Cold Kinyoun Method- Modified AFB Staining Procedure

Principle:
Cryptosporidium and Isospora species have been recognized to cause severe diarrhea in immunocompromised hosts, but it can also cause diarrhea in immunocompetent host. Modified acid-fast stains are recommended for demonstrating these organisms.Unlike the Ziehl-Neelsen modified acid-fast stain, the modified Kinyoun acid-fast stain does not require heating the reagents used for staining.

Procedure:

• Heat fix the slide. 
• Flood slide with Carbol fuchsin stain for 4 minutes. 
• Wash with H2O. 
• Decolorize using 1% aqueous sulfuric acid until all pink color disappears. 
• Washe again with H2O. 
• Counterstain with Methylene blue for 3 minutes. 
• Washe with H2O. 
• Air dry and examine the slide. 

Interpretation:

• With this cold Kinyoun acid-fast method, C. cayetanensis and the oocysts of sporozoites may be visible in the Cryptosporidium oocysts. Some of the Isospora immature oocysts (entire oocyst) will stain, while in oocysts that are mature, the two sporocysts within the oocyst wall will usually stain pink to purple and there will be a clear area between the stained sporocysts and the oocyst wall. The background will stain blue or green, depending on the counterstain used. 
• If Cyclospora oocysts are present (uncommon), they tend to be approximately 10 µm, they resemble C. parvum but are larger, and they have no definite internal morphology; the acid-fast staining will tend to be more variable than that seen with Cryptosporidium or Isospora spp. Modified acid-fast stains stain the Cyclospora oocysts from light pink to deep red, some of which will contain granules or have a bubbly appearance, often being described as looking like “wrinkled cellophane.” Even with the 1% acid decolorizer, some oocysts of Cyclospora may appear clear or very pale. If the patient has a heavy infection with microsporidia (immunocompromised patient), small (1 to 2 µm) spores may be seen but may not be recognized as anything other than bacteria or small yeast cells.