FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

Auramine-Rhodamine Solution

Auramine 0, C.I. 41000 ---------------------------------------------------------        0.45 gm

Rhodamine B, C.I. 45170 -------------------------------------------------------        0.03 gm

Glycerine ------------------------------------------------------------------------        90.00 ml

Phenol, liquid, approximately 90% -----------------------------------------------        13.20 ml

Ethyl alcohol ---------------------------------------------------------------------        15.00 ml

Distilled water -------------------------------------------------------------------        60.00 ml

       Combine the liquids in a 250 ml flask and place on a magnetic stirrer.  Add the dyes to the solution, apply gentle heat, and allow to mix for about 30 minutes.  Filter through #4 Whatman filter paper, while warm, before use.

0.5% Acid Alcohol Solution

Alcohol, 70% ---------------------------------------------------------------------        1000.0 ml

Hydrochloric acid, concentrated -----------------------------------------------        ----        5.0 ml

0.3% Eriochrome Black T

Eriochrome black T, C.I. 14645 -------------------------------------------------        0.3 gm

Distilled water --------------------------------------------------------------------        100.0 ml

Use Positive and negative control slides.

1.        Deparaffinize and hydrate to distilled water.

2.        Place in 45 ml of auramine-rhodamine solution in a glass Coplin jar.  Place in a microwave oven and microwave at power level 1 (60W) for 3 minutes.  Agitate for about 15 seconds and allow to set in the warm solution for 2 minutes.

3.        Rinse with three changes of distilled water.

4.        Differentiate in two changes of acid alcohol, 1 minute in each change.

5.        Rinse in four changes of distilled water.

6.        Place in 0.3% eriochrome black T solution for 20 seconds.

7.        Rinse with three changes of distilled water.

8.        Stand slide on end and thoroughly air dry.

9.        Dip in xylene and mount with synthetic resin.

Acid fast bacteria, including leprosy and M. avium intracellulare, will fluoresce orange-yellow on a dark background.

Always use a positive and a negative control slide.  The negative control slide is important because tap water, and on occasion staining solutions, may contain acid fast bacilli.  Look at the slide away from the tissue section for such contaminants.  False positive staining in our laboratory was found on culture to be due to Runyon Group II scotochromogenic acid fast bacteria present in tap water.

Remember that this stain is not specific for M. tuberculosis; it will stain several types of acid fast organisms.  Also, it does not appear to be affected by the state of viability of the organisms.  Dead or dying organisms will stain.

The use of heat is necessary to obtain satisfactory staining results.  Most texts recommend staining for 10 minutes in the auramine-rhodamine solution, which has been preheated to 60˚ C.  For many years we did the staining in a 58˚ C water bath for 30 minutes with good results.  This microwave oven method saves time and produces consistently good staining results.

Usually a plastic Coplin jar is used when staining in the microwave oven.  This is because there is a tendency for glass Coplin jars to break when solutions in them are rapidly heated with microwaves.  However, when the lowest power setting is used the solution heats up slowly and there is no danger of the Coplin jar cracking.  The main reason for using a glass Coplin jar in this method is that it is easier to clean than a plastic Coplin jar.  Cleaning is accomplished with the acid alcohol solution.

Truant, J.P.:  Fluorescence microscopy of tubercle bacilli stained with auramine and rhodamine.  Henry Ford Hospital Med. Bull. 10:287-296, 1962.

Churukian, C.J.:  Demonstration of mycobacteria:  a brief review with special emphasis on fluorochrome staining.  J. Histotechnol. 14:117-121, 1991.