38 Questions
What is the primary purpose of using permanent stain in stool smear preparation?
To provide a permanent record of a patient's specimen for review by consultants
How many fields must be reviewed before a slide can be considered negative?
300 fields
What is the sample of choice for permanent staining?
Thinly prepared slide made of PVA-preserved sample
What is the limitation of using permanent stain?
It is not useful in detecting helminth eggs or larvae
What is the preferred stain for Schaudinn-preserved samples?
Iron hematoxylin
What is the first step in preparing a smear for staining?
Place the prepared smear in a coplin jar containing Schaudinn's fixative
What is the purpose of using several layers of paper towel in preparing the PVA-preserved stool sample for staining?
To remove excess PVA from the stool sample
What is the advantage of using Trichrome stain (Wheatley modification)?
It uses reagents with a relatively long shelf life and is easy to perform
What should be done to a fresh sample submitted for staining?
Allow it to dry and then fix it
What is the recommended temperature for drying the smear in preparing the PVA-preserved stool sample for staining?
Room temperature
What is the preferred stain for SAF-preserved samples?
Iron hematoxylin
What is the purpose of adding a few drops of permount to the SAF-preserved specimen?
To let it dry
What is the purpose of using an applicator stick in preparing the PVA-preserved stool sample for staining?
To apply the sample to the slide
What is the purpose of using 70% ethanol in preparing the PVA-preserved stool sample for staining?
To remove excess PVA from the sample
What is the recommended method for examining the SAF-preserved specimen under a microscope?
Add one drop of oil immersion to the slide
What is the purpose of letting the smear dry overnight at room temperature or 2-3 hours at 35 degrees Celsius?
To prepare the sample for examination
What is the purpose of centrifuging the sample at 1,500 rpm for 1 minute?
To concentrate the sediment
What is the function of albumin in the staining procedure?
To mix with the sediment
Why are acid-fast stains used for certain bacteria?
Because they have a high lipid content
What is the purpose of using Kinyoun's carbolfuchsin in the acid-fast staining procedure?
To stain the bacteria
Why do bacteria with high lipid content require acid-fast stains?
Because they are resistant to decolorization
What is the purpose of using Malachite green in the acid-fast staining procedure?
To counterstain the specimen
What is the advantage of using acid-fast stains?
They can detect bacteria with high lipid content
How many fields should be examined under the microscope?
200 to 300 fields
Why is it important for laboratory technicians to be knowledgeable about specimen collection?
To explain the procedure to patients, families, or other health care professionals
What is the primary purpose of emphasizing proper hygiene and preventive measures during specimen collection?
To prevent contamination of the laboratory environment
What is the characteristic of mycobacteria?
Resistance to decolorization with acid alcohol
What is the purpose of using 100x oil immersion objective?
To confirm internal morphology
What type of tape should be avoided when collecting a specimen using the cellophane tape method?
Frosted tape
What is the composition of acid alcohol?
10ml Sulfuric Acid + 90ml Absolute Ethanol
What is the recommended way to apply the adhesive side of the tape during specimen collection?
Using a rocking motion
What is the stain used for detection of oocyst of Cryptosporidium, Isospora & Cyclospora?
Modified acid fast stain
What is the purpose of using a low power objective during the microscopic examination?
To reduce the intensity of the light
What is the purpose of cellophane tape preparation?
Detection of Enterobius vermicularis
What is the purpose of using a clean glass microscopic slide during the specimen collection procedure?
To ensure the eggs are not obscured by debris
Why is it important to collect specimen in the morning for cellophane tape preparation?
Because the gravid female worm lays numerous eggs in the perianal region at night
What is the benefit of using a modified acid fast stain?
Detection of oocyst of Cryptosporidium, Isospora & Cyclospora which may be difficult to detect with routine stain
What is the significance of non-acid fast organisms?
They lack cell walls fortified with mycolic acid
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.
This quiz covers the preparation and permanent staining of stool smears in clinical parasitology laboratory, including the use of oil immersion objective and detection of protozoan cysts and trophozoites.
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