Stool Microscopic Examination PDF

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Parasitology Laboratory...

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1. DIRECT FECAL SMEAR Direct fecal smear technique is the simplest and easiest technique to facilitate detection of intestinal parasites that infected subjects pass in their feces. The presence of intestinal protozoa (trophozoites or cysts) or helminth eggs can be observed directly with a light microscope. A small amount of fresh feces is mixed with either saline (to detect the protozoa motility) or lugol/iodine solution (to reveal the parasite structure). 

Materials required Wooden applicator sticks/match, object glass, cover-slip, isotonic saline solution (0.85%; 8.5g/l), Lugol's iodine (1% solution), pipettes, microscopic slide, light microscope

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Samples Fresh stool (please refer to the SOP for stool collection)

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Procedure 1. Place a drop of saline on the centre of the left half of the slide and a drop of Lugol's solution on the centre of the right half of the slide on a microscopic slide, which has been labeled with a NIDIAG patient number (see Fig. 1).

2. With a wooden applicator stick or match, pick up a small portion of the stool specimen (size of match head) and mix with a drop of saline to form suspension (see Fig. 2).

3. Similarly, pick up a small portion of the stool specimen and mix with Lugol's solution to form suspension. 4. Cover each drop with a cover slip by holding the cover slip at an angle, touch the edge of the drop and gently lower the cover slip onto the slide to reduce the possibility of air bubbles in the smear (see Fig. 3).

Mukaram, Shaira R. | BSMT 2-A

5. Put the slide under a light microscope with 10x objective. Examine the entire cover slip area by moving the slide systematically backwards and forwards, or up and down (see Fig.4).

6. Switch to 40x objective lenses when suspected parasites are seen.

2. KATO-KATZ THICK SMEAR TECHNIQUE Kato-Katz Method was developed in 1954 by the Japanese physician, Dr. Kan Kato (1913-2011), together with his adviser, Dr. Momoshige Miura (1891-1989), a renowned Japanese medical researcher & psychiatrist. The technique was modified for use in field studies in 1972 by a Brazilian team of researchers led by the Brazilian Parasitologist, Naftale Katz and this modification was adopted by the WHO as a gold standard for multiple helminth infections. Its purpose is to detect Helminth egg in feces, including those soil-transmitted helminth and intestinal Schistosoma. 

Materials required Pinset, microscope slide, oil paper, wooden applicator stick, a holed (6mm) cardboard, wire net 3x3 cm/mesh, thick hydrophilic cellophane 7x2.5 cm

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Sample Fresh stool (sample)

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Solution Malachite green solution (1 mL Malachite-green 3% + 100 mL glycerin + 100 mL aquadest) Note: Soak the cellophane in malachite-green solution for 24 hours

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Procedure 1. Wash your hands and wear mask & gloves. 2. Write down patient’s identity (name, gender, age) & sample’s collection date on the left side of a clean slide. 3. Put +/- 5 gr of the fecal sample on an oil paper and place wire net above it.

Mukaram, Shaira R. | BSMT 2-A

4. Gently press a piece of wire screen at the top using a spatula. 5. Scrape the sieved fecal material through the screen so that only the debris remains. 6. Put a holed cardboard on the slide. Scrape up some of the sieved feces to fill the hole in the template, avoiding air bubbles and levelling the feces off to remove any excess.

7. Take the holed cardboard and put the filtered feces on the slide. 8. Place 1 piece of the cellophane which has been soaked overnight in malachite green over the fecal sample.

9. Place a clean applicator stick over the top and press it evenly downwards to spread the feces in a circle.

10. Carefully remove the stick by gently sliding it sideways to avoid separating the cellophane strip. 11. Put the slide under room temperature for 30 minutes until it becomes transparent. 12. Put the slide under the microscope. NOTE: The slide should be read within 30-60 minutes. After that time, the hookworm eggs disappear. 13. Examine systematically with low-power objective (10x) or high-power objective (40x) if needed. 14. Determine the egg’s species and count the eggs found under the microscope.

Mukaram, Shaira R. | BSMT 2-A

15. N from 30 mg of feces, so the number of eggs found per gram of feces (egg per gram or epg) = 1000/30 x N 16. The calculation & interpretation of this examination must be different for each species. 17. All unused materials are soaked in liquid chlorine to prevent infection. 18. Wash your hands after and dry it properly. 19. Complete the patient report form according to WHO Proposed Grade of Infection.

3. SCOTCH TAPE TECHNIQUE The clear-cellulose tape preparation is the most widely used procedure for the detection of human pinworm infections. Adult Enterobius vermicularis worms inhabit the large intestine and rectum; however, the eggs are not normally found in fecal material. During period of extraintestinal migration, the adult female migrates out the anal opening and deposits the eggs on the perianal skin, usually during the night. The eggs are not commonly found in feces though they may occasionally found on the surface of stool specimen or on underclothing, pajamas, or sheets about 2 to 3 hours after falling asleep. Stool is not an appropriate specimen for the diagnosis of pinworm. It is preferable to collect sample at night. Specimens can however be obtained in the morning before the patient bathes or goes to the bathroom. Itching during the night in a child’s perianal area strongly suggests pinworm infection. Diagnosis is made by identifying the worm or its eggs. The eggs, and occasionally the adult female worms stick to the glued (sticky) surface of the cellulose tape. These cellulose tape preparations are submitted to the laboratory, where they are examined under microscope. Commercial collection systems ‘pinworm paddles’ are also available.

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Materials Glass slide, scotch tape, stick/tongue depressor/popsicle stick,

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Sample Fresh stool

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Procedure 1. The tape should be looped (adhesive side outward) over a wooden tongue depressor.

2. Spread the buttocks and press the tape firmly several times against the right and left perianal fold with wooden applicator.

Mukaram, Shaira R. | BSMT 2-A

3. Place the tape on the slide and press gently to make sure there are no bubbles.

4. Put the slide under the microscope. 5. Examine with low power objective (10x) or dry objective (40x) if necessary.

4. HARADA MORI FILTER PAPER STRIP CULTURE To detect light infections with hookworm, S. stercoralis, and Trichostrongylus spp., as well as to facilitate specific identification of Necator Americanus and Ancylostoma species, the Harada-Mori filter paper strip culture technique is very useful. This filter paper test tube culture technique was initially introduced by Harada and Mori in 1955 (10) and was later modified by others. The technique requires filter paper to which fresh fecal material is added and a test tube into which the filter paper is inserted. Moisture is provided by adding water to the tube, which continuously soaks the filter paper by capillary action. Incubation under suitable conditions favors hatching of ova and/or development of larvae. Fecal specimens to be cultured should not be refrigerated, since some parasites (especially Necator americanus ) are susceptible to cold and may fail to develop after refrigeration. Also, caution must be exercised in handling the filter paper strip itself, since infective Strongyloides larvae may migrate upward as well as downward on the paper strip. Always observe standard precautions and wear gloves when performing these procedures. 

Materials Filter Paper, cotton ball, applicator stick, 1 cc sterile water.

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Sample Fresh stool

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Procedure 1. Smear 0.5 to 1 g of fresh feces in the center of a narrow strip of filter paper (3/8 by 5 in. [1 in. x 2.54 cm], slightly tapered at one end). 2. Add 3 to 4 ml of distilled water to a 15-ml conical centrifuge tube; identify the specimen on the tube. 3. Insert the filter paper strip into the tube so that the tapered end is near the bottom of the tube. The water level should be approximately 1/2 in. below the fecal spot. It is not necessary to cap the tube. However, a cork stopper or a cotton plug may be used.

Mukaram, Shaira R. | BSMT 2-A

4. Maintain the tube upright in a rack at 25 to 28°C. Add distilled water to maintain the original level (usually evaporation takes place over the first 2 days, and then the culture becomes stabilized). 5. After 10 days, remove the filter paper and discard it properly. 6. Add a few drops of 10% formalin to immobilize the larvae so they won’t be moving anymore. 7. Prepare a smear on a glass slide, cover the slide with a coverslip, and examine the smear with the 10x objective or high power objective if necessary. 6. Examine the larvae for motility and typical morphological features to reveal whether hookworm, Strongyloides, or Trichostrongylus larvae are present.

5. CONCENTRATION (FLOATATION AND SEDIMENTATION) Fecal concentration has become a routine procedure as a part of the complete ova and parasite examination for parasites; it allows the detection of small numbers of organisms that may be missed by using only a direct wet smear. There are two types of concentration procedures, sedimentation and flotation, both of which are designed to separate protozoan organisms and helminth eggs and larvae from fecal debris by centrifugation and/or differences in specific gravity.

A. Sedimentation methods (by centrifugation) lead to the recovery of all protozoa, oocysts, eggs, and larvae present; however, the concentration sediment that will be examined contains more debris. Although some workers recommend using both flotation and sedimentation procedures for every stool specimen submitted for examination, this approach is impractical for most laboratories. If one technique is selected for routine use, the sedimentation procedure is recommended as being the easiest to perform and the least subject to technical error. 

Materials

Stool sample, the fine mesh of pore size 350 to 415 micrometer, fifteen mL centrifuge tube, a glass applicator for emulsifying the stool sample, the beaker; for the filtrates, the ten percent Formol-saline for mixing the stool, then the diethyl ether. 

Procedure 1. Add ten percent Formaline to the stool, about a gram of the stool sample. 2. Emulsify the stool sample with the applicator. Emulsify it very well to get a smooth substance. 3. Sift the stool sample through the fine mesh into the beaker. 4. Discard the stool sample. 5. Transfer 7mL of stool filtrate into the 15mL centrifuge tube. Rinse whatever is left in the beaker into the centrifuge tube. 6. Add three mL of the diethyl ether to the 7mL of the stool filtrate in the 15mL centrifuge tube making it to a total of 10mL. 7. And we will see two layers: the ether layer and then the Formalin layer.

Mukaram, Shaira R. | BSMT 2-A

8. Cover the centrifuge tube with a lid and mix the two layers very well. And thus the ether is to dissolve in the fat, which is present in the stool sample, to release the parasites. 9. Put it in the centrifuge tube balancing it. 10. Centrifuge at thousand five hundred (1500) RPM for two to five minutes. 11. Take out the centrifuge tube and you will see four layers. The first layer is the ether layer, the debris layer, which is the insoluble pad, and then formalin layer, and then the sediment which contains the parasite. 12. Break through the debris layer and discard the first three layers. The sediment will contain the parasites of the egg or the larvae and then cysts were suspended with part of the Formol-saline or saline. 13. Use a disposable pipet to transmit it very well and transfer a drop onto the microscope slide. 14. Cover the drop of smear with the cover slip and examine it under the microscope using the times ten objective (10X). 15. Low light allows better to see the parasite of the egg, the cyst, and the larvae. Summary 1. 2. 3. 4. 5. 6. 7.

Mix stool with 10% formalin. Filter through a 350-450 micrometer mesh. Pour 7ml of filtrate into 15ml centrifuge tube. Add 3ml of diethyl ether and mix. Centrifuge at 1500 rpm for 2-5 mins. Discard the top three layers. Place a drop of the sediment on a glass slide under a coverslip and examine under low power.

Ma’am Tan 1. 2. 3. 4. 5.

Mix stool sample with distilled water, then centrifuge. Decant supernatant liquid. Repeat 1 and 2 until the supernatant liquid is clear. Add acid ether solution (formalin and ether mixture) After centrifugation with acid ether sol’n, pour out the supernatant liquid leaving the sediment behind. 6. Place a drop of the sediment on a glass slide under a coverslip and examine under low power. B. The Flotation Procedure permits the separation of protozoan cysts and eggs of certain helminths from excess debris through the use of a liquid (zinc sulfate) with a high specific gravity. The parasitic elements are recovered in the surface film, and the debris and some heavy parasitic elements remain in the bottom of the tube. This technique yields a cleaner preparation than does the sedimentation procedure; however, some helminth eggs (operculated and/or very dense eggs, such as unfertilized Ascaris eggs) do not concentrate well in the flotation method; a sedimentation technique is recommended to detect these infections. When the zinc sulfate solution is prepared, the specific gravity should be 1.18 for fresh stool specimens; it must be checked with a hydrometer. This procedure may be used on formalin-preserved specimens if the specific gravity of the zinc sulfate is increased to 1.20; however, this usually causes more

Mukaram, Shaira R. | BSMT 2-A

distortion in the organisms present and is not recommended for routine clinical use. To ensure detection of all possible organisms, both the surface film and the sediment must be examined. For most laboratories, this is not a practical approach.

1. Mix the stool sample with distilled water and centrifuge. 2. Repeat the step for several times until the supernatant liquid becomes clear. 3. The last time we decant, we refill the tube with the zinc sulfate sol’n with a specific gravity of 1.180. 4. Use a cover slip to remove one or two drops of the surface film, and place them on a slide. Pasteur pipette or a freshly flamed can also be used to recover drops of the surface film. Put it in a slide then add coverslip.

Mukaram, Shaira R. | BSMT 2-A...