Acid fast bacteria and acid fast staining

1. ACID FAST BACTERIA AND ACID FAST
STAINING
 BY – MOHIT HINSU

2. INTRODUCTION
In nature many microorganism are present, they have some special
characters.
 Generally bacteria stained by simple staining method like gram staining
, bur some bacteria are not stained by this type of staining method or
procedure , like Acid fast bacteria.
WHY ACID FAST BACTERIA ARE NOT STAINED BY SIMPLE
STAINING METHOD?
 Acid-fast bacteria, also known as acid-fast bacilli or simply AFB, is a
group of bacteria sharing the characteristic of acid fastness. Acid
fastness is a physical property that gives a bacterium the ability to
resist decolorization by acids during staining procedures. This
means that once the bacterium is stained, it cannot be decolorized using
acids routinely used in the process. This important and unique feature
of certain bacteria gives us the ability to classify and detect them using
relatively easy laboratory procedures such as microscopy.

3.  This type acid fast bacteria have waxy covering on the surface. If
anyhow they get stained they don’t get decolorized even by strong
decolorizing agent , This type bacteria called acid fast bacteria.
 For this type bacteria required a special type of staining method, we use
acid fast staining method to Distinguish acid fast bacteria to non
acid fast bacteria.
 It is the differential staining techniques which was first developed by
Ziehl and later on modified by Neelsen. So this method is also called
Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s
carbol-fuchsin and heat then decolorized with an acid alcohol,
and counter stained with methylene blue. Thus Ziehl-Neelsen
staining techniques was developed.
 The main aim of this staining is to differentiate bacteria into acid
fast group and non-acid fast groups.
 This method is used for those microorganisms which are not staining by simple or
Gram staining method, particularly the member of genus Mycobacterium,
are resistant and can only be visualized by acid-fast staining.

4. PRINCIPLE OF ACID FAST STAINING
 When the smear is stained with Ziehl-Neelsen carbol
fuchsin[ZNCF], it solubilizes the lipoidal material present in the
Mycobacterial cell wall but by the application of heat, Ziehl-Neelsen
carbol fuchsin further penetrates through lipoidal wall and enters
into cytoplasm. Then after all cell appears red.
 Then the smear is decolorized with decolorizing agent (3% HCL in 95%
alcohol) but the acid fast cells are resistant due to the presence of large
amount of lipoidal material in their cell wall which prevents the
penetration of decolorizing solution.
 The non-acid fast organism lack the lipoidal material in their cell wall
due to which they are easily decolorized, leaving the cells colorless. Then
the smear is stained with counterstain, methylene blue. Only decolorized
cells absorb the counter stain and take its color and appears blue while
acid-fast cells retain the red color.

5. Structure of an Acid-Fast Cell Wall

6. Requirement
 Primary Stain: Ziehl-Neelsen carbol fuchsin [ZNCF]
 Decolorization Solution: Acid alcohol
 Counterstain: Methylene blue
 Why is Ziehl-Neelse carbol Fuchsin used in acid fast staining?
-Carbol fuchsin is used as the primary stain dye to detect acid-fast
bacteria because it is more soluble in the cells wall lipids than
in the acid alcohol.

7. PROCEDURE:
 1.Prepare and fix the specimen smear prior to staining.
 2.Place a small strip of blotting or filter paper over the top of the
specimen, and place the slide over a boiling hot water bath on a mesh
surface.
 3.Cover the filter paper with the primary stain, carbolfuchsin. Leave the
slide on the water bath for 3 to 5 minutes. Continue to apply stain if the
filter paper begins to dry.
 4.Remove the filter paper and rinse the slide with water until the
solution runs clear.
 5.Run acid-alcohol decolorizer over the slide for approximately 10 to 15
seconds.
 6.Rinse the slide with water.
 7.Cover the smear with the secondary or counterstain, methylene blue,
for 1 minute.
 8.Gently rinse the slide with water.
 9.Blot the slide dry with bibulous paper.

8. Summary of Acid-Fast Stain
Application of Reagent
Cell colour
Acid fast Non-acid fast
Primary dye
Ziehl-Neelsen carbon
fuchsin
Red Red
Decolorizer Acid alcohol Red Colorless
Counter stain Methylene blue Red Blue

9. Result Interpretation of Acid Fast Stain
 Acid fast: Bright red to intensive purple, Red, straight or slightly curved
rods, occurring singly or in small groups, may appear beaded
 Non-acid fast: Blue color; In addition, background material should stain
blue.

10. What is the purpose of the acid fast stain?
 The acid-fast stain is a laboratory test that
determines if a sample of tissue, blood, or other
body substance is infected with the bacteria that
causes tuberculosis (TB) and other illnesses.
 Belove acid fast bacteria example given.
 Eg. Genus Mycobacterium – M. leprae, M. Tuberculosis, M.
smegmatis, M. Avium complex, M. kansasii.
 Genus Nocardia – N. brasiliensis, N. cyriacigeorgica, N.