles coated with purified and highly reactive anti-HBsAg antibodies; there would be no agglutination when the serum sample does not contain HBsAg. 5.2.2 MATERIALS AND REAGENTS Commercial kits for this test are available in India. They contain the following reagents and accessories. Reagent 1: HBsAg latex reagent - 1 vial Reagent 2: Positive control serum - 1 vial Reagent 3: Negative control serum - 1 vial Accessories: Disposable plastic slides Disposable applicator sticks Disposable plastic droppers Rubber teats. All the reagents are stable and active, till the expiry date mentioned, provided they are stored in a refrigerator at 2-8oC. Do not freeze the reagents. 5.2.3 SPECIMEN The test is performed on serum harvested from the patient's/donor's blood. Do not heat inactivate the test or the control sera samples. If delay in testing, store test serum samples in a refrigerator or deep freezer, taking care to avoid repeated freezing and thawing of the specimens. 5.2.4 TEST PROCEDURE Allow the reagents to attain room temperature, and shake the vials gently to make sure that the latex reagent is completely in suspension. Place one drop (50 ìL) of undiluted serum in one of the circles on the slide. More circles to be filled if more than one test sera samples are to be tested. Use separate droppers for each specimen. Add one drop (50 ìL) of latex reagent on to each specimen drop in circles, using a disposable dropper. Mix the content of each circle, using separate disposable applicator sticks for each circle, and spread the mixture uniformly over the entire area of the circle. Rock the slide gently, to and fro, for 5 minutes, and watch for agglutination. Precautions: 1. To avoid contamination of reagents, make sure that the cap of each vial is properly and promptly applied to the same vial. Interchanging of caps and droppers lead to contamination and erroneous results. 2. Improper mixing and interchange of applicator sticks also lead to erroneous results. 3. Vigourous rocking of slides may lead to impaired agglutination. Use of Controls: Positive and negative controls should be put up simultaneously, as quality control measures. 5.2.5 Interpretation Visible agglutination in < 5 minutes - HBsAg Positive No agglutination - HBsAg Negative 5.2.6 LIMITATIONS: Probability of FALSE POSITIVITY = 1% of all samples, due to presence of other antigens (Rheumatoid Factor). FALSE NEGATIVE results may be encountered with specimens containing very high titres of HBsAg (Prozone effect). In such cases the characteristic syndrome (severe jaundice, GPT/GOT elevation) will be apparent. In that case repeat the test after diluting the specimen 1:40, with normal saline. 5.3 VDRL SLIDE FLOCCULATION TEST FOR SYPHILIS: This is a test with high sensitivity and specificity and can be used for rapid and exact quantitative titration of the reactive sera samples. 5.3.1 PRINCIPLE