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

Stool Examination:- Part 3 – Stool Smear Preparation, Stains,  Handling, and Preservatives
March 25, 2021Lab TestsParasitology

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. 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.
  1. Take a drop of Lugol’s iodine solution.
  2. Take a small amount of stool and mix it well.
  3. Make a thin smear.
  4. Put the cover glass on it and gently press it to get an evenly thin smear.
  5. 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

      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 concentration by formalin-ethyl acetate method

    Stool formalin-ethyl acetate after centrifugation

    Stool formalin-ethyl acetate after centrifugation

  1. Remove the debris with a wooden applicator stick. Decant the upper three layers carefully and leave the sediments in the test tube.
  2. Clean the sides of the test tube with a swab.
    1. Giardia cyst may stick to the side of the test tube.
  3. Add a few drops of the formalin and mix the sediment thoroughly. This will preserve the sediment.
    1. Now, we can make the smears in saline and iodine wet preparation.
  4. 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

    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:
Stool polyvinyl alcohol formula

Stool polyvinyl alcohol formula

      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.
  1. 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

      Stool SAF formula

  2. 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

        Stool preservatives

  3. 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.
  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.

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
  1. Now seal the slide with the fixative and keep it for some time.
  2. Examine the slide under the oil immersion lens.
Possible References Used
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Comments

Zahra Reply
February 11, 2020

Thank you.

Dr. Riaz Reply
February 21, 2020

Thanks for the encouraging comments.

Abel Chukwuebuka Reply
March 8, 2020

Thanks so much

Dr. Riaz Reply
March 8, 2020

Great for the encouraging remarks

mel Reply
March 10, 2020

very nice presentatation.

Dr. Riaz Reply
March 10, 2020

Thanks for the encouraging remarks.

Abdidek Ahmed Jama Reply
March 24, 2020

I need this stool examination book

Dr. Riaz Reply
March 25, 2020

This is a comprehensive study on the stool. Please consult this website labpedia.net

Dr. Riaz Reply
March 25, 2020

There is no such book like this on the stool. This is my own research and I have put it on website labpedia.net

Sumesh. P Reply
August 21, 2020

It is very good note. Present in very well. I study this topic in simply and properly. It is realy usefull in all students and Others. Thank u

Dr. Riaz Reply
August 23, 2020

Thanks.

amira Reply
April 2, 2020

thank you for your great effort

Dr. Riaz Reply
April 2, 2020

Thanks for the encouraging remarks.

md asgar Reply
April 8, 2020

stool examination

Dr. Riaz Reply
April 8, 2020

Thanks

md asgar Reply
April 8, 2020

stool examination good

Dr. Riaz Reply
April 8, 2020

Thanks

Zeinab Reply
April 9, 2020

Can i do the test with just saline and no preservatives? Also, can i use the sedimentation method(leave the tube in room temperature for an hour or 40 minutes then discard the supernatant and examine the sediment)? Thank u so much i like your article it is very helpful.

Dr. Riaz Reply
April 9, 2020

Thanks for the question. The purpose of the preservative is to preserve the ova and parasite. In saline, there may be deterioration.You can use a concentrated saline solution and then make the smear from the top layer. Moreover you can do practically experiment and see the results.

khai Reply
April 25, 2020

thank you dr diaz, very helpful

khai Reply
April 25, 2020

i mean *dr riaz

Dr. Riaz Reply
April 25, 2020

Thanks

Dr. Riaz Reply
April 25, 2020

Thanks for the encouraging remarks

Davis Kirui Reply
May 1, 2020

I normally examine using ×10 and cover slip when using×40….otherwise nice presentation.

Dr. Riaz Reply
May 1, 2020

I think screening in x1o is the best option if you are experienced to find ova, cyst, and parasite. While x40 needed to confirm the diagnosis.
I agree with you, thanks.

Santosh Kumar Roy Reply
May 8, 2020

Nice compilation.
THANK YOU. Sir!

Dr. Riaz Reply
May 8, 2020

Thanks for the encouraging remarks

T- Robert Reply
May 21, 2020

Thanks so much
Dr. If I find Bacteria in stool smear microscopically and I don’t do Gram stain to classify the bacteria also stool culture not done,
Should I report the diagnosis as bacteria Infection and being clinical manifestation which needs Treatment?

Dr. Riaz Reply
May 21, 2020

I don’t think so because normally you can see the bacteria. You should get the history of the patient, and also you will see WBcs and maybe a few RBCs. The presence of WBCs indicates infection. Only bacteria may be ignored.

mahya Reply
June 2, 2020

Thanks very much .
so complete so useful .

Dr. Riaz Reply
June 2, 2020

Thanks for the encouraging remarks.

Vollrath Reply
June 4, 2020

Dr. Riaz, Thank you all your information. You made it easier to understand.

Dr. Riaz Reply
June 4, 2020

Thanks for the comments.

Samiullah Reply
June 24, 2020

Thank you for your efforts to create this website, I have been searching for long time for such easy access to any lab topics, previously I have to go to some text books for any info about lab. This will help me in diagnosing any samples as i am a lab technician. I would like you to improve further.

Dr. Riaz Reply
June 24, 2020

Thanks for the comments. Actually we are continously improving the various topics.

Sara Reply
July 14, 2020

Nice one kindly email me the pdf sarahsikede96@gmail.com

Dr. Riaz Reply
July 14, 2020

Thanks. Please let me know which topic pdf you want.

Edson A.Jackson Reply
July 24, 2020

May God bless you for this articles,lets me be together with you for more research.

Dr. Riaz Reply
July 24, 2020

Thanks a lot. You are welcome and let me know how you can help and improve it.

Havon Reply
July 30, 2020

Thank you so much. The article has answered all my questions.

Dr. Riaz Reply
July 30, 2020

Thanks, we are trying our best to update and add more material to each subject.

Mussa S.N Reply
August 4, 2020

Thank you so much,i understand and learn more and more in this lesson keep it up

Dr. Riaz Reply
August 4, 2020

Thanks. We are updating most of the topics.

kunjal gatecha Reply
August 15, 2020

Thank you so much for this site. I am a pathology technician and this is quite informative and helpful in my forthcoming interview. Thanks a lot again.

Dr. Riaz Reply
August 15, 2020

Thanks. You are welcome.

Kelli Stemmler Reply
August 23, 2020

I agree with you

Dr. Riaz Reply
August 23, 2020

Thanks.

Leoanthony Reply
August 27, 2020

I like this work well analyzied

Dr. Riaz Reply
August 27, 2020

Thanks.

Dr. Md. Reazuddin Reply
August 29, 2020

Dr. Riaz, thanks for your endeavor
Myself also Reaz

Really a nice piece of work, actually what I am looking for

Dr. Riaz Reply
August 29, 2020

Thanks.

Dr. Md. Reazuddin Reply
August 29, 2020

Can you please refer any ideal reference book for stool examination?

Dr. Riaz Reply
August 29, 2020

Dear, There are so many reference books. I can recommend a few of these:
Medical parasitology by Markell, John, and Krotoski (W.B. Saunders Company).
Foundation of parasitology by Gerald, Schmidt, and Roberts (Wm. C. Brown publishers).
Lynch’s Medical laboratory technology by Raphael (W.B. Saunders Company).
I hope these will help you.

Adam Lawal Reply
August 31, 2020

The topic is highly appreciable. Immeasurable thanks for the full explanation

Dr. Riaz Reply
August 31, 2020

Thanks.

pradeep patil Reply
September 10, 2020

nice information. thanks a lot

Dr. Riaz Reply
September 15, 2020

Thanks.

cm.salis144@gmail.com Reply
October 3, 2020

Please what’s the normal pus cell in stool?

Dr. Riaz Reply
October 3, 2020

I don’t think there is any normal number of WBC in the stool. Their presence is abnormal.

ah young kim Reply
October 18, 2020

what’s the interpretation if there is presence of e.coli and pus cells in stool of an acute gastroenteritis patient?

Dr. Riaz Reply
October 19, 2020

This depends upon the age of the patients. In children, E.coli can cause diarrhea. The pus cell presence indicates infection.

Zoyeb Zia Reply
October 27, 2020

thank you doc, nice and useful info

Dr. Riaz Reply
October 27, 2020

Thanks.

Abdi Reply
October 28, 2020

Very informative Article Doctor. What is the Publication date ?

Dr. Riaz Reply
October 28, 2020

Updated on October 28, 2020. Thanks for the remarks.

Julius Bamanyisa Reply
October 30, 2020

Good worker done God bless you

Dr. Riaz Reply
October 30, 2020

Thanks.

ken Reply
October 31, 2020

thank you sir for this piece it is very helpful

Dr. Riaz Reply
November 1, 2020

Thanks.

Macrina Reply
November 5, 2020

5. While doing the microscopic examination of stool specimen, you have noticed that there are many epithelial cells and few calcium crystals found per field. What action you will do?

Dr. Riaz Reply
November 5, 2020

If there are no WBCs, then no problem. Calcium oxalate crystals may be seen after taking the calcium-rich vegetables.

Macrina
November 6, 2020

A 25 year old adult patient came to the laboratory to submit urine specimen. The specimen was 1 mL only. What will you do?

Macrina Reply
November 5, 2020

While doing the microscopic examination of stool specimen, you have noticed that there are many epithelial cells and few calcium crystals found per field. What action you will do?

Mohammed hussian Reply
December 14, 2020

sir Can you name other cysts seen in stool.

Dr. Riaz Reply
December 14, 2020

I am always updating the subject, soon I will add more cysts.

Mohammed hussian Reply
December 15, 2020

Ok sir 👍
Masha Allah this website is amazing 🧿
Thanks for helping us

Dr. Riaz Reply
December 15, 2020

Thanks a lot.

Dr Dipak Reply
January 18, 2021

Really extensive, precise, and excellent information Loved it

Dr. Riaz Reply
January 18, 2021

Thanks, Dr. Dipak

Emmanuel Reply
January 25, 2021

Nice write- up, please I need the parasite chart

Dr. Riaz Reply
January 25, 2021

Please let me know the details of the parasite chart which you want. I will try my best for its presentation on labpedia.net

alisenmbambagwa@gmail.com Reply
January 30, 2021

Can i report ova of stool per HPF like this Ascariasis lumbricoide ova/ HPF

Dr. Riaz Reply
January 31, 2021

I don’t think any problem in reporting ova /HPF. Also, you can report mild, moderate, and severe infestation. You have to explain to the physician what you are trying to explain.

Joshua N. Ngaima Reply
February 3, 2021

Thanks so much, I find your article helpful

Dr. Riaz Reply
February 3, 2021

Thanks.

A. O. Johnson Reply
February 13, 2021

That was a very helpful article. Thank you.

Dr. Riaz Reply
February 13, 2021

Thanks for the appreciation.

NIAZI Reply
February 22, 2021

THANKS A LOT SIR FOR INFORMATIVE ARTICLE-JAZAKALLAHKHAIR.IF THERE ARE 2- 4 RBC,IS IT SIGNIFICANT FINDING ?OCCULT BLOOD NEGATIVE IN THE SAME SAMPLE.PT HAVING H/O GI BLEED 2 MONTHS AGO.THANKS A LOT SIR.

Dr. Riaz Reply
February 22, 2021

I think 2 to 4 RBCs need further workup. Occult blood is negative. If the RBCs are intact in shape, then these may be from the rectum like hemorrhoids.

NIAZI Reply
February 23, 2021

FINE SIR.THANKS A LOT FOR YOUR VALUABLE ADVISE.

EID Reply
February 27, 2021

thanks a lot sir

can we report bacteria in stool microscopically, with out a bacterial culture

Dr. Riaz Reply
February 27, 2021

I don’t think there is any need to report bacteria in the stool because these are normally seen in the stool.

noble Reply
March 25, 2021

Please Dr Riaz I would want to know more about stool microscopy and stool mcs separately.

Dr. Riaz Reply
March 25, 2021

You are welcome.

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