Stool Examination:- Part 2 – Stool Smear Preparation, Stains, Handling, and Preservatives
- Can take a random stool sample.
- More than 2 grams of the stool is needed, ideally 2 to 5 grams, sometimes referred to as pigeon’s egg.
- To rule out worm infestation, three consecutive stools are tested.
- Collect three stools in the span of 10 days.
- Two samples on alternate days.
- The hospitalized patient can take a stool sample every day.
- Multiple samples are needed to rule out the parasitic infestation.
- One sample after purgation.
- Collect the sample in a clean, water-tight, dry, urine-free container with a tight lid.
- In the case of Infants, collect from the diaper.
Cellophane tape method for kids:
- This is also known as Scotch tape preparation.
- This is the best method for infants and children to diagnose pinworms (Enterobius vermicularis).
- 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.
- Put the tape at night and collect the cellophane tape in the morning.
- Take 10 cm of the transparent adhesive tape.
- Fold it on the tongue depressor with the adhesive side out.
- Now press the adhesive side of tape over the perianal area and cover the maximum area. Should apply this tape at night.
- In the morning, remove the tape and put the adhesive side on the slide.
- See under the microscope.
Methods to prepare the stool smears:
Saline wet preparation:
- Take one drop of 0.85% saline.
- Take a small amount of stool and mix well.
- The smear should be thin so that you can see the newsprint under the slide.
- Put cover glass and see under the microscope 100x and 400x objective.
- This is best to see helminth eggs, larva, and trophozoites.
Iodine wet preparation:
- This is also called wet preparation.
- Iodine solution consists of:
- Potassium iodide (KI) = 10 grams
- Iodine powder crystals = 5 grams
- Distilled water = 100 ml
Procedure to make Lugol’s iodine solution:
- Dissolve KI in D.water.
- Slowly add iodine crystals.
- Shake the solution gently until they dissolve.
- Filter the solution before use, and this is the stock solution.
- Dilute the stock solution 1:5 with D. water. Make this working solution before use.
- Take a drop of Lugol’s iodine solution.
- Take a small amount of stool and mix it well.
- Make a thin smear.
- Put the cover glass on it and gently press it to get an evenly thin smear.
- See under 100 x and 400 x objective lenses.
- Too weak iodine solution; in that case, organisms will not stain properly.
- Too strong iodine solution will clump the stool.
Concentration method for stool:
- Purpose of the 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:
- Formalin-ethyl-acetate concentration method.
- Zinc-floatation method.
- Sheather sugar floatation method.
The formalin-ethyl-acetate concentration method for stool:
- 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 settle down at the bottom as sediments. Debris is lighter and rises to the upper layers.
- It is easy to prepare the solution.
- This is inexpensive.
- The procedure is easy to perform.
- There is a rare distortion of parasite forms (eggs).
The procedure of formalin-ethyl-acetate concentration:
- The stool should be fixed in formalin for at least 30 minutes.
- Take 2 to 5 grams of the stool and mix thoroughly in the 10% formalin.
- Filter the above stool in the formalin.
- This can be done by two layers of gauze or a wire screen and collecting around 3 mL.
- Add 10 to 12 mL of 0.85% saline and mix it well.
- Centrifuge for 2 minutes at 2000 RPM (or 2500 RPM).
- Discard the supernatant and leave 1 to 1.5 mL of the sediment.
- If the supernatant is cloudy, then repeat the above steps of saline.
- Add 9 mL of 10% formalin to the sediment.
- Now add 3 mL of ethyl acetate.
- Cap the test tube and shake well for 30 seconds.
- Centrifuge the tubes for 1 minute at 2000 RPM.
- Four layers will form. The bottom is the sediment that is needed to prepare the smear.
- Remove the debris with a wooden applicator stick. Decant the upper three layers carefully and leave the sediments in the test tube.
- Clean the sides of the test tube with a swab.
- Giardia cyst may stick to the side of the test tube.
- Add a few drops of the formalin and mix the sediment thoroughly. This will preserve the sediment.
- Now, we can make the smears in saline and iodine wet preparation.
- Examine under the microscope.
Zinc sulfate floatation method:
- Some authors believe it is a superior method for concentrating and identifying eggs and protozoan cysts.
- The parasites are lighter and float on the surface, while the debris settles at the bottom.
The procedure of Zinc sulfate floatation:
- Fix the stool in the formalin.
- Make a dilution of the above specimen (1 mL) with tap water from 1:10 to 15.
- Pour above suspension through a funnel with two layers of gauze in a small test tube.
- Add 2 mL of ether to the test tube with a stopper and gently shake.
- Now add water to the above test tube to the top just 1 cm from above.
- Centrifuge at 2500 rpm for 45 seconds.
- Decant the supernatant.
- Add 2.5 ml of the water to the sediment, shake well to resuspend the sediment—repeat steps 5 and 6.
- Add 2.5 ml of zinc sulfate (ZnSO4) to the sediment to resuspend it.
- Add zinc sulfate solution to the top of the test tube, leaving only 1 to 0.5 cm open on top.
- Centrifuge the test tube for 2 minutes at 2000 RPM.
- Take the surface material with a wire loop; this is the material where you can see the parasites.
- Make wet preparation with saline and iodine.
- Examine under the microscope.
Collection, Precautions, and Handling of the Stool samples
Precautions before the collection:
- Advise patients for the following things for at least 48 hours before the collection of the stool:
- Avoid mineral oils.
- Do not take bismuth.
- Don’t take antibiotics like tetracyclines.
- Don’t take anti-diarrheal drugs that are non-absorbent.
- Avoid anti-malarial drugs.
- The patient should not have a barium swallow examination before the stool examination.
- For occult blood, stop iron-containing drugs, meat, and fish 48 hours before the collection.
- In the case of antibiotics or contrast media, either take a sample before or after one week.
- These substances produce unknown objects or mask the parasites.
- Warm stools are better for the ova and parasites.
- Don’t refrigerate the stool for ova and parasites.
- Stools for ova and parasites can be collected in formalin and polyvinyl alcohol. These are used as a fixative.
- If there is blood or mucus, that should be included in the stool because most of the pathogens are found in this substance.
- Examine the stool before giving antibiotics or other drugs.
- The semi-formed stool should be examined within 60 minutes of collection.
- The liquid stool should be examined within the first 30 minutes.
- The solid stool should be examined within the first hour of collection.
- Trophozoites degenerate in liquid stool rapidly, so exam the stool within 30 minutes.
- In the case of constipated cases, use non-residual purgative on the night before collecting the stool.
- Avoid urine contamination because some of the parasites are destroyed by the urine.
- Water destroys some of the parasites like Schistosome eggs and amoebic trophozoites.
- Toilet paper in the stool makes it difficult to examine the stool and may mask the parasites.
Stool preservatives are:
In routine, stool preservatives used are:
- 5% is ideal for protozoan cysts.
- 10% preserves eggs and cysts.
- Advantages are:
- It is easy to prepare.
- It can preserve the stool for several years.
- It has a long shelf life.
- Disadvantages are:
- It is not good for a permanent smear.
- Details of eggs and cysts fade away.
- Trophozoites can not be recovered.
- This is an effective parasitic fixative.
- This can be used along with Schaudinn’s solution.
- Advantages are:
- This is easy to do with stool.
- This helps in gluing the sample on the slide.
- It has a long shelf life when stored at room temperature.
- Smears can stain with trichrome and iron, hemotoxylin.
- Disadvantages are:
- There is a large amount of mercury present in the solution, which is hazardous to health.
- It is not easy to prepare in the lab.
The formula of Polyvinyl alcohol:
- Procedure: Add glycerol to PVA in a large beaker.
- Blend it with a glass rod by stirring.
- Gradually add water and keep on mixing.
- Leave it overnight before use.
Sodium-acetate formalin mixture (SAF)
- There is increased interest in the use of this fixative.
- Advantages are:
- This is good for intestinal protozoa and coccidia-like bodies.
- This fixative eliminates the use of mercury compounds.
- This is an inexpensive fixative.
- This is easy to prepare in the lab.
- It has a buffering effect to decrease the distortion of the protozoa.
- This can be used in a concentration of the stool smear.
- It has good results when used with iron and hematoxylin permanent stain.
- Disadvantages are:
- This does not have adhesive properties, and you may need albumin for this purpose.
- It is diluted with water.
- The formula of Sodium acetate formalin:
- If the sample needs delay, then must use stool preservatives; otherwise, reject the sample.
- Send sample in two vials:
- One contains 5% or 10% formalin.
- The second vial contains either polyvinyl or sodium acetate formalin.
Preservatives for the wet preparation are:
- 10% formol-saline for the wet preparation. This is the best preservative as it kills the bacteria and preserves the protozoa and helminths.
- Another preservative is Sodium acetate formalin (SAF).
- Methionate iodine formalin. This is a good preservative for the field collection of the stool.
- For staining, use Polyvinyl alcohol.
- Avoid preservatives for the culture of stool.
- 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:
- Wheatley trichrome.
- Iron hematoxylin.
- Modified acid-fast stain.
- The most commonly used is the Wheatley trichrome.
Wheatley trichrome procedure:
- Take the following Coplin jar and put the chemicals in those jars:
|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|
- Now seal the slide with the fixative and keep it for some time.
- Examine the slide under the oil immersion lens.