Modified Acid Fast Staining

Introduction

Cryptosporidium and Isospora species have been identified as causes of severe diarrhea in immunocompromised hosts, but they can also cause diarrhea in the immunocompetent host. Oocysts in clinical specimens may be difficult to identify with out special staining. Various cyanobacterium-like bodies (CLBS) have also been reported to be acid- fast. Modified acid-fast (partial acid-fast) stains are recommended for identifying these organisms.

Reagents

PRODUCT # DESCTRIPTION
PACKAGING
482K 

Partial AFB Stain Kit

4 x 8 oz. bottles

483A-8oz

Kinyoun Carbol Fuchsin

8 oz.  

483A-1gl  

1 gallon

341A-8oz Sulfuric acid, 1% v/v

8 oz.

341A-1gl

  1 gallon

675A-8oz

Methylene Blue 1% 8oz.

675A-1gl

  1 gallon  

3715A-8oz

Reagent Alcohol, 50% v/v 8oz
3715A-1gl   1 gallon  

Specimen Collection

Concentrated sediment of fresh or formalin preserved stool may be used. Other types of clinical specimens such as bile , duodenal fluid, pulmonary fluid (induced sputum, bronchial washings, biopsy specimens may also be used to stain for organisms.

Results

With this method the background will stain blue CLBs and the oocysts of Cryptosporidium sp. and Isospora sp. will stain pink to red to deep purple. All or some of the sporozoites may be visible in the Cryptosporidium oocysts. Some of the two sporocysts within the mature Isospora oocyst wall will stain pink to purple with a clear area between the stained sporocysts and the oocysts wall. The oocysts in the immature Isospora will sometimes stain completely.

Procedure

  1. Place the fixed smear on a staining rack and flood with Kinyoun carbol fuchsin for about 3 minutes.
  2. Rinse briefly with 50% v/v reagent alcohol.
  3. Rinse with tap water and decolorize for about 2 minutes with 1% v/v sulfuric acid.
  4. Rinse thoroughly with distilled water and drain.
  5. Counterstain for 0.5 minute to 1 minute with 1% w/v methylene blue.
  6. Examine under high dry magnification and verified under oil immersion. Partially acid fast organisms show reddish to purple filaments as opposed to non-acid fast organisms, which stain only blue.

Note: Staining times may vary to suit the individual.

Notes

  1. Some Nocardia spp., and acid-fast bacteria will stain positive.
  2. Smears should not be to thick. After air drying, examine under a microscope. If there are no areas of bacteria separation, more water should be added to dilute the smear.
  3. After staining it is essential that the back surface of the slide is wiped clean.
  4. If washing with distilled water is not done adequately, crystallization of the stain may appear on the slide.