Trichrome Staining Procedure

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