E XP ERIMENT
11
Acid-Fast Stain
LEARNING OBJECTIVES
Once you have completed this experiment,
you should be able to
1. Explain the chemical basis of the acid-fast
stain.
2. Perform the procedure to differentiate
bacteria into acid-fast and non–acid-fast
groups.
Principle
While the majority of bacterial organisms are stainable by either simple or Gram staining procedures,
a few genera, particularly the members of the genus
Mycobacterium, are visualized more clearly by the
acid-fast method. Since Mycobacterium tuberculosis and Mycobacterium leprae represent bacteria
that are pathogenic to humans, the stain is of diagnostic value in identifying these organisms.
The characteristic difference between mycobacteria and other microorganisms is the presence of a
thick, waxy (lipoidal) wall that makes penetration
by stains extremely difficult. Mycobacteria tend to
clump together, and it is difficult to identify individual cells in stained preparations if this clumping
effect occurs. Avoiding or minimizing this phenomenon requires careful preparation of the smear. Place
a small drop of water on the slide, suspend the culture in the water, and mix the suspension thoroughly
to dislodge and disperse some of the cells. Once the
stain has penetrated, however, it cannot be readily
removed even with the vigorous use of acid-alcohol
as a decolorizing agent (unlike the 95% ethyl alcohol
used in the Gram stain). Because of this property,
these organisms are called acid-fast, while all other
microorganisms, which are easily decolorized by
acid-alcohol, are non–acid-fast. The acid-fast stain
uses the three different reagents listed below along
with a description of their purpose.
Primary Stain
Carbol fuchsin: Unlike cells that are easily stained
by ordinary aqueous stains, most species of
mycobacteria are not stainable with common
dyes such as methylene blue and crystal violet.
Carbol fuchsin, a dark red stain in 5% phenol that
is soluble in the lipoidal materials that constitute
most of the mycobacterial cell wall, does penetrate
these bacteria, and is retained. Applying heat
enhances penetration further, which drives the
carbol fuchsin through the lipoidal wall and into the
cytoplasm. This application of heat is used in the
Ziehl-Neelsen method. The Kinyoun method, a
modification of the Ziehl-Neelsen method, circumvents the use of heat by adding a wetting agent
(Tergitol®), which reduces surface tension between
the cell wall of the mycobacteria and the stain.
Following application of the primary stain, all cells
appear red.
Decolorizing Agent
Acid-alcohol (3% HCl + 95% ethanol): Prior
to decolorization, the smear is cooled, which
allows the waxy cell substances to harden. On
application of acid-alcohol, acid-fast cells are
resistant to decolorization, since the primary stain
is more soluble in the cellular waxes than in the
decolorizing agent. In this event, the primary stain
is retained and the mycobacteria will stay red.
This is not the case with non–acid-fast organisms,
which lack cellular waxes. The primary stain is
more easily removed during decolorization,
leaving these cells colorless or unstained.
Counterstain
Methylene blue: This is used as the final reagent
to stain previously decolorized cells. As only non–
acid-fast cells undergo decolorization, they may
now absorb the counterstain and take on its blue
color, while acid-fast cells retain the red of the
primary stain.
F U RT H E R RE A DI N G
Refer to the section in your textbook discussing
bacterial stains for further information on when
to use the acid-fast stain instead of the widely
used Gram stain. In your textbook’s index, search
under “Acid-Fast Stain,” “Mycolic Acid,” and “Soil
Microbes.”
79
PROCEDURE
or
1a Heat method: Apply carbol fuchsin and steam over a beaker of
boiling water that is placed on a hot plate for 5 minutes. Do not
allow the stain to evaporate.
2 Cool and wash off stain with
tap water.
3 Add acid-alcohol drop by drop
until the alcohol runs almost clear.
4 Wash off the acid-alcohol with
tap water.
5 Counterstain with methylene blue
for 2 minutes.
6 Wash off the methylene blue with
tap water.
7 Blot the slide dry with bibulous
paper.
Figure 11.1 Acid-fast staining procedure
80
1b Heatless method: Apply carbol fuchsin
with Tergitol for 5 to 10 minutes.
Experiment 11
C L I N I C A L A P P L I C AT I O N
Diagnosing Leprosy and Lung Infections
The cell walls of bacteria belonging to the genera
Mycobacterium and Nocardia contain mycolic acid
and are resistant to penetration by water-soluble
stains such as the Gram stain, which can lead to a
false gram-positive result. Acid-fast stains are medically important in diagnosing the Mycobacterium
species, which cause tuberculosis, leprosy, and
other infections. The genus Nocardia, which is the
causative agent for lung infections, is also identified
by the acid-fast staining method.
AT THE B EN C H
Materials
Cultures
❏ 72- to 96-hour Trypticase soy broth culture of
Mycobacterium smegmatis
❏ 18- to 24-hour culture of Staphylococcus
aureus BSL -2
Reagents
❏ Carbol fuchsin
❏ Acid-alcohol
❏ Methylene blue
Equipment
❏ Microincinerator or
Bunsen burner
❏ Hot plate
❏ 250-ml beaker
❏ Inoculating loop
❏
❏
❏
❏
❏
Glass slides
Bibulous paper
Lens paper
Staining tray
Microscope
Acid-Fast Staining
Figure 11.1 shows steps 1–7.
1. a. Flood smears with carbol fuchsin and
place over a beaker of water on a warm hot
plate, allowing the preparation to steam
for 5_minutes. Note: Do not allow stain to
evaporate; replenish stain as needed. Also,
prevent stain from boiling by adjusting
the hot-plate temperature.
b. For a heatless method, flood the smear with
carbol fuchsin containing Tergitol for 5 to
10 minutes.
2. Wash with tap water. Heated slides must be
cooled prior to washing.
3. Decolorize with acid-alcohol, adding the
reagent drop by drop until the alcohol runs
almost clear with a slight red tinge.
4. Wash with tap water.
5. Counterstain with methylene blue for
2 minutes.
6. Wash the smear with tap water.
7. Blot dry with bibulous paper and examine
under oil immersion.
8. In the chart provided in the Lab Report,
complete the following:
a. Draw a representative microscopic field for
each preparation.
b. Describe the cells according to their shapes
and arrangements.
c. Describe the color of the stained cells.
d. Classify the organisms as to reaction: acidfast or non–acid-fast.
Refer to Figure 11.2 for a photograph of an acidfast stain.
Procedure
Smear Preparation
1. Obtain three clean glass slides.
2. Using aseptic technique, prepare a bacterial
smear of each organism plus a third mixed
smear of M. smegmatis and S. aureus BSL -2 .
3. Allow smears to air-dry and then heat fix in the
usual manner.
Figure 11.2 Acid-fast stain of mycobacteria
Experiment 11
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EXPERIMENT
11
Name:
Date:
Lab Report
Section:
Observations and Results
E. coli
B. cereus
S. aureus
Mixture
Draw a
repre entati e eld.
Cell morphology:
Shape
Arrangement
Cell color
Gram reaction
Review Questions
1. Why must you use heat or a surface-active agent when applying the primary stain during acid-fast
staining?
2. Why do you use acid-alcohol rather than ethyl alcohol as a decolorizing agent?
Experiment 11: Lab Report
83
3. What is the specific diagnostic value of this staining procedure?
4.
5.
84
Why is the application of heat or a surface-active agent not required during the application
of the counterstain in acid-fast staining?
A child presents symptoms suggestive of tuberculosis, namely a respiratory infection with a
productive cough. Microscopic examination of the child’s sputum reveals no acid-fast rods.
However, examination of gastric washings reveals the presence of both acid-fast and non–acid-fast
bacilli. Do you think the child has active tuberculosis? Explain.
Experiment 11: Lab Report