SOP 6: Acid Fast Stain for Cryptosporidium oocysts

As the oocysts of Cryptosporidium spp. are very small and difficult to detect by inexperienced examiners, this method provides specific staining and allows an easier detection.

Reagents

  • Absolute methanol
  • Kinyoun’s carbol fuchsin
  • 10% sulfuric acid solution (H2SO4)
  • 3% Malachite green

Procedure

  1. Make a thin faecal smear and allow to air dry
  2. Fix with absolute methanol for 10 min and allow smear to dry
  3. Stain with cold Kinyoun’s carbol fuchsin strong stain (filtered) for 5 min
  4. Wash thoroughly in tap water until no further stain comes out (very important step that can take 3-5 min)
  5. Decolourise in 10% H2SO4 (for very thin smears a rapid dip in Coplin jar of acid followed by an immediate rinse in tap water is sufficient)
  6. Counterstain with 3% Malachite green for 2-5 min
  7. Wash in tap water and blot dry
  8. Examine under a light microscope at high power (40x) for oocysts

Results

  • Oocysts are seen as acid fast (bright pink) oval to round bodies (4 to 6 mm in diameter), surrounded by a colourless halo. Bacteria and yeasts stain green.

Safety precautions

  • Wear lab coat and disposable gloves
  • Wash hands thoroughly when finished

Clean up procedures

  • Dispose of all disposable equipment in clinical waste bin or sharps as appropriate