Trichrome Staining Procedure

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Questions and Answers

What is the formula to calculate the number of microns equal to each unit on the ocular micrometer?

(No.of stage micrometer units x 100)/(no.of ocular micrometer units)

(No.of stage micrometer units x 1000)/(no.of ocular micrometer units) (correct)

(No.of ocular micrometer units x 100)/(no.of stage micrometer units)

(No.of ocular micrometer units x 1000)/(no.of stage micrometer units)

How many microns are equal to 2 ocular units if 1 ocular unit is equal to 7.5 micrometers?

12 microns

7.5 microns

15 microns (correct)

10 microns

What type of parasite stages may be observed in direct wet preparation?

Only Oocyst

Helminth eggs and larvae, Oocyst

Protozoan cyst, Helminth eggs and larvae

Protozoan cyst, Oocyst, Helminth eggs and larvae (correct)

What is the concentration of saline used in direct saline wet preparation?

0.85% saline Signup and view all the answers

What is the purpose of mixing a small portion of unfixed stool with saline in direct saline wet preparation?

To create a uniform mixture for observation Signup and view all the answers

What is the primary advantage of using fixatives in stool sample preservation?

To prevent the further development of certain helminth eggs and larvae Signup and view all the answers

Why is the diagnostic yield of direct wet preparation considered low?

Because parasites may not be present in the sample Signup and view all the answers

What tool is used to mix the stool with saline in direct saline wet preparation?

Wooden applicator stick Signup and view all the answers

What is the disadvantage of using Schaudinn's solution as a fixative?

It does not preserve parasite morphology adequately for permanent smears Signup and view all the answers

What is the purpose of the plastic powder in Polyvinyl Alcohol (PVA)?

To act as an adhesive for the stool specimen when preparing slides for staining Signup and view all the answers

What is the purpose of direct saline wet preparation?

To observe parasites in the stool sample Signup and view all the answers

What is the recommended ratio for PVA?

2 I BSMDT 2 Signup and view all the answers

What is the advantage of using PVA as a fixative?

It acts as a mounting media where the specimen adheres to the slide Signup and view all the answers

What is the disadvantage of using PVA as a fixative?

It does not preserve morphologic details of cysts and eggs Signup and view all the answers

What is the importance of immediate processing and analysis of freshly collected stool samples in the laboratory?

To maintain the integrity of the sample and prevent degradation Signup and view all the answers

What is the primary advantage of using Polyvinyl Alcohol (PVA) over other fixatives?

It can recover trophozoite Signup and view all the answers

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

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

What should be done to the stool sample except for hookworms eggs before examining it under the microscope?

Keep the slide for one or more hours at ambient temperature Signup and view all the answers

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

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

How should the smear be examined under the microscope?

In a systematic manner Signup and view all the answers

What is the purpose of the xylene step in the Modified Acid Fast Staining procedure?

To remove excess water from the slide Signup and view all the answers

What is the recommended magnification for examining the stained slide?

40x or higher Signup and view all the answers

What is the unit of measurement used to report the Kato Katz technique?

Eggs per gram Signup and view all the answers

How many hours should the slide be kept at ambient temperature for hookworms eggs?

Less than 60 minutes Signup and view all the answers

What is the function of the malachite green counterstain in the Modified Acid Fast Staining procedure?

To counterstain the background of the slide Signup and view all the answers

What is the recommended time for the xylene step in the Modified Acid Fast Staining procedure?

10 minutes Signup and view all the answers

What is a characteristic of Ascaris and Trichuris in the Kato Katz preparation?

They remain visible and recognizable for many months Signup and view all the answers

What is the factor by which the number of eggs counted from the slide should be multiplied?

24 Signup and view all the answers

What is the alternative method for mounting the slide?

Let the slide air dry and add one drop of oil immersion Signup and view all the answers

What is the recommended temperature for drying the slide on a slide warmer?

60oC Signup and view all the answers

What is the purpose of using iodine in wet preparations?

To kill any trophozoite present Signup and view all the answers

What adjustment is crucial for the detection of protozoa in wet preparations?

Reducing the light adjustment of the microscope Signup and view all the answers

What is the purpose of using saline in wet preparations?

To demonstrate the motility of protozoan trophozoites Signup and view all the answers

Why is lowering the condenser recommended in microscopy?

To reduce the light and allow transparent structures to be seen Signup and view all the answers

What is the importance of proper adjustment of the microscope in reading and interpreting wet preparations?

It is essential for the successful reading and interpreting of wet preparations Signup and view all the answers

Why is experience necessary for screening a slide using a microscope?

Because it requires a high level of expertise and experience Signup and view all the answers

What is the characteristic of protozoans that makes them difficult to detect in microscopy?

They are translucent and colorless Signup and view all the answers

What is the term for a slide made by mixing a small portion of unfixed stool with saline or iodine and the mixture?

Direct Wet Preparation Signup and view all the answers

Study Notes

Specimen Preservation

A freshly collected stool sample must be immediately processed and analyzed in the laboratory, but possible samples must refer to maintain integrity and use fixative or preservatives.
Fixatives are substances that preserve the morphology of protozoa and prevent further development of certain helminth eggs and larvae.
Advantages of fixatives:
- Detects trophozoite, cysts, helminth eggs
- Long shelf life at room temp
- Recovery of certain parasites is possible

Polyvinyl Alcohol (PVA)

Trophozoite can be recovered
Has plastic powder that acts as an adhesive for the stool specimen when preparing slides for staining
Acts like a mounting media where the specimen adheres to the slide
PVA: Schaudinn's solution contains zinc sulfate, copper sulfate, or mercuric chloride

Ocular Micrometer

The number of microns (um) equal to each unit on the ocular micrometer may be calculated by using the following formula: (No.of stage micrometer units x 1000/(no.of ocular micrometer units) = no.of microns
Example: 2 ocular unit (parasite) x 7.5 micrometer = 15 microns

Direct Saline Wet Preparation

Made by placing a drop of 0.85% saline on glass slide and mixing a small portion of unfixed stool
Used to demonstrate the motility of protozoan trophozoites
Also known as Direct Wet Preparation

Iodine Wet Preparation

Defined as a slide made by mixing a small portion of unfixed stool with saline or iodine
The purpose of this technique is to demonstrate the motility of protozoan trophozoites
Iodine kills any trophozoite present, thus it is recommended to use direct saline and direct wet preparations on each sample that requires this component of testing

Microscope Adjustment

Proper adjustment of microscope is essential to the successful reading and interpreting wet preparations
The light adjustment of microscope is crucial for detection of protozoa
Protozoans are often translucent and colorless, therefore light in microscope should be reduced using the iris diaphragm to provide contrast between cellular elements in the specimen
Lowering the condenser is recommended to lower the light and allow transparent structures to be seen

Trichrome Stain

Steps involved:
1. 4 minutes in mixture of 95% ethanol and 5% acetic acid
2. 7 minutes in trichrome stain
3. 5-10 seconds in 90% acidified alcohol
4. Quick rinse in 100% ethanol
5. 5 minutes in 100% ethanol
6. 10 minutes in xylene
7. 10 minutes in xylene
Add few drops of permount and let it dry
Alternative method: let the slide air dry, and prior to examination, add one drop of oil immersion to the slide

Modified Acid Fast Staining

Used to stain bacteria that have a high lipid (long chain of mycolic acid) and wax content in their cell walls
Steps involved:
1. 5 minutes in carbol fuchsin
2. 2 minutes in 3% acid alcohol
3. 2 minutes in 0.5% acid alcohol
4. 2 minutes in distilled water
5. Counterstain with malachite green for 2 minutes
6. Dry on slide warmer at 60oC for about 5 minutes
7. Mount with coverslip using desired mounting media
Examination:
- Examine 200 to 300 fields using 40x or higher objectives
- To confirm internal morphology, use 100x oil immersion objective

Kato Katz Technique

Materials needed:
- Kato set (template with hole, scree, nylon or plastic, plastic spatula)
- Newspaper
- Microscope slides
- Cellophane soaked in glycerol-malachite green or glycerol-methylene blue solution
- Stool sample
- Applicator sticks
Reporting:
- The smear should be examined in systematic manner
- Reporting of Kato Katz technique is # of eggs per gram (EPG) of stool
- Multiply the number of eggs counted from the slide by the factor of 24

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Description

This quiz covers the step-by-step process of trichrome staining, including the use of acidified alcohol, ethanol, and xylene, and the alternative method of air drying the slide.