Fluorescent - Auramine Rhodamine Staining For Mycobacterium

Friday, November 21, 2003

Description
The best known and distinctive property of the genus, Mycobacteria, depends upon their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. Once stained the cells resist decolorization with acidified organic solvents and are therefore called ACID FAST.

Procedure
STEP 1:

Flame slides to heat fix

STEP 2:

Flood the slide with Auramine Rhodamine stain (BBL, Difco) and allow to stain for 20 minutes.

Be sure that the stain stays on the smear.

Do NOT heat.

Do NOT use paper strips.

STEP 3:

Rinse the slide with water.

Auramine-Rhodamine is very "sticky" and should be washed off by "peeling" the stain off the slide. Aim the flow of water at the edge of the slide and slowly "peel" the stain from the slide.

STEP 4:

Flood the slide with 0.5% Acid Alcohol and allow to decolorize for 5 minutes.

Ensure that the slides are flooded thoroughly with Acid-Alcohol. Continue to add acid-alcohol until NO auramine-rhodamine stain remains visible to the naked eye.

To the right are examples of insufficiently and sufficiently flooded slides.

STEP5:

Rinse off the 0.5% Acid Alcohol with water

STEP 6:

Flood each slide with Potassium Permanganate (MSDS) and allow to quench for 1 minute.

Note: It is critical that the Potassium Permanganate remain on the slides for no longer than 2 minutes as over quenching of fluorescence can occur.

STEP 7:

Wash off the Potassium Permanganate

If all steps have been completed successfully then your slides when examined with 25X objective using a microscope that has an HBO L2 bulb heat filter, a BG 12 primary filter, and OG 1 barrier filter should look like this.

Quality Control Parameters

A positive and negative control slide should be included with each run of stains. This will verify the correct performance of the procedure as well as the staining intensity of the acid-fast organisms.

Control slides should be reviewed before patient smears are read to confirm that the Mycobacteria stain acid-fast. If the results of the QC slides are acceptable, go on to the patient smears. If, however, the control slide(s) are unacceptable, review procedures and reagent preparations. When the problem has been identified and corrected, remake and stain all of the patient's slides from the problem run along with a new set of controls.

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Fluorescent - Auramine Rhodamine Staining For Mycobacterium