THE ACID-FAST STAIN

Robert Koch was the first person to isolate and identify Mycobacterium tuberculosis
from a patient with tuberculosis. He developed a stain for the bacterium, although it was
not very effective for visualizing this slender bacillus. Paul Ehrlich is the first to
describe the acid-fast properties of the bacterium. In the 1890s, Friedrich Neelsen and
Franz Ziehl modified the stain by adding phenol (carbolic acid) and basic fuschin. The
name of the dye carbol fuschin comes from the phenol and basic fuschin ingredients of
the stain.

The ability of the bacteria to resist decolorization with ACID alcohol confers acid
fastness to the bacterium. Acid-fast bacteria, of which there are very few---the major
genus Mycobacterium, have a high concentration of mycolic acid, a lipid, in their walls.
Although difficult to stain, once the stain goes into the wall, the cell will not de-stain or
decolorize easily. The ability of the bacteria to resist decolorization with acid (1%)
alcohol confers acid -fastness to the bacterium. It is thought that the phenol in the
carbon fuschin facilitates the dye going into the waxy wall of the bacterium. Although
gram positive, acid-fast bacteria do not take the crystal violet into the wall well,
appearing very light purple rather than the deep purple of normal gram positive bacteria
(the waxy lipid in the acid-fast wall repels the aqueous crystal violet stain).

As in the spore stain, steam is used as a gimmick to get the carbol fuschin primary dye
to go into the wall. Once in, it will not come out: But the acid alcohol decolorizer WILL
take it out of the nonacid-fast wall since the primary dye does not bind strongly to the
cell wall. Nonacid-fast bacteria will also take up the carbol fuschin, but the acid alcohol
decolorizer will remove it from wall since the primary dye does not bind strongly to the
cell wall.

Acid-fastness is an uncommon characteristic

shared by the genera Mycobacterium and
Nocardia (weakly acid-fast). Because of this
feature, this stain is extremely helpful in
identification in diseases caused by acid-fast
bacteria, particularly tuberculosis and leprosy.
In addition, the stain is used to determine the
presence of AF bacteria from lung tissue in
patients undergoing antibiotic therapy.

Acid-fast bacteria
 Nonacid-fast bacteria

OBJECTIVES:

Learn to perform the acid-fast stain.

Differentiate between acid-fast and nonacid-fast bacteria.

MATERIALS NEEDED:

dye kit
stain rack
hot plate and beaker
paper towel (cut the size of the slide)
cultures: Mycobacterium and E. coli

THE PROCEDURE (Ziehl-Neelsen method):

1. Prepare the bacterial smears---air-dry, and heat-fix. (or the Mycobacterium

smear may be given to you already air-dried on a slide.)
2. Put a beaker of water on the hot plate and boil until steam is coming up from the
water. Then turn the hot plate down so that the water is barely boiling.
3. Place the wire stain rack over the beaker which now has steam coming up from
the boiled water.
4. Cut a small piece of paper towel and place it on top of the smear on the
slide. The towel will keep the dye from evaporating too quickly, thereby giving
more contact time between the dye and the bacterial walls.
5. Flood the smear with the primary dye, carbol fuschin, and leave for 5 minutes.
Keep the paper towel moist with the carbol fuschin. DO NOT let the dye dry on
the towel.
6. Remove the piece of paper towel and discard in the garbage. Wash the slide
really WELL with water.
7. Add the decolorizer acid alcohol (1% HCl + ethanol) and decolorize for 15-20
seconds. (The slide can be held, titled at an angle, and decolorizer dripped
down the slide.)
 8. Wash WELL with water.
9. Flood the smear with the counterstain dye, methylene blue, and leave for 1
minute.
10. Rinse well with water. Blot dry with bibulous paper.

ALTERNATE COLD METHOD ACID FAST STAIN (Kinyoun)

The only difference is that without heat you have to stain the
smear with carbol fuschin for 10 minutes.

INTERPRETATION:

Acid-fast bacteria are hot pink or fuschia. Nonacid-fast bacteria are light blue.

QUESTIONS:

1. What is chemically unique about the Mycobacterium genus that causes it to be

acid-fast?

2. How is this stain procedure similar to the spore stain procedure?

Fall 2011 - Jackie Reynolds, Richland College, BIOL 2421