Modified Kinyoun Acid-Fast Stain

Specimen requirements: unfixed stool or stool preserved in 10% formalin (including FEA concentrates), SAF, or EcoFix. Procedure Prepare a thin film of stool on a glass microscope slide. Allow to completely air dry (do not use heat blocks to speed up drying). Fix in absolute methanol for 30 seconds. Stain with carbol-fuschin for 1 minute. Rinse and drain with distilled water. Decolorize in 10% sulfuric acid (10 mL sulfuric acid in 90 mL absolute ethanol) for 2 minutes. Rinse quickly and drain with distilled water. Counterstain with 3% malachite green (3 g malachite green in 100 mL distilled water) or methylene blue for 2 minutes. Allow to dry (air dry or on a slide warmer at 60°C). Mount with no. 1 thickness coverslip using Permount (or other sealing reagent). Procedural notes Do not use a slide warmer for the first step (drying of specimen); it cooks the oocysts of Cryptosporidium, and they do not retain stain. Drain slides between each reagent. All reagents should be changed monthly or earlier as needed.

Hot Safranin Stain

Specimen requirements: unfixed stool or stool preserved in 10% formalin (including FEA concentrates), SAF, or EcoFix. Procedure Prepare a thin film of stool on a glass microscope slide. Allow to completely air dry (do not use heat blocks to speed up drying). Fix in acid alcohol (3 mL hydrochloric acid in 97 mL absolute methanol) for 5 minutes. Boil in safranin (1% aqueous solution in distilled water) for 1 minute. Counterstain with 3% malachite green (3 g malachite green in 100 mL distilled water) for 1 minute. Allow to dry (air dry or on a slide warmer at 60°C). Mount with no. 1 thickness coverslip using Permount (or other sealing reagent). Procedural notes Do not use a slide warmer for the first step (drying of specimen); it cooks the oocysts of Cryptosporidium, and they do not retain stain. Drain slides between each reagent. All reagents should be changed monthly or earlier as needed. Safranin stain should be boiling when slides are added.