Modified Trichrome

Modified trichrome stains are variations of the standard trichrome stain that are used to enhance the visualization of specific microorganisms, particularly microsporidia. Microsporidia are obligate intracellular parasites that can cause a range of infections, especially in immunocompromised individuals. Because they are small and often difficult to see with standard staining methods, modified trichrome stains have been developed to improve their detection

Principle of Modified Trichrome Staining

• Enhanced Visualization of Microsporidia: The primary goal of modified trichrome stains is to improve the detection and visualization of microsporidia spores in clinical specimens, such as stool, urine, and respiratory secretions
• Dye Composition: Modified trichrome stains typically contain a mixture of dyes, including chromotrope 2R, light green SF, and phosphotungstic acid, similar to the standard trichrome stain. However, the concentrations of the dyes and the staining procedure are optimized to enhance the staining of microsporidia spores
• Mechanism
  1. Fixation: The staining process typically begins with fixation, either with formalin or another suitable fixative, to preserve the morphology of the spores
  2. Dye Penetration: The dyes in the modified trichrome stain penetrate the spores and bind to different cellular components based on their chemical properties
  3. Color Differentiation: The dyes stain different structures within the spores and background material in distinct colors:
     • Microsporidia spores: Typically stain pinkish-red to red-purple, with a distinct polar filament (a characteristic structure of microsporidia)
     • Background material: Stains green or blue-green

Procedure (Weber’s Modified Trichrome Stain)

• This is one of the most commonly used modified trichrome stains for microsporidia

1. Smear Preparation: Prepare a thin smear of the specimen (e.g., stool concentrate, duodenal aspirate) on a clean microscope slide. Allow the smear to air dry completely
2. Fixation: Fix the smear in 10% formalin for 10 minutes
3. Ethanol Wash: Wash the smear in 70% ethanol for 5 minutes
4. Staining: Flood the smear with Weber’s modified trichrome stain and heat at 50-60°C for 10 minutes, or leave at room temperature for 60 minutes
5. Acetic Acid Rinse: Briefly rinse the smear in 1% acetic acid for a few seconds to remove excess stain and enhance color differentiation
6. Dehydration: Dehydrate the smear in a series of graded alcohols (95% and 100%) for 2 minutes each
7. Clearing: Clear the smear in xylene or xylene substitute for 2 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 (100x oil immersion) to identify microsporidia spores

Interpretation

• Microsporidia Spores: Look for small, oval-shaped spores that stain pinkish-red to red-purple. A key feature to look for is the polar filament, which appears as a diagonal or coiled line within the spore. The polar filament is a characteristic structure of microsporidia and helps to differentiate them from other organisms or artifacts
• Background Material: The background material typically stains green or blue-green, providing contrast to the microsporidia spores
• Artifacts: Be aware of potential artifacts, such as yeast cells, bacteria, and debris, that can resemble microsporidia spores. Use higher magnification and careful focusing to confirm the presence of true spores and to identify the polar filament
• Reporting: Report the presence or absence of microsporidia spores and identify them to the genus level whenever possible. Describe the morphology of the spores observed, including the presence and appearance of the polar filament. Estimate the quantity of spores observed (e.g., rare, few, moderate, many)

Common Problems and Troubleshooting

• Poor Staining: Poor staining can result from inadequate fixation, old or contaminated reagents, improper staining time, or incorrect temperature. Ensure that all reagents are fresh, the staining procedure is followed carefully, and the temperature is controlled
• Artifacts: Artifacts can be difficult to distinguish from microsporidia spores. Use higher magnification and careful focusing to confirm the presence of true spores and to identify the polar filament. Consult reference materials and experienced colleagues for assistance
• Safety: Xylene is a hazardous chemical and should be used in a well-ventilated area or under a fume hood

Advantages

• Enhances the visualization of microsporidia spores, making them easier to detect and identify
• Relatively simple and inexpensive to perform
• Can be used to stain a variety of clinical specimens

Disadvantages

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

Key Terms

• Modified Trichrome Stain: A variation of the standard trichrome stain used to enhance the visualization of specific microorganisms, particularly microsporidia
• Microsporidia: Obligate intracellular parasites that can cause a range of infections, especially in immunocompromised individuals
• Spore: The infective form of microsporidia
• Polar Filament: A characteristic structure of microsporidia spores, appearing as a diagonal or coiled line within the spore
• Weber’s Modified Trichrome Stain: A commonly used modified trichrome stain for microsporidia
• Artifact: A structure or substance that is not part of the original specimen but may resemble a microorganism under the microscope
• Formalin: A fixative solution used to preserve the morphology of cells and microorganisms in clinical specimens
• Chromotrope 2R, Light Green SF, and Phosphotungstic Acid: Dyes used to stain cellular components in trichrome staining