Cat. no. AF900 With Methylene Blue Counterstain
Cat. no. 483KB With Brilliant Green Counterstain


The Acid-Fast Stain Kit is used as a differential method to detect the difference between those organisms which decolorize with treatment of acid alcohol, and those that do not, such as the mycobacteria.


Based on the Kinyoun modified staining technique, the acid-fast stain is a very useful tool for differentiating acid-fast bacteria (AFB), such as Mycobacterium spp., from those that do not resist the acidified decolorization step. The mechanism for acid-fastness is not clearly understood, however it is suspected that it is determined by selective permeability of the cytoplasmic membrane. The brilliance of red coloration is due to retention of the carbol fuchsin dye within the cell membrane. Should the cell be mechanically disrupted, its acid-fast property is lost.

After staining, decolorizing, and counterstaining, any acid-fast bacteria present are brilliant pink to red in color, and other bacteria present are seen as blue (methylene blue counterstain) or green (brilliant green counterstain). No heating or time dependent decolorization steps are required.


Carbol Fuchsin Stain Caustic, Poison
Basic Fuchsin 569-61-9
Phenol, Liquid 108-95-2
Ethyl Alcohol 64-17-5
Penetrating Agent (as needed)
Deionized Water
Acid Alcohol Decolorizer Flammable, Poison
Hydrochloric Acid 7647-01-0
Ethyl Alcohol 64-17-5
Methylene Blue Counterstain
Methylene Blue 7220-79-3
Deionized Water
Brilliant Green (optional counterstain)
Brilliant Green 633-03-4
Deionized Water


Storage: Upon receipt store at 2-30ºC. Products should not be used if there are any signs of deterioration or if the expiration date has passed.

After prolonged storage, phenol may be seen to separate from the carbol fuchsin stain. Shake the tightly capped bottle well to remix before use.



Specimen Collection: Consult listed references for information on specimen collection.(2-4)

Specimen Preparation, Sputum: Using a portion of blood, caseous, or purulent material, spread a loop full of the specimen over a small area of clean, dry glass slide in a thin smear. When dry, heat fix and proceed with staining.

Urine and Gastric Samples: To concentrate organism, centrifuge 10-50ml of the specimen. Follow smear preparation instructions as stated above using the resulting sediment.

Histology Sections: Prepare the sample following published techniques.

Staining Procedure:

Note: All times are approximate. The user may wish to adjust staining times as appropriate to achieve desired results.

1. Flood the heat fixed smear with the carbol fuchsin stain for 2-3 minutes. No heat is required.

2. Rinse slide with water.

3. Using the acid alcohol decolorizer, direct a gentle stream on the smear. When no more color is seen coming off, stop decolorizing.

4. Rinse with water again.

5. Flood the smear with the methylene blue (or brilliant green, if preferred) counterstain for 2 minutes.

6. Rinse with water, and allow slide to dry or blot gently with absorbent paper.

Histology Sections:

1. In the normal manner, section the paraffin tissue blocks.

2. Process the sections through deparaffinization.

3. Rehydrate to water.

4. Stain slides individually as described within the staining procedure above, with the exception of adding occasional gentle agitation to the staining steps.

5. Finish processing the slides for permanent mounting as follows: Acetone for 10 seconds, acetone:xylene (1:1) for 1 minute, xylene for 2 minutes, and mount the specimen.


The appearance of bright red stained bodies that are slender, slightly curved, long or short rods, and sometimes granular or beaded are typically acid-fast bacteria (AFB). Some atypical forms appear thick or diptheriod, sometimes coccoid, or are very long. Other non-acid-fast organisms will stain blue (or green when using brilliant green counterstain), as will background material.


If the cell walls of the bacteria in the stained sample are disrupted, the Acid-Fast Stain will yield false-negatives. Therefore, care must be taken to avoid this type of damage. In stained preparations, observation of acid-fast bacilli is only presumptive evidence for the presence of Mycobacterium organisms. Cultures for Mycobacterium should also be carried out, in conjunction with other biochemical tests to identify the organism. Tap water used for rinsing slides may contain mycobacteria, which could produce false-positives.


Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


User Quality Control

It is recommended that each new lot or shipment of reagent be tested with known positive and negative controls and retested each day of use thereafter.(1)

Physical Appearance

Acid-Fast Stain Kit

Acid-Fast Stain Kit with Methylene Blue Counterstain (Cat. no. AF900)


Acid-Fast Stain Kit

Cat. no. AF900 contains 8oz. of each (with dropper caps):

Cat. no. 483KB contains 8oz. of each (with dropper caps):

Also available as individual components as follows:

Component, 8oz. Cat. no.
Carbol Fuchsin CF008
Acid Alcohol AA008
Methylene Blue MB008
Brilliant Green BG008


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.