Intestinal Protozoa Staining Techniques

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Questions and Answers

What is the purpose of adding paper towel layers when preparing a PVA-preserved stool sample for staining?

To spread the sample from edge to edge

To absorb the excess PVA (correct)

To mix the stool sample with PVA

To strain the stool sample

What is the preferred stain for SAF-preserved samples?

Iron hematoxylin (correct)

Trichrome stain

Xylene

Iodine

How long should the smear be allowed to dry at room temperature?

1-2 hours

Several days

2-3 hours

Overnight (correct)

What is the purpose of adding permount to a SAF-preserved specimen?

To prepare the specimen for examination Signup and view all the answers

What is the purpose of using an applicator stick in preparing a PVA-preserved stool sample?

To apply the stool sample to the glass slide Signup and view all the answers

What is the purpose of letting the slide air dry before examination?

To add oil immersion to the slide Signup and view all the answers

What is the purpose of straining the SAF-preserved specimen through gauze?

To remove excess liquid from the specimen Signup and view all the answers

What is the purpose of using 70% ethanol in the staining process?

To dehydrate the specimen Signup and view all the answers

What is the primary advantage of using a permanent stain in laboratory testing?

It allows for the creation of a permanent record of a patient's specimen for future review. Signup and view all the answers

What is the minimum number of fields that must be reviewed before a slide can be considered negative?

300 fields Signup and view all the answers

Which of the following stains is limited to use with SAF-preserved samples?

Iron hematoxylin Signup and view all the answers

What is the preferred fixative for fresh stool samples submitted for staining?

Schaudinn's fixative Signup and view all the answers

What is the primary advantage of the Trichrome (Wheatley modification) stain?

It uses reagents with a relatively long shelf life. Signup and view all the answers

What type of sample is best suited for permanent staining?

Thin, PVA-preserved smear Signup and view all the answers

What is the purpose of adding PVA to a smear prior to staining?

To fix the sample Signup and view all the answers

Why are permanent stains not useful in detecting helminth eggs or larvae?

They stain too dark or appear distorted. Signup and view all the answers

Which type of bacteria lacks cell walls fortified with mycolic acid?

Non-acid fast organism Signup and view all the answers

What is the purpose of using a modified acid fast stain?

To detect oocyst of Cryptosporidium, Isospora & Cyclospora Signup and view all the answers

What is the composition of acid alcohol used in the staining procedure?

10ml Sulfuric Acid + 90ml Absolute Ethanol Signup and view all the answers

What is the purpose of cellophane tape preparation?

To detect Enterobius vermicularis Signup and view all the answers

What is the ideal time to collect specimen for cellophane tape preparation?

In the morning before the patient washes or defecates Signup and view all the answers

What is the purpose of using 100x oil immersion objective?

To confirm internal morphology Signup and view all the answers

What is the characteristic of some non-mycobacterial bacteria and coccidian parasites?

Some degree of acid-fastiness Signup and view all the answers

What is the composition of 3% malachite green?

3g of malachite green in 100ml of distilled water Signup and view all the answers

What is the primary purpose of acid fast staining in microbiology?

To detect bacteria with high lipid content in their cell walls Signup and view all the answers

What is the function of mycolic acid in the cell walls of bacteria?

To make the cells resistant to decolorization Signup and view all the answers

What is the initial step in the modified acid fast staining of stool smear?

Preparing a smear with 1 or 2 drops of specimen on the slide Signup and view all the answers

What is the purpose of counterstaining with Malachite green in the modified acid fast staining of stool smear?

To enhance the color of the acid fast bacteria Signup and view all the answers

What is the final step in the modified acid fast staining of stool smear?

Mounting with a coverslip using desired mounting media Signup and view all the answers

What is the purpose of centrifuging the sample at 1,500 rpm for 1 minute?

To separate the sediment from the supernatant Signup and view all the answers

What is the temperature at which the slide is dried on a slide warmer?

60°C Signup and view all the answers

What is the purpose of adding 2 drops of albumin to the glass slide?

To mix the sample with the sediment Signup and view all the answers

What is the primary reason why laboratory technicians should be knowledgeable about specimen collection?

To explain the procedure to patients and healthcare professionals Signup and view all the answers

What type of tape should be avoided when collecting a specimen?

Frosted tape Signup and view all the answers

What is the purpose of pressing the adhesive side of the tape firmly against the perianal region?

To collect a sufficient sample Signup and view all the answers

Why is reduced light recommended when microscopically examining the slide?

To enhance egg visibility Signup and view all the answers

What is the purpose of using a tongue depressor?

To facilitate the collection of the specimen Signup and view all the answers

What is the primary importance of emphasizing proper hygiene and preventive measures during specimen collection?

To reduce the risk of contamination Signup and view all the answers

Study Notes

Stool Smear Preparation & Permanent Staining

Permanent stain preparation provides a permanent record of a patient's specimen, allowing for review and consultation if difficulties arise in identification.
Permanent stain is designed for analysis using the oil immersion objective (100x), and 300 fields must be reviewed before the slide can be considered negative.
Permanent stain is used to detect protozoan cysts and trophozoites by making their intracellular organelles visible.

Staining Techniques

Two common stains used in routine O&P testing are:
- Trichrome (Wheatley modification)
- Iron hematoxylin
Wheatley trichrome stain is the most widely used permanent stain, preferred by many users due to its relatively long shelf life and ease of performance.
Iron hematoxylin stain reveals excellent morphology of intestinal protozoa, with nuclear details stained clearer and sharper compared to trichrome.

Smear Preparation for Staining

For fresh stool samples, allow the sample to dry and then fix it in Schaudinn's fixative.
For PVA-preserved samples, mix the stool sample with PVA and apply it to a glass slide.
For SAF-preserved samples, mix the specimen well, add a few drops of permount, and let it dry.

Acid Fast Staining

Acid fast stains are used to stain bacteria that have a high lipid (long chain of mycolic acid) and wax content in their cell walls, making them resistant to decolorization with acid alcohol decolorizers.
The acid fast stain takes advantage of the waxy content of the cell walls to maximize detection.
Mycobacteria are the most commonly encountered acid-fast bacteria.
Coccidian oocyst and other acid-fast organisms will appear pink or red, while the background may appear blue or green depending on the counterstain used.

Cellophane Tape Preparation

Cellophane tape preparation is the specimen of choice for the detection of Enterobius vermicularis.
The adult female worm may also be seen in the perianal region.
Specimen collection is critical, and laboratory technicians must be knowledgeable regarding collection procedures.
Emphasize the importance of exercising proper hygiene and preventive measures during specimen collection to avoid spreading infectious eggs into the environment.

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Description

This quiz covers the steps involved in preparing a stool sample for microscopic examination, specifically for identifying intestinal protozoa. The process involves mixing the stool with PVA and staining the slide to reveal clear morphology and nuclear details of the organisms.