Stool Examination

What Sample is Needed for Stool Examination?

1. Can take a random stool sample.
2. More than 2 grams of stool is needed, ideally 2 to 5 grams, which is sometimes called a pigeon’s egg.
3. To rule out worm infestation, three consecutive stools are tested.
   1. Collect three stools in a span of 10 days.
   2. Two samples on alternate days.
   3. The hospitalized patient can take a stool sample every day.
   4. Multiple samples are needed to rule out the parasitic infestation.
   5. One sample after purgation.
4. Collect the sample in a clean, water-tight, dry, urine-free container with a tight lid.
5. In the case of Infants, collect from the diaper.

How will you describe Concentration methods?

1. It is used for:
   1. Protozoan cysts and helminth eggs.
2. It consists of the following:
   1. Sedimentation method.
   2. Floatation technique.

How will you describe the Qualitative method for fats?

1. Fat in the feces is stained with Sudan III.
2. Neutral fat is seen as bright orange droplets.
3. Fatty acids usually do not stain.

How will you discuss a quantitative method for fats?

1. It will quantify fat contents in the stool in patients with steatorrhea.
2. Method:
   1. Collect stool for 3 consecutive days.
   2. The patient must have a diet containing roughly 50 to 150 grams/day of fat 2 days before stool collection.
3. It is important to be sure that all stool samples are collected and that the patient is not incontinent of feces.
4. Patient noncompliance with complete stool collection is the common cause of false negative results.
5. In constipated patients, give bedtime laxatives.

What are the normal fat contents in the stool?

1. In a normal person = <7 grams of fats in the stool/24 hours.
2. Pancreatic insufficiency = >9.5 grams/100 grams of stool. (it is also seen in bacterial overgrowth or biliary tract disease).
3. Nontropiocal sprue and celiac disease (24 hours fats) = <9.5 grams/100 grams of the stool favors.

Describe the Cellophane tape method for kids with pinworms (Enterobius vermicularis)?

1. This is also known as Scotch tape preparation.
2. This is the best method for diagnosing infant and child pinworms (Enterobius vermicularis) infestation.
3. The female (pinworm) at night, when the child is resting, a female (pinworm) comes out of the rectum and lays eggs in the perianal area at night.
4. Put the tape at night and collect the cellophane tape in the morning.

What is the procedure for pinworms in infants?

1. Take 10 cm of the transparent adhesive tape.
2. Fold it on the tongue depressor with the adhesive side out.
3. Now press the adhesive side of the 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.

What are the methods to prepare the stool smears?

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 to see the newsprint under the slide.
4. Put cover glass and see the 100x and 400x objective under the microscope.
   1. This is best to see helminth eggs, larvae, and trophozoites.

Iodine wet preparation:

1. This is also called wet preparation.
2. The stool is mixed with an iodine solution.

How will you prepare Lugol’s iodine solution?

What are the contents of the Iodine solution?

1. Potassium iodide (KI) = 10 grams
2. Iodine powder crystals = 5 grams
3. Distilled water = 100 ml

How will you prepare Lugol’s iodine solution?

1. Dissolve KI in D.water.
2. Slowly add iodine crystals.
3. Shake the solution gently until it dissolves.
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.

How do you make a stool smear with Lugol’s Iodine?

1. Take a drop of Lugol’s iodine solution.
2. Take a small amount of stool and mix it well.
   1. Make a thin smear.
3. Put the cover glass on it and gently press it to get an evenly thin smear.
4. See under 100 x and 400 x objective lenses.
   1. Too weak iodine solution; in that case, organisms will not stain properly.
   2. Too strong an iodine solution will clump the stool.

How will you discuss Concentration methods for stool?

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

What are the possibilities of the concentration of stool method?

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

How would you discuss the formalin-ethyl-acetate concentration method for stool?

1. The formalin-ethyl-acetate concentration method is most commonly used.
2. This method recovers the helminth eggs and larvae and, to a lesser extent, trophozoites.
3. Principle:
4. This is based on specific gravity. After centrifugation, the stool’s parasites are heavier and settle at the bottom as sediments.
5. Debris is lighter and rises to the upper layers.
6. Advantages are:
7. It is easy to prepare the solution.
8. This is inexpensive.
9. The procedure is easy to perform.
10. There is a rare distortion of parasite forms (eggs).

What is the procedure of formalin-ethyl-acetate concentration?

1. The stool should be fixed in formalin for at least 30 minutes.
   1. Mix 2 to 5 grams of the stool 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 collecting 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 method by ethyl-formalin method

Stool examination: Stool formalin-ethyl acetate method

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.

How will you discuss the Zinc sulfate floatation method?

• Some authors believe it is superior for concentrating and identifying eggs and protozoan cysts.
• The parasites are lighter and float on the surface, while the debris settles at the bottom.

What is the procedure for Zinc sulfate floatation?

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

Stool examination: Stool zinc sulfate concentration method

Collection, Precautions, and Handling of the Stool samples

What are the precautions before collecting the stool?

1. Advise patients about 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.
2. Stop iron-containing drugs, meat, and fish 48 hours before the collection of occult blood.
   1. Take a sample before or after one week for antibiotics or contrast media.
   2. These substances produce unknown objects or mask the parasites.
3. Warm stools are better for the ova and parasites.
4. Don’t refrigerate the stool for ova and parasites.
5. Stools for ova and parasites can be collected in formalin and polyvinyl alcohol, which are used as fixatives.
6. If there is blood or mucus, it should be included in the stool because most pathogens are found in this substance.
7. Examine the stool before giving antibiotics or other drugs.
8. 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.
9. Trophozoites degenerate in liquid stool rapidly, so examine the stool within 30 minutes.
10. In constipated cases, use non-residual purgatives the night before collecting the stool.
11. Avoid urine contamination because the urine destroys some of the parasites.
12. Water destroys some of the parasites like Schistosome eggs and amoebic trophozoites.
13. Toilet paper in the stool makes it difficult to examine the stool and may mask the parasites.

What are the Stool preservatives?

• In routine, stool preservatives used are:

How will you preserve the stool by 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.

How will you preserve the stool with 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 do 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, iron, and hemotoxylin.
4. Disadvantages are:
   1. A large amount of mercury is present in the solution, which is hazardous to health.
   2. It is not easy to prepare in the lab.

What is the formula of Polyvinyl alcohol?

4. How will you perform the PVA Procedure?
   1.  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.

How will you preserve the stool using a 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 to concentrate 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, so you may need albumin.
   2. It is diluted with water.

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

What are the preservatives for the wet preparation of stool?

1. 10% formol-saline for the wet preparation. This is the best preservative as it kills bacteria and preserves 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.

Stool preservatives

4. For staining, use Polyvinyl alcohol.
5. Avoid preservatives for the culture of stool.
   1. Usually, three parts of the preservatives and one part of the stool are used.

What are the permanent stains for 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 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:

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

Questions and answers: