Lab 3 LUS21 Acid fast stain

ACID FAST STAIN

INTRODUCTION:
The Acid fast stain is a differential stain that detects the presence or absence of mycolic acids in the
bacterial cell wall. Mycolic acid is a waxy material that is found in the genera Mycobacterium and some
Nocardia in lesser amounts. The mycolic acid is 50% of the dry weight of the Mycobacterium cells and
is sometimes referred to as “cord factor” as it results in clumping and aggregating of the bacterial cells. It
is a virulence factor, allowing survival of Mycobacterium in phagocytes, and contributing to resistance to
disinfectants and antimicrobial therapy and may make a mycobacterial infection more difficult to treat.

The Kinyoun method is a cold acid fast stain method using a phenolic compound, carbol fuchsin as the
primary dye which is a lipid soluble, concentrated stain. This stain penetrates the waxy mycolic acid in
acid fast bacteria and is retained as a fuchsia pink complex. Mycolic acid gives lipids a higher affinity
for the primary dye, carbol fuchsin, and therefore resists decolorization with acid alcohol. Bacteria
that are not acid-fast are easily decolorized by the acid fast decolorization step and stain with the
counterstain methylene blue.

The acid fast stain is used as a presumptive test for the presence of acid fast bacteria in clinical
specimens where infections with Mycobacterium are suspected. Mycobacterium tuberculosis causes
tuberculosis, infecting about 1/3 of the earth’s human population. However, only those with the disease
are producing the bacterium and are infectious to others. Tuberculosis is a re-emerging infection. The
bacterium has gained resistance to a number of commonly used antibiotics and now is classified as multi-
drug resistant and extensively drug resistant. Mycobacterium leprae causes leprosy and is also acid fast
but is much less common than tuberculosis.

Materials, Supplies and Equipment:

Kinyoun stain kit: carbolfuchsin, acid alcohol, methylene blue
Glass slides, slide staining tray and pad, clothespin, bibulous paper, wash bottle
Incinerator,bacteriological loop,

Goggles, Gloves
Bacterial cultures
Agar plate cultures of Mycobacterium smegmatis and Staphylococcus aureus (BSL-2)

PROCEDURE:
Note: you will be watching a video on this technique. The procedure is given for
your reference.
1. Prepare a bacterial smear using aseptic technique as follows:
a. Draw a circle on the underside of a glass slide and add a drop of saline to the center of
the circle.
Lab 3 LUS21 Acid fast stain

b. Flame a loop and let it cool. Obtain a small amount of a Mycobacterium smegmatis with
the loop and spread out into the saline on the slide. Flame the loop.
c. In the same smear, mix a small amount of Staphylococcus aureus obtained with a
sterilized loop. Re-flame the loop and return it to the lab bench.
d. NOTE: the smear now contains two different bacteria.
2. Let the slide air dry or dry. Pass slide in front of incinerator to heat fix sample.
3. Place the slide on the slide staining tray and cover the smear area with carbol fuchsin. Let it
stay on the smear for at least five minutes.
4. Wash the stain off the slide with the wash water bottle.
5. Decolorize with the acid alcohol. Add the decolorizer drop by drop for 5-30 seconds until there is
no more dye being removed. NOTE: This is the critical step. You can over-decolorize the
smear if you add too much alcohol.
6. Immediately add water to the smear to stop the decolorization step.
7. Add the methylene blue counterstain and let it stand for 30 seconds.
8. Wash the counterstain off and blot the slide dry with bibulous paper.
9. Observe under oil immersion.
10. Acid fast Mycobacterium will appear fuchsia pink. They are sometimes pleomorphic, long rods
and coccoid shapes. You may see only a few clumps of acid-fast shapes in the entire slide.
11. Non-acid fast Staphylococcus will appear as light blue cocci throughout the slide.
12. False positive and false negative results may also be observed.

RESULTS

1. Read pink rods as acid fast positive. Read blue cocci as acid fast negative.
2. Draw your results in the circle provided below.
3. Note: If you have endospores or free spores, they will appear as false positive acid fast.
Lab 3 LUS21 Acid fast stain

QUESTIONS FOR DISCUSSION:

1. What is the cellular target of the acid fast stain?

The absence of mycolic acids in the bacterium cell wall.

2. What genera of bacteria is typically acid fast?

Mycobacterium and some Nocardia in lesser amounts

3. Explain how the acid fast property of the bacterium is a virulence factor?

Allowing survival of Mycobacterium in phagocytes and contributing to resistance to disinfectants and antimicrobial
therapy and may make a mycobacterial infection more difficult to treat.

4. Explain the meaning of tuberculosis as a re-emerging infection.

Tuberculosis has gained resistance to a number of commonly used antibiotics and now is classified as
multi- drug resistant and extensively drug resistant. The disease is producing the bacterium and are
infectious to others