1. Improper decontamination and concentration procedure will yield erroneous results.

2. Treat the specimens and used slants by immersing in

2% activated glutaraldehyde for at least 2 hours before

incineration and disposal.

3. Good laboratory practices and hazard precautions

must be observed at all times.

4. Observe stain timings, which are essential to obtain

correct staining results.

5. Understain or over decolorization may give false

results.

6. The stains stored between 25 and 30o

C should not show

precipitation.

7. Artefacts could be mistaken for Acid Fast Bacilli.

The fluorochrome based stains for AFB comprise of

Auramine O, sometimes used in combination with a

second fluorochrome stain, Rhodamine.

Smears stained with Auramine O can be scanned using a

25 × objective. Fluorochrome-stained Mycobacteria appear

bright yellow against a dark background obtained by

counterstaining with potassium permanganate, thereby

permitting the slide to be scanned under the lower

magnification without losing sensitivity. The sharp visual

contrast between the bright colored mycobacteria and the

dark background offers a distinct advantage in scanning

a much larger area of the slide during the same time

necessary for looking at the carbol fuchsin stain.

When using the Auramine stain, a significantly larger

area of the smear can be scanned in the same period of

time used to scan a carbol fuschin-stained smear.

Enthusiasm for the carbol fuchsin and fluorochrome

staining methods varies between laboratories, with

different professionals strongly partial to one method or

the other. Specificity for mycobacteria seems to be the

same for both.

The crucial factors in maximizing smear sensitivity and

specificity are:

¾ Centrifugation of digested fluid specimen at a minimum

of 3000 g

¾ The smear should be prepared on a new clean

undamaged glass slide

¾ Scanning of at least 30 fields per slide

¾ The reporting of the AFB smear should be preferably

done according to the CDC, USA method, or as per the

National Reference Institution norms.