2.
06 Laboratory: Acid Fast Staining
September 27, 2017
MICROBIOLOGY
OUTLINE
I. Acid-fast Stain
A. Ziehl-Neelsen Method
B. Kinyoun’s Method
C. Results
II. Guide Questions
LEGEND
Remember Textbook Trans Editor Previous Trans
   
ACID-FAST STAIN
 Certain organisms, i.e. members of the genus
Mycobacterium and Nocardia are acid-fast
 These organisms possess a high content of lipid-like waxes
called mycolic acids
o These lipids make the cells very resistant to
penetration by ordinary stains or dyes
 Carbol fuchsin is used to stain these bacteria by steaming.
 Carbol fuchsin − basic fuchsin dissolved in a
phenol-alcohol-water mixture
o Steaming allows dye to penetrate and combine
strongly with any mycolic acids in the cells
 Once combined with the mycolic acids, the dye is bound
lightly and the cell resists decolorization
o Decolorizing agent: acidified solution of alcohol
(hydrochloric or sulfuric acid in alcohol)
 Potent decolorizer that easily removes
carbol fuchsin from non-acid fast
organisms but not from acid-fast ones
 After decolorization:
o ACID-FAST cells are RED
o NON-ACID-FAST cells are COLORLESS
 To visualize non-acid fast cells, methylene blue is used as
counterstain.
 After methylene blue addition:
o ACID-FAST cells are RED
o NON-ACID-FAST cells are stained BLUE
 All acid-fast organisms are not consistent in their reactions.
o Young Mycobacterium cultures may not be as
acid-fast as older ones since they have not yet
accumulated as much lipid
 Acid-fast stain is used in clinical microbiology where is it
very useful in the identification of the tubercle and leprosy
bacilli.
Two types of acid-fast staining method:
1. Hot staining method (Ziehl-Neelsen Method)
2. Cold staining method (Kinyoun’s Method)
o Used in the experiment
REAGENTS
 Carbol fushsin solution (initial stain)
 Acid alcohol (acid decolorizer)
 Alkaline methylene blue (counterstain)
GROUP 9: Guillermo, Hernandez, K., Hernandez, S., Hilario, Ibalio
EDITOR/S: Manalastas (09425524436)
A. ZIEHL-NEELSEN METHOD
 Hot staining method
 Involves steaming/heating
 Steaming will break down the cell wall and allow entry of
carbol fuchsin and binding with mycolic acids.
PROCEDURE:
1. Place the slides on the staining rack and cover the smears
with carbol fuchsin. Allow to stand for 30-60 seconds before
heating.
2. Heat the preparations gently by passing the Bunsen burner
flame under the slides (or over) on the staining rack.
3. Maintain steaming for 5 minutes. Add more dye as needed
to prevent the smear from drying out.
4. Allow the slides to cool on the staining rack for 10 seconds.
Rinse with running water.
5. Apply the acid alcohol decolorizer drop-wise and slowly.
Continue to add this reagent until the dye does not run off
from the smear.
6. Rinse with running water. You may repeat Step 5 if
decolorization is not yet complete.
7. Cover the smears with methylene blue for 1 minute.
8. Rinse again with water.
9. Air dry or blot out but do not rub the smear.
10. View under the microscope.
B. KINYOUN’S METHOD
 USED IN THE EXPERIMENT
 Cold staining method
 No steaming/heating required
 Entry of stain is through diffusion. Prolonged exposure of the
cell to carbol fuschin will allow the stain to diffuse.
PROCEDURE
1. Place fixed slides on the staining rack with the smear facing
upward.
2. Flood the slides with carbol fuchsin for not less than 10
minutes.
3. Wash the slide with tap water.
4. Decolorize with acid alcohol until pink color disappears from
thinner portion of smear.
5. Lightly wash away the free stains with tap water. Tip slides
to drain.
6. Counterstain with alkaline methylene blue for 1 minute.
7. Gently wash with tap water and tip to drain.
8. Air-dry slides by placing them tilted in a slide tray.
9. View under a microscope.
Note:
Make sure to scan the whole slide so as not to miss AFB (+)
cells.
C. RESULTS
 Acid-fast cells RED or PINK= AFB (+)
 Non-acid-fast cells BLUE= AFB (-)
 *AFB=Acid-fast bacilli
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 MINI-QUIZ
1. What is the primary stain used in acid-fast staining?
2. What component of acid-fast cells does the primary stain
adhere to?
3. What reagent is used as decolorizer in acid-fast staining?

ANSWERS:
1. Carbol fuchsin
2. Mycolic acid
3. Acid alcohol

Figure 1. Non-acid-fast cells are stained BLUE while acid-fast cells

(pointed arrow) are stained RED or PINK

GUIDE QUESTIONS
1. What is the purpose of the heat in the Ziehl-Neelsen
procedure?
In Zeil-Neelsen procedure, also known as Hot staining
method, it involves steaming or heating because it will
breakdown the cell wall of the bacterium/ bacteria. It allows
the carbol fuchsin dye to penetrate the bacterial cell wall to
bind with mycolic acids.
2. What is the function of the counterstain in the acid-fast
staining procedure?
The counterstain, methylene blue, is added to stain the non-
acid-fast cells. Methylene blue is unable to penetrate the
waxy mycolic acid but is easily able to penetrate the non-
acid-fast cells. So at the end of the procedure acid-fast cells
will appear red and non-acid-fast cells will appear blue.
3. What structure in the cell is responsible for the acid-fast
property of Mycobacteria?
The mycolic acid (lipid-like waxes) in the cell wall makes
mycobacterial cells resistant to penetration by ordinary
stains or dyes. Mycolic acids are long-chain fatty acids
(C78-C90); they are contained in the inner leaflet of the cell
wall.
4. Are acid-fast bacteria gram-positive or gram-negative?
Explain your answer.
Acid-fast bacteria are mostly considered gram-positive
because they contain peptidoglycan in their cell walls.
However, they have an additional thick outer layer of
glycolipids, composed mainly of mycolic acid, that
differentiate them from gram-positive bacteria. Acid-fast
bacteria generally don’t take up the crystal violet stain as
well as gram-positive bacteria.
5. List five acid-fast pathogens and the diseases they
produce.
a. Myobacterium tuberculosis – Tuberculosis
b. Myobacterium leprae – Leprosy
c. Myobacterium ulcerans – Buruli ulcer
d. Myobacterium bovis – Tuberculosis-like disease
e. Myobacterium kansasii – Pulmonary infection

REFERENCES
 Lab Manual
 Jawetz
 2019 Trans

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