Histology FAQ
Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
London, Canada
Malachite green
in stain for Cryptosporidium
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)