SOP 6: Acid Fast Stain for Cryptosporidium oocysts

Method

  • Make a thin faecal smear and allow to air dry
  • Fix in methanol for 10 min and allow smear to dry
  • Stain with cold kinyoun carbol fuchin strong stain (filtered) for 5 min
  • Wash thoroughly in tap water until no further stain comes out (very important step that can take 3 to 5 min)
  • Decolourise in 10% H2SO4 [For very thin smears a rapid dip in koplin jar of acid followed by an immediate rinse in tap water is sufficient]
  • Counterstain with Malachite Green for 2 to 5 min
  • Wash in tap water and blot dry
  • Examine under x40

Results

  • Oocysts  acid fast (bright pink) oval to round bodies 4 to 6 mm in diameter                                   surrounded by a colourless halo
  • Yeasts red and white cells
  • Bacteria  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