Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
in stain for Cryptosporidium
How do you do a malachite green stain for Cryptosporidium?
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.
Concentrated carbol fuchsine
10 ml 95% ethyl
0.3 gm Basic fuchsine
6 ml Liquid
94 ml Distilled
Combine in the listed
10% Sulfuric Acid
10 ml Sulfuric acid
90 ml Distilled water
5% Malachite Green
95 ml Distilled water
5 gm Malachite
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.
Penn State University