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 The VDRL antigen particles, which are seen as small fusiform needles under the microscope, floculate into clumps (small, medium and large), when they come in contact with a reactive (+ve) serum. 5.3.2 MATERIALS VDRL antigen It consists of a mixture of cardiolipin, lecithin and cholesterol in definite proportions and is commercially available. Each sealed glass ampoule contains 0.5 ml (with sufficient excess for convenient withdrawal). Antigen amouples should be stored in a cool, dark place. Ampoules showing precipitate should be discarded. Buffered saline solution l0 ampoules containing 5 ml each are supplied with each package of VDRL antigen. Buffered saline is required for preparing the antigen emulsion for the test. SLIDES Glass slides, 2"x3", with 12 paraffin rings of 14 mm inner diameter are used for the test. Slides of same size, with permanently fixed ceramic rings are also available commercially and may be used. The following points regarding the slides are to be noted. New slides, as well as the used slides should be cleaned thoroughly. Slides should be handled by the edges only, to avoid any greasy finger prints. Serum within the circles will spread evenly, within the rings, only if the slides are absolutely clean. Parafin rings can be made on slides by transferring molten paraffin on to slide using a suitable mould or threaded wire rings. 5.3.3 PROCEDURE Preparation of serum Inactivate serum by heating at 56oC for 30 minutes. On removal from water bath, centrifuge the serum sample if it shows particulate debris. Test serum sample need to be reheated (at 56oC for 10 min.), if they are >4 hr. old since original inactivation. 0.05 ml of each sample is required for testing. Preparation of antigem emulsion Pipette out 0.4 ml of buffered saline on to the bottom of a 1 oz.reagent bottle with flat or concave inner bottom surface. Add 0.5 ml of VDRL antigen, drawn out from an ampoule, using a graduated pipette, directly on to saline in the reagent bottle, while rotating the bottle on a flat surface. The antigen should be added drop by drop, but rapidly, so that it takes approximately 6 seconds to complete the delivery of antigen. Blow the last drop of the antigen and continue rotation of the bottle for l0 more seconds. Add 4.1 ml. of buffered saline, using a graduated 5 ml. pipette. Stopper the bottle and shake it vigorously for about l0 seconds. Take care to see that the temperature of buffered saline solution and that of VDRL antigen is maintained within the range of 23-29oC, during preparation of the antigen emulsion. Maturation of antigen is important for increased sensitivity. Maturation is complete in 15-30 minutes, after preparation. Store the antigen emulsion in a refrigerator, if necessary. It should be brought to room temperature and shaken gently before use. 5.0 ml of antigen emulsion would suffice for 250 serum tests. Each batch of antigen emulsion prepared must be pre-tested with known ractive and non-reactive serum samples, in order to confirm that exact pattern of distribution of antigen particles, typical of reactive and non-reactive serum samples, would result on testing. 5.3.4 TEST PROCEDURE Qualitative Test Pipette out 0.05 ml of inactivated serum into one paraffin/ceramic ring on the glass slide; serum should spread. Add one drop (1/60 ml) of antigen emulsion on the serum within the ring. Rotate the slide for 4 minutes, by hand on a flat surface (+ or 120 times per minute covering circle of 2"dia.) Always include a positive and negative control with each testing. 5.3.5 READING AND REPORTING OF RESULTS Read the test results immediately after rotation. Observe the slide under micrscope, using low power objective( 100 x magnification) Antigen particles appear as small fusiform needles, they are more or less evenly spread in case of a non-reactive serum sample, and aggregated into clumps (flocculation) in the case of a reactive serum. Grade the observations as under: No clumps or very slight roughness Small clumps Medium and large clumps ... ... ... NON-REACTIVE (N) WEAKLY REACTIVE (W) REACTIVE (R) Zone reactions are possible; they are recognizable by irregular clumping. The clumps are not compact and very small and large clumps may be seen within the same microscopic field. In such cases, the results are reported on the basis of quantitative test done on the same serum. Quantitative test Quantitative test is performed on all positive (reactive) serum samples and on samples which show weak(W) or "rough" reaction in the qualitative tests. Prepare successive two-fold dilution (1:1, 1:2, 1:4, 1:8,etc.) of serum sample to be tested, using 0.9% saline. Each serum dilution sample thus prepared is treated as an individual sample and tested as described under "qualitative" test. Results are read and graded under the microscope as before. Reporting of results: Results are reported in terms of the highest dilution of the serum that produces a definite positive (or Reactive, R) reaction as below. Weakly reactive is not acceptable. Serum dilution 1:1 R R R W N W W 1:2 W R R W W N W 1:4 N W R R R N N 1:8 N N W R R N N 1:16 N N N W R N N 1:32 N N N N N N N 1:64 N N N N N N N Report R1 dil R2 dil R4 dil R8 dil R16 dil WO dil R1 dil 5.4 RAPID PLASMA REAGIN (RPR) TEST FOR DIAGNOSIS OF SYPHILIS: This test detects antibodies formed, in the blood of syphilitic patients, against cardiolipin. These antibodies are called "Reagin". Two advantages of this test over the previously described VDRL Slide flocculation test are - (a) It does not require a microscope to read the test results; (b) The test sera/plasma sample need not be inactivated prior to testing. 5.4.1 PRINCIPLE Reagin formed in the blood of syphilitic patients cause flocculation of the antigen, which co-agglutinates with the charcoal particles, giving small black clumps that are readily visible without a microscope. Commercial kits, designed for carrying out 50 tests per kit, are available in India. 5.4.2 REAGENTS AND MATERIALS Provided in the Kit RPR antigen ... l vial Positive control serum ... l vial Negative control serum ... l vial RPR antigen dropper ... l Specimen droppers (disposable) Rubber teats Mixing sticks (disposable) Plastic test cards ... 9 Materials required, but not provided in the kit: Micropipette (capable of delivering 0.05 ml of test sample) Stop watch Saline solution (0.9%) - Only for quantitative test. Container with disinfectant (for discard) Storage : The RPR antigen and control sera will remain stable and active, till the expiry date printed on the label, provided they are stored in a refrigerator between 2-8oC. They should not be frozen. 5.4.3 THE SPECIMENS Serum: Use fresh serum harvested from patient's blood sample. If the test cannot be conducted immediately due to some reason, store the serum sample between 2-8oC in a refrigerator, BUT NOT LONGER THAN 48 hr., after collection. Plasma: Collect patient's blood into a tube/vial containing one of the anticoagulants (EDTA, Heparin, Oxalate, Sodium Flouride etc.) Avoid excess of coagulant. Centrifuge the blood sample, to separate the cells. Use the plasma sample within 18 hr. of collection. Inactivation of serum/plasma samples is not necessary. PRECAUTIONS Blood samples should be collected from fasting patients, since very lipaemic samples may give false +ve reactions. Do not use grossly haemolysed samples. Discard contaminated samples. 5.4.4 TEST PROCEDURE A. Qualitative test: Allow all reagents to attain room temperature. Place one drop of (0.05 ml) test serum or plasma, positive control and negative control sera on to separate circles on the plastic test card, using disposable specimen droppers provided. Shake the RPR antigen suspension gently, to resuspend the particles. Place one drop (0.015-0.02 ml) of the antigen suspension, on each of the circles containing test samples and the positive and negative control sera drops, using the antigen dropper provided. Mix the contents of each circle, using the disposable mixing sticks provided, and spreading the reagent mixture over the entire area of the circle. Gently rock the card, to and fro, for 6 minutes, either manually or on a mechanical shaker at 100 rpm, to ensure thorough mixing. Read the results at the end of 6 minutes, using a high intensity light source. Interpretation of Results REACTIVE (POSITIVE) NON-REACTIVE(NEGATIVE) Development of clearly visible clumps of black particles, within the test circles. No development of clumps, the charcoal particles remain in a (NON-REACTIVE) HOMOGENEOUS GREY SUSPENSION. A quantitative estimation is further recommended for all samples positive in the qualitative test. B. Quantitative test: Dispense 0.05 ml (50 ìl) of saline solution on to each of the circles (No.1-5) on the test card, using a micropipette. Dispense 0.05 ml (50ìl) of the specimen (test serum/plasma) onto circle l and mix the two (saline and test sample) thoroughly by drawing the mixture into the micropipette, up and down several times. Transfer 0.05 ml (50ìl) of the mixture in Circle-1, on to the drop of saline in Circle-2. Repeat the mixing action, several times, as explained above. Repeat transferring and mixing actions from Circle-2, through circle-5. Discard 0.05 ml (50 ìl) from Circle-5, after mixing. The dilutions of specimen obtained in different circles on the test are as under: CIRCLE SALINE (mL) SPECIMEN (Serum/Plasma mL) MIX & TRANSFER DILUTION 1 0.05 0.05   0.05 1:2 2 0.05 -   0.05 1:4 3 0.05 -   0.05 1:8 4 0.05 -   0.05 1:16 5 0.05 -   0.05 1:32 Using the disposable mixing sticks, spread the specimen dilutions in the circles to cover the area of the circle. Start with circle 5 and end with Circle 1. Wipe the sticks clean between circles. Gently shake the RPR antigen vial to resuspend the particles, and add one drop (0.l5-0.20 ml.) of antigen, on to each circle, using the antigen dropper. Gently rock the card to and fro for 6 minutes (manually or on a mechanical shaker), to ensure thorough mixing. Read results at the end of 6 minutes, as described above under Qualitative testing. 5.4.5 INTERPRETATION The highest dilution of the sample, giving a definite positive reaction, is considered as the titre of the specimen. In case the titre exceeds 1:32, continue with double dilutions beyond that point,till the titre is obtained. LIMITATIONS OF THE TESTS (RPR and VDRL Slide Flocculation): Both these tests are considered as "non-treponemal antibody tests", which are primarily meant as screening tests. If the tests are positive when there is no clinical evidence of syphilis, they must be repeated; if positivity persists, verifications by more specific tests (for anti-treponemal antibody) would be necessary to confirm syphilis. In RPR and VDRL slide flocculation tests, false positive results may be obtained in diseases such as leprosy, malaria, toxoplasmosis, infectious mononucleosis and lupus erythematosus, and also in specimens having bacterial contamination. 5.5 WIDAL TEST FOR DIAGNOSIS OF ENTERIC FEVER: (TYPHOID AND PARATYPHOID) Widal test is an agglutination test for detection of antibodies against Salmonella typhi and Salmonella paratyphi, the common causal agents of enteric fevers. 5.5.1 Principle When serum sample containing antibodies against S.typhi and S.paratyphi A, B are mixed with respective antigens, agglutination will take place. In S.typhi and S.paratyphi A and B, two types of antigens are recognised as diagnostically important: (a) 'O' antigen or "Somatic' antigen. (b) 'H' antigen or 'Flagellar'antigen. 'O' antigens of various species have components in common and hence only one 'O'antigen i.e. that of S.typhi is employed; the 'H' antigens of Salmonella spp. are species specific, and hence the 'H' antigens of all three, viz. S.typhi, S.paratyphi A and S.paratyphi B, are employed in the test. Commercial test kits for WIDAL test are available in India, and using them both quantitative and qualitative tests can be put up on suspected sera samples. 5.5.2 Materials and reagents Test kit contains the following reagents and materials Reagent 1: S.typhi ('H') - 5 ml Reagent 2: S.typhi ('O') - 5 ml Reagent 3: S.paratyphi A ('H') - 5 ml Reagent 4: S.paratyphi B ('H') - 5 ml Reagent 5: Positive control - 1 ml Glass slide - 1 No. Product Insert - 1 No. Materials required, but not supplied in the kit: Small, dry and clean glass tubes 8/specimen (for quantitative tube test) Normal saline solution Water bath Micropipette/dropper 5.5.3 Specimen Fresh serum (patient) free from contamination should be used. In case of delay in testing, store the sera samples at 2-8oC in a refrigerator. Note: Specimen is used undiluted. Do not use haemolysed specimen. Do not heat or inactivate the specimen. 5.5.4 Test procedure A. Qualitative slide test for screening Clean the glass slide provided and wipe it dry. Place a drop of undiluted serum sample to be tested in each of the first four circles. Add one drop of Reagent 1, Reagent-2, Reagent-3 and Reagent 4, on to the specimen drop in Circles 1-4 respectively. Mix the contents of each circle with separate mixing sticks, and spread the mixture to cover the whole circle. Rock the slide gently for 1 minute. Read the results at the end of one minute. Interpretation A positive reaction shows agglutination, visible to naked eye, in the respective circle. Then proceed for quantitative slide test or quantitative tube test for the appropriate antigen. B. Quantitative slide test Clean the glass slide supplied in the kit and proceed as follows: CIRCLE NO. 1 2 3 4 5 SERUM VOLUME 0.08ml 0.04 ml 0.02 ml 0.01 ml 0.005 ml Appropriate antigen 1 drop 1 drop 1 drop 1 drop 1 drop titre 1:20 1:40 1:80 1:160 1:320 Mix the contents of each circle, starting with circle 5 and through Circle-1, wiping the mixing stick clean between circles. Rotate the slide for one minute and observe for agglutination. Interpretation Titre of the serum is the highest dilution of the serum giving a positive reaction. C. Quantitative tube test Take a set of 8 clean glass tubes, per specimen, per antigen. Prepare dilutions of serum specimen and add appropriate antigen as below: TUBE 1 2 3 4 5 6 7 8 Serum dilution 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 Saline control Normal saline 1.9 ml 1.0ml 1.0ml 1.0ml 1.0ml 1.0ml 1.0ml 1.0ml Patient serum 0.1 ml               Transfer diluted serum ê 1ml ê 1ml ê 1ml ê 1ml ê 1ml ê 1ml ê 1ml (discard ml) ê Appropriate antigen 1 drop 1 drop 1 drop 1 drop 1 drop 1 drop 1 drop 1 drop Mix well and incubate at 37oC for l6-20 hr. and observe for agglutination. Repeat steps (ii) and (iii) with all antigens which showed agglutination in the screening test. Note the highest dilution showing clearly visible agglutination with naked eye. 'O' antigen shows granular agglutination. 'H' antigen shows flocular appearance. Saline control should remain unchanged as it is a negative control. Interpretation Agglutination titre of 1:160 is suggestive of infection. (These titres would depend upon prevalence of antibodies in the local population, and if possible, data about base-line titres should be generated). Factors affecting WIDAL Test Effect of antibiotic administration: There is evidence that early treatment with antibiotics suppresses the antibody response by suppressing the multiplication of organisms. This may result in a low titre in WIDAL test. Effect of past infection or typhoid vaccination: þ It has been seen that the `H' antibodies persist for a long time upto many years after typhoid vaccination. Also, many years after recovering from enteric fever, any gram-negative bacterial infection can trigger a Salmonella `H' antibody production, thereby giving a false positive result in WIDAL test. þ Cross reaction of `O' antigen with other enteropathogens such as Proteus Time of collection of blood sample: This is a very important parameter affecting the results of the WIDAL test. A single blood sample collected during the first week of the illness may give a negative WIDAL result, whereas in the same patient, a sample collected during the second or third week of illness may show a very high titre. Accordingly, paired samples should be collected; the first sample being taken as early as possible and the second, 10-14 days later, for optimum results. Top

CHAPTER - 6

BACTERIOLOGICAL ANALYSIS OF WATER

Although it is not possible to lay down fixed standards, as various types of water are examined, from a public health point of view it is generally sufficient to say that no faecal contamination has occured. Coliform bacteria present in water may not be harmful, but they indicate that water supply is contaminated with faecal matter and water is, therefore, liable to contamination with more dangerous organisms. The coliform bacilli of human origin are the most reliable indicators of faecal pollution. The method of quantitative test for all coliform bacilli known as the 'presumptive coliform count is described below. 6.1 Collection of specimen Collect water in presterilized bottles of 230 ml capacity with ground glass stoppers, having an over hanging rim. Sterilise the bottles by autoclaving. Alternatively, auto Clavable plastic bottles with a tight screw copped lid may be used. Tap water: When water is taken from tap, flame the mouth of the tap and allow the water to run for five minutes before filling the bottle. Stream, river and lake water Insert the bottle with its mouth closed with the stopper, a foot below the surface of water and fill with water. Bring the bottle to the surface and replace the stopper. Avoid the collection of surface water as it contains organic matter. Precautions: During collection of water, avoid the contamination of the sample. Test the water samples as soon as possible after collection. If delay of more than 3 hours is expected, pack the water sample in ice for transport to laboratory. When sampling chlorinated water, add a quantity of sodium thiosulphate to the sample bottle before sterilising. This will neutralize the chlorine present in the water. Presumptive Coliform count: Requirements: Sample of water Sterilized test tubes. 1 ml and 50 ml pipettes. Double strength MacConkey's fluid medium. Single strength MacConkey's fluid medium. Method: Invert the water sample 25 times to mix. Flame the mouth of the bottle and discard 1/3 of the contents and mix thoroughly, Using sterile graduated pipettes, the following amounts of water are added. - One 50 ml quantity of water to 50 ml double strength MacConkey medium in a flask. - Five l0 ml quantity each to l0 ml double strength MacConkey medium in test tubes. - Five 1 ml quantities each to 5 ml single strength MacConkey medium. Incubate all tubes at 37oC for l8-24 hours. All tubes showing acid and gas are regarded as presumptive positives. Reincubate negatives for further 24 hrs. Using McCrady's statistical tables the probable number of coliform organisms present in 100 ml of sample can be calculated. Interpretation: Water samples are classified based on the presumptive count in the following way: Class 1. Excellent 2. Satisfactory 3. Suspicious 4. Unsatisfactory Presumptive coliform count/100 ml 0 1-3 4-10 >10 Faecal Coliform Count: From the tubes showing acid and gas in presumptive coliform count, subculture into fresh single strength MacConkey's broth or Incubate Most Probable Number (MPN) values/100 ml of sample, for a set of tests of one 50 ml, five 10 ml, and five 1 ml volumes. (McCrady's Statistical Table) No. Of tubes giving positive reactions 1x50 ml 5x10 ml 5x1 ml MNP 100ml 0 0 0 <1 0 0 1 1 0 0 2 2 0 1 0 1 0 1 1 2 0 1 2 3 0 2 0 2 0 2 1 3 0 2 2 4 0 3 0 3 0 3 1 5 0 4 0 5 1 0 0 1 1 0 1 3 1 0 2 4 1 0 3 6 1 1 0 3 1 1 1 5 1 1 2 7 1 1 3 9 1 2 0 5 1 2 1 7 1 2 2 10 1 2 3 12 1 3 0 8 1 3 1 11 1 3 2 14 1 3 3 18 1 4 4 21 1 4 0 13 1 4 1 17 1 4 2 22 1 4 3 28 1 4 4 35 1 5 5 43 1 5 0 24 1 5 1 35 1 5 2 54 1 5 3 92 1 5 4 161 1 5 5 >180 at 44º C in a water bath. Tubes showing both acid and gas should be taken as positive for Faecal coliform. Using McCrady's tables compute the number of faecal coliform as in presumptive test. Water showing even one faecal coliform is unfit for human consumption. 6.2 H2S-Strip method: In recent years a simple, reliable and easy-to-perform (by even untrained personell), `Yes-No' test for bacteriological quality of water has been devised. This test, which is currently under field evaluation and quality standardization is expected to be adopted as the field test for water quality monitoring in the hands of peripheral health workers and community participants. Principle: Presence of coliform bacteria in drinking water is associated with hydrogen sulphide (H2S)- producing organisms, and faecal pollution of water can be established by demonstration of H2S production. It has been claimed, by various workers, that the H2S-strip method shows 80% agreement with the conventional MPN test described above. Description of the test device (kit): It simply consists of a pre-calibrated 20 ml glass bottle (McCartney bottle) with a screw-cap lid, from which a strip of specially treated/coated tissue paper hangs down, internally. The whole system is sterile and needs to be opened at the time of water testing. The paper strip inside the glass bottle (80 cm2,folded) is pre-soaked in a concentrated medium containing peptone (20g), dipotassium hydrogen phosphate (1.5g), ferric ammonium citrate (0.75g), sodium thiosulphate (1g), Teepol (1ml) and water (50 ml); 1 ml of the concentrated medium is absorbed on to the folded tissue paper strip and dried at 500 C under sterile conditions. It is then introduced into the sterile bottle. Test procedure: o Pour the water sample to be tested for faecal pollution into the bottle, upto the precalibrated level (20 ml). Incubate at 370C or allow to stand at ambient temperature (30-370C); no incubator is necessary under field conditions, as the bottles can be held in the pockets and body temperature can be made use of. Faecal pollution is indicated if the contents of the bottle turn black. Advantages of H2S-Strip Test: No need to measure the volume of water to be tested; No need to dechlorinate the water sample, since it instantaneously dechlorinates thr sample; The end point (reading) is very clear, due to development of black colour; No incubator is necessary; The test starts immediately on collection into the bottle, unlike other methods which start after the sample is transported to the laboratory.