Question.

How do you do a malachite green stain for Cryptosporidium?

Answer.

The Cryptosporidia are stained by carbol fuchsine; malachite green is a counterstain for the background.

This is the procedure I use. (I also do the parasitology here.)  It works fairly well but is not the best diagnostic technique for Cyrptosporidia. There are Meriflour commercial kits that are better than this stain.

A MODIFIED ZIEHL-NEELSEN TECHNIQUE FOR CRYPTOSPORIDIUM

This is used on fecal smears.

Solutions.

  Concentrated carbol fuchsine
         10 ml     95% ethyl alcohol
         0.3 gm   Basic fuchsine
         6 ml       Liquid Phenol
         94 ml     Distilled water
         Combine in the listed order.

  10% Sulfuric Acid
        10 ml    Sulfuric acid
        90 ml    Distilled water

  5% Malachite Green
       95 ml     Distilled water
       5 gm     Malachite green

Procedure.

    1. Make a thin smear from the fecal sample.
    2. Dry the smear at room temperature.
    3. Fix the smear in absolute methanol for 2-5 minutes.
    4. Dry at room temperature
    5. Fix briefly in a flame.
    6. Stain with concentrated carbol fuchsine for 20-30 minutes without heating.
    7. Rinse in tap water.
    8. Differentiate with 10% sulfuric acid for 20-60 seconds.
       (Concentrations from 0.25 to 10% can be used; we use 10% sulfuric acid.)
    9. Rinse in tap water.
   10. Counterstain with 5% malachite green for 5 minutes.
   11. Rinse in tap water.
   12. Dry at room temperature.
   13. Examine under oil.
   14. Cryptosporidia will stain bright red with a blue-green background.

Roberta Horner
Penn State University
(rjr6[AT]psu.edu)