Stool Examination:- Part 3 – Stool Smear Preparation, Stains, Handling, and Preservatives

Sample

1. Can take a random stool sample.
   1. More than 2 grams of the stool is needed, ideally 2 to 5 grams, and sometimes referred to as pigeon’s egg.
2. To rule out worm infestation, three consecutive stools are tested.
   1. Collect three stools in the span of 10 days.
   2. Two samples on alternate days.
      1. In the hospitalized patient can take a stool sample every day.
      2. Multiple samples are needed to rule out the parasitic infestation.
   3. One sample after purgation.
3. Collect the sample in a clean, water-tight dry urine free container with a tight lid.
4. In the case of Infants, collect from the diaper.
5. Or cellophane tape method:
   1. This is also known as Scotch tape preparation.
   2. This is the best method in infants and children to diagnose pinworms (Enterobius vermicularis).
   3. The female at night, when the child is resting, comes out of the rectum and lays eggs in the perianal area.
   4. Put the tape at night and collect the tape in the morning.
   5. Procedure:
      1. Take 10 cms of the transparent adhesive tape.
      2. Fold it on the tongue depressor with the adhesive side out.
      3. Now press the adhesive side of tape over the perianal area and cover the maximum area. Should apply this tape at night.
      4. In the morning, remove the tape and put the adhesive side on the slide.
      5. See under the microscope.

Method to prepare the stool smears:

Saline and iodine Direct wet preparations:

Saline wet preparation:

1. Take one drop of 0.85% saline.
2. Take a small amount of stool and mix well.
3. The smear should be thin so that you can see the newsprint under the slide.
4. Put cover glass and see under the microscope 100x and 400x objective.
   1. This is best to see helminth eggs, larva, and trophozoites.

Iodine wet preparation:

This is also called wet preparation.

Iodine solution consists of:

1. 1. Potassium iodide (KI) = 10 grams
   2. Iodine powder crystals = 5 grams
   3. Distilled water = 100 ml
   4. Procedure to make Lugol’s iodine solution:
      1. Dissolve KI in D.water.
      2. Slowly add iodine crystals.
      3. Shake the solution gently until they dissolve.
      4. Filter the solution before use, and this is the stock solution.
      5. Dilute the stock solution 1:5 with D. water. Make this working solution before use.
2. Take a drop of Lugol’s iodine solution.
3. Take a small amount of stool and mix it well.
4. Make a thin smear.
5. Put the cover glass on it and gently press it to get an evenly thin smear.
6. See under 100x and 400x objective lenses.
   1. Too weak iodine solution; in that case, organisms will not stain properly.
   2. Too strong iodine solution will clump the stool.
      

      Stool wet preparation, saline, and iodine

Concentration method

The main aim of the concentration method is to remove the debris.  Also, when the parasite is low in number.

There are three methods used for the concentration of stool:

1. Formalin-ethyl-acetate concentration method.
2. Zinc-floatation method.
3. Sheather sugar floatation method.

The formalin-ethyl-acetate concentration:

The formalin-ethyl-acetate concentration method is most commonly used.

This method recovers the helminth eggs and larvae, to a lesser extent, trophozoites.

Principle: This is based on specific gravity. After centrifugation, the stool’s parasites are heavier and settles down at the bottom as sediments. Debris is lighter and rises to the upper layers.

Advantages are:

1. It is easy to prepare the solution.
2. This is inexpensive.
3. The procedure is easy to perform.
4. There is a rare distortion of parasite forms (eggs).

The procedure of formalin-ethyl-acetate concentration:

1. The stool should be fixed in formalin for at least 30 minutes.
   1. Take 2 to 5 grams of the stool and mix thoroughly in the 10% formalin.
2. Filter the above stool in the formalin.
   1. This can be done by two layers of gauze or a wire screen and collect around 3 mL.
3. Add 10 to 12  mL of 0.85% saline and mix it well.
4. Centrifuge for 2 minutes at 2000 RPM ( or 2500 RPM).
5. Discard the supernatant and leave 1 to 1.5 mL of the sediment.
   1. If the supernatant is cloudy, then repeat the above steps of saline.
6. Add 9 mL of 10% formalin to the sediment.
7. Now add 3 mL of ethyl acetate.
8. Cap the test tube and shake well for 30 seconds.
9. Centrifuge the tubes for 1 minute at 2000 RPM.
10. Four layers will form.  The bottom is the sediment that is needed to prepare the smear.
    

    Stool concentration by formalin-ethyl acetate method

    

    Stool formalin-ethyl acetate after centrifugation

11. Remove the debris with a wooden applicator stick. Decant the upper three layers carefully and leave the sediments in the test tube.
12. Clean the sides of the test tube with a swab.
    1. Giardia cyst may stick to the side of the test tube.
13. Add a few drops of the formalin and mix the sediment thoroughly. This will preserve the sediment.
    11. Now, we can make the smears in saline and iodine wet preparation.
14. Examine under the microscope.

Zinc sulfate floatation method:

Some authors believe it a superior method for concentration and identifying eggs and protozoan cyst.

The parasites are lighter and float on the surface, while the debris settles at the bottom.

Procedure:

1. Fix the stool in the formalin.
2. Make a dilution of the above specimen (1 mL) with tap water as 1:10 to 15.
3. Pour above suspension through a funnel that has two layers of gauze in a small test tube.
4. Add 2 mL of ether to the above test tube with a stopper and gently shake.
5. Now add water to the above test tube to the top just 1 cm from above.
6. Centrifuge at 2500 rpm for 45 seconds.
7. Decant the supernatant.
8. Add 2.5 ml of the water to the sediment, shake well to resuspend the sediment—repeat steps 5 and 6.
9. Add 2.5 ml of the zinc sulfate (ZnSO4) to the sediment to resuspend it.
10. Add zinc sulfate solution to the top of the test tube, leaving only 1 to 0.5 cm open top.
11. Centrifuge the test tube for 2 minutes at 2000 RPM.
12. Take the surface material with a wire loop; this is the material where you can see the parasites.
13. Make wet preparation with saline and iodine.
14. Examine under the microscope.
    

    Stool zinc sulfate concentration method

Collection, Precautions, and Handling of the Stool samples

1. Advise patients for the following things for at least 48 hours before the collection of the stool:
   1. Avoid mineral oils.
   2. Do not take bismuth.
   3. Don’t take antibiotics like tetracyclines.
   4. Don’t take anti-diarrheal drugs that are non-absorbent.
   5. Avoid anti-malarial drugs.
   6. The patient should not have a barium swallow examination before the stool examination.
   7. For occult blood, stop iron-containing drugs, meat, and fish 48 hours before the collection.
      1. In the case of antibiotics or contrast media, either take a sample before or after one week.
      2. These substances produce unknown objects or mask the parasites.
2. Warm stools are better for the ova and parasites.
3. Don’t refrigerate the stool for ova and parasites.
4. Stool for ova and parasites can be collected in formalin and polyvinyl alcohol. These are used as a fixative.
5. If there is blood or mucus, that should be included in the stool because most of the pathogens are found in this substance.
6. Exam the stool before giving antibiotics or other drugs.
7. The semi-formed stool should be examined within 60 minutes of collection.
   1. The liquid stool should be examined within the first 30 minutes.
   2. The solid stool should be examined within the first hour of collection.
8. Trophozoites degenerate in liquid stool rapidly, so exam the stool within 30 minutes.
9. In the case of constipated cases, use non-residual purgative on the night before collecting the stool.
10. Avoid urine contamination because some of the parasites are destroyed by the urine.
11. Water destroys some of the parasites like Schistosome eggs and amoebic trophozoites.
12. Toilet paper in the stool makes it difficult to exam the stool and may mask the parasites.

Stool preservatives are:

In routine, stool preservatives used are:

1. Formalin
   1. 5% is ideal for protozoan cysts.
   2. 10% preserves eggs and cysts.
   3. Advantages are:
      1. It is easy to prepare.
      2. It can preserve the stool for several years.
      3. It has a long shelf life.
   4. Disadvantages are:
      1. It is not good for a permanent smear.
      2. Details of eggs and cysts fade away.
      3. Trophozoites can not be recovered.
2. Polyvinyl alcohol
   1. This is an effective parasitic fixative.
   2. This can be used along with Schaudinn’s solution.
   3. Advantages are:
      1. This is easy to with stool.
      2. This helps in gluing the sample on the slide.
      3. It has a long shelf life when stored at room temperature.
      4. Smears can stain with trichrome and iron, hemotoxylin.
   4. Disadvantages are:
      1. There is a large amount of mercury present in the solution, which is hazardous for health.
      2. It is not easy to prepare in the lab.
   5. The formula of Polyvinyl alcohol:

1. 1. 1. Procedure: Add glycerol to PVA in a large beaker.
      2. Blend it with a glass rod by stirring.
      3. Gradually add water and keep on mixing.
      4. Leave it overnight before use.

3. Sodium-acetate formalin mixture (SAF)
   1. There is increased interest in the use of this fixative.
   2. Advantages are:
      1. This is good for intestinal protozoa and coccidia-like bodies.
      2. This fixative eliminates the use of mercury compounds.
      3. This is an inexpensive fixative.
      4. This is easy to prepare in the lab.
      5. It has a buffering effect to decrease the distortion of the protozoa.
      6. This can be used in a concentration of the stool smear.
      7. It has good results when used with iron and hematoxylin permanent stain.
   3. Disadvantages are:
      1. This does not have adhesive properties, and you may need albumin for this purpose.
      2. It is diluted with water.
   4. The formula of Sodium acetate formalin:
      

      Stool SAF formula

4. If the sample needs delay, then must use stool preservatives, otherwise reject the sample.
   1. Send sample in two vials:
      1. One containing 5% or 10% formalin.
      2. The second vial contains either polyvinyl or sodium acetate formalin.
         

         Stool preservatives

5. Preservatives for the wet preparation are:
   1. 10% formol-saline for the wet preparation. This is the best preservatives as it kills the bacteria and preserves the protozoa and helminths.
   2. Another preservative is Sodium acetate formalin (SAF).
   3. Methionate iodine formalin. This is a good preservative for the field collection of the stool.
6. For staining, use Polyvinyl alcohol.
7. Avoid preservatives for the culture of stool.
   1. Usually, three parts of the preservatives and one part of the stool are used.

The permanent stain of the stool smears:

The sample of choice for stains is a thinly prepared slide from a PVA preservative (polyvinyl alcohol).

There are three methods for the permanent stain:

1. Wheatley trichrome.
2. Iron hematoxylin.
3. Modified acid-fast stain.
4. The most commonly used is the Wheatley trichrome.

Wheatley trichrome procedure:

1. Take the following Coplin jar and put the chemicals in those jars:

	1	2	3	4	5	6	7	8	9
Reagents	Iodine +70% alcohol	70% alcohol	70% alcohol	Trichrome stain	90% acidified alcohol	95% alcohol	95% alcohol	Carboxylene	Xylene
Purpose of the reagents	remove the HgCl and hydration	remove iodine and hydration	Rinse, hydration	Stain	Destain	Stop staining	Dehydration	Removes debris and dehydration	Removes debris
Time for each jar	10 minutes	5 minutes	5 minutes	7 minutes	5 to 10 seconds	Quick rinse	5 minutes	10 minutes	10 minutes

7. Now seal the slide with the fixative and keep it for some time.
8. Examine the slide under the oil immersion lens.