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Microbiology

Trichrome

Trichrome stains are essential in the clinical microbiology lab, particularly in parasitology. These stains allow us to visualize and identify intestinal parasites in stool specimens

Principle of Trichrome Staining

  • Differential Staining: Trichrome staining is a differential staining technique that uses multiple dyes to stain different cellular components of parasites and background material in stool specimens. This allows for clear visualization and identification of parasites based on their color and morphology
  • Mechanism
    1. Fixation: The staining process typically begins with fixation, either with Schaudinn’s fixative or a similar solution, to preserve the morphology of the parasites
    2. Dye Penetration: The trichrome stain contains a mixture of dyes, including chromotrope 2R, light green SF, and phosphotungstic acid. These dyes penetrate the cells and bind to different cellular components based on their chemical properties
    3. Color Differentiation: The dyes stain different structures within the parasites and background material in distinct colors:
      • Protozoan cytoplasm: Typically stains blue-green or purple-pink
      • Nuclear structures (nuclei, karyosomes): Stain red or purple-red
      • Background material: Stains green

Procedure

  1. Smear Preparation: Prepare a thin smear of the stool specimen on a clean microscope slide. Allow the smear to air dry completely
  2. Fixation: Fix the smear by placing it in Schaudinn’s fixative for 30-60 minutes (or according to the manufacturer’s instructions). Schaudinn’s fixative typically contains mercuric chloride, which is toxic and must be handled with care. Some labs use non-mercury fixatives
  3. Ethanol Wash: Rinse the fixed smear in 70% ethanol containing iodine for 5-10 minutes to remove excess mercuric chloride. Then, rinse in 70% ethanol for another 5-10 minutes to remove the iodine
  4. Staining: Stain the smear with trichrome stain for 10-15 minutes (or according to the manufacturer’s instructions). Ensure the stain covers the entire smear
  5. Acetic Acid Rinse: Briefly rinse the smear in 90% ethanol containing 1% acetic acid for a few seconds to remove excess stain and enhance color differentiation. This is a critical step to sharpen the colors
  6. Dehydration: Dehydrate the smear by passing it through a series of graded alcohols (95% and 100%) for 2-3 minutes each
  7. Clearing: Clear the smear in xylene or xylene substitute for 2-3 minutes
  8. Mounting: Mount the smear with a coverslip using a permanent mounting medium
  9. Microscopy: Examine the slide under a microscope, starting with low power (10x) to locate areas of interest and then increasing to higher power (40x and 100x oil immersion) to identify parasites

Interpretation

  • Protozoa: Identify protozoa based on their size, shape, and staining characteristics. Look for key features such as nuclei, karyosomes, cytoplasm, and inclusions
    • Entamoeba histolytica/dispar: Look for trophozoites (irregular shape, single nucleus with a central karyosome) and cysts (round shape, 1-4 nuclei with central karyosomes)
    • Giardia lamblia: Look for trophozoites (pear shape, two nuclei, prominent flagella) and cysts (oval shape, 2-4 nuclei)
    • Cryptosporidium parvum: Look for small, round oocysts that stain pink to red
  • Helminth Eggs and Larvae: Identify helminth eggs and larvae based on their size, shape, and internal structures
    • Ascaris lumbricoides: Look for large, oval eggs with a thick, mamillated shell
    • Hookworm: Look for oval eggs with a thin shell and developing larvae inside
  • Artifacts: Be aware of potential artifacts, such as yeast cells, pollen grains, and fecal debris, that can resemble parasites. Use higher magnification and careful focusing to confirm the presence of true parasitic structures
  • Reporting: Report the presence or absence of parasites and identify them to the species level whenever possible. Describe the morphology of the parasites observed and estimate their quantity (e.g., rare, few, moderate, many)

Common Problems and Troubleshooting

  • Poor Staining: Poor staining can result from inadequate fixation, old or contaminated reagents, or improper timing. Ensure that all reagents are fresh and that the staining procedure is followed carefully
  • Over-decolorization: Over-decolorization can cause parasites to appear pale or washed out. Reduce the rinsing time in the acetic acid solution
  • Artifacts: Artifacts can be difficult to distinguish from parasites. Use higher magnification and careful focusing to confirm the presence of true parasitic structures. Consult reference materials and experienced colleagues for assistance
  • Safety: Schaudinn’s fixative contains mercuric chloride, which is toxic. Handle with care and dispose of properly. Use a fume hood when working with xylene

Advantages

  • Allows for clear visualization and identification of intestinal parasites
  • Relatively simple and inexpensive to perform
  • Can be used to stain both protozoa and helminth eggs and larvae

Disadvantages

  • Requires experience to differentiate parasites from artifacts
  • The use of toxic chemicals (e.g., mercuric chloride, xylene) requires careful handling and disposal
  • Can be time-consuming to perform

Modified Trichrome Stain

  • There are some modified trichrome stains that are used for other specimens, such as modified trichrome for microsporidia

Key Terms

  • Trichrome Stain: A differential staining technique used to visualize and identify intestinal parasites in stool specimens
  • Schaudinn’s Fixative: A fixative solution containing mercuric chloride, used to preserve the morphology of parasites in stool specimens
  • Trophozoite: The active, motile feeding stage of a protozoan parasite
  • Cyst: The dormant, non-motile stage of a protozoan parasite
  • Oocyst: The environmentally resistant, infective stage of certain protozoan parasites, such as Cryptosporidium
  • Helminth: A parasitic worm, such as a roundworm, tapeworm, or fluke
  • Artifact: A structure or substance that is not part of the original specimen but may resemble a parasite under the microscope
  • Karyosome: The mass of chromatin within the nucleus of a cell, and a key feature in parasite identification
  • Chromotrope 2R, Light Green SF, and Phosphotungstic Acid: Dyes used to stain cellular components in trichrome staining