Smart Parasitology Lab
Easy laboratory techniques
Modified Ziehl-Neelsen (Acid-fast staining)
Aims: Given the small size of Cryptosporidium spp. oocysts, this staining technique allows for easier recognition and identification of this parasite in the copromicroscopic examination.
Materials:
- Faecal sample
- Absolute methanol (100-proof methanol)
- Ziehl's carbol fuchsin stain
- Acid ethanol 3% (3 mL of HCl in 100 mL of absolute ethanol)
- Methylene blue
- Glass microscope slides and coverslips
- Staining trays
- Mounting resin (Permount type)
Procedure:
Place a very thin smear of faeces on an alcohol-degreased glass slide and allow it to air dry. Fix the smear with methanol and let it to air dry for 2 minutes. Fully immerse the slide in Ziehl’s carbol fuchsin stain for at least 5 minutes. Gently rinse the slide with running water. Once fully rinsed, decolorize the slide by immersing it in 3% acid ethanol for a few seconds until the excess dye is removed. Rinse the slide again with running water. To contrast the smear, immerse the slide in Methylene Blue dye for 30 seconds. Rinse the slide under running water once more and let it air dry completely. Mount the resin slide and cover the smear with a coverslip.
Results:
Examine the mounted slide under an optical microscope at a high-magnification (20x to 40x objectives).
If the sample is positive, Cryptosporidium spp. oocysts will appear to have a red-pink cytoplasm and a blue-purple nuclei.
Reference Technique:
Angus, K.W. (1987) Cryptosporidiosis in domestic animals and humans. Practice, 9, pp. 47-49.