AFB STAINING

Objective:
 
 To study and gain expertise on differential and cytological staining techniques.

 
Theory:
 
Differential staining is a technique that helps to characterize the microorganisms depending on the
difference in the physical and chemical nature of the microorganism. The differential and cytological
staining techniques discussed in this chapter help to differentiate between acid fast and non acid fast
cells and to visualize the intracellular constituents of the microbial cells including endospores,
capsules, metachromatic granules, and flagella. In this technique the differential stains applied on the
bacterial smear reveals the different types of cells at one point, reveals one part of the cell with one
color and other parts a different color.
 

Differential Staining
Gram stain technique is a differential staining technique, which separates bacteria into two groups
(discussed in earlier chapters), Gram-positive bacteria and Gram-negative bacteria. Another
differential staining technique is acid-fast technique which differentiates species of Mycobacterium
from other bacteria. Mycobacterium species due to its special cell wall resist the effect of the
decolorizer acid-alcohol and retain the color of the primary stain carbolfuchsin stain and stains the acid
fast cells in bright red color. Other bacteria lose the stain and take on the subsequent color of the
counter stain methylene blue stain and stain the cell as blue. Endospore staining is a special staining
technique, to observe bacterial spores, where the spores take the color of the primary stain Malachite
green, while the counterstain, safranin, give color to the non-spore forming bacteria. Specific stains
such as nigrosine, Indian ink etc help to visualize the bacterial stains which cannot be stained by usual
staining methods. The metachromatic granules, the characteristic feature of Cornybacterium
diphtheriae, can be differentiated from the bacterial cells with the help of Albert staining techniques.
Flagella are the thin delicate structure for bacterial motility. The thin structures of the bacterial flagella
make it difficult to observe under bright field microscope. Special stains and mordents such as
Leifson’s stain are required for staining the bacterial flagella.
 
 

Endospore Stain:
 
Schaeffer-Fulton Method
 
Endospore staining is used to visualize specialized cell structures. The endospore stain is used to
determine the highly resistant spores of certain microorganisms within their vegetative cells. The
multiple thick coats of the spore made the endospore resistant to stain with most dyes. In Schaeffer-
Fulton method, the primary stain, Malachite Green, is added over the heat fixed bacterial smear and
heated over a steam bath for few minutes. This will soften the hard outer coverings of the spore and
the primary stain gets stick to the spore. When taken from the steam bath followed by further cooling
hardens the outer layer of the spore. During this stage both the spore and vegetative cells appear as
green in color. But later the thick outer layer makes the spore resistant to the action of decolorizing
agent (water), but however, water can easily decolorize the vegetative cells.  When counterstained
with Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink
color.
 
Dorner Method
 
Dorner method is an alternative method for staining the endospores. In this staining process, 2- 3ml
broth culture of microorganism and equal volume of  Basic fuchsin is heated in a water bath at 100 o C
for 10 minutes. The extensive boiling softens the structure of the bacterial spores and the basic
fuschin get into the spores. After the boiling process, the microbial culture-basic fuschin stain is
allowed to cool for sometime which hardens the outer covering of the spore. In order to give a color to
the background and to differentiate between the vegetative cells and endospores, a thin film of loopful
of the microbial culture-basic fuschin stain and 2 nigrosin on a clean glass slide. Since nigrosin is
negative charged, the bacterial cells cannot easily taken up by the cells. After staining the vegetative
cells appear become colorless, the endospores stains as red which can be present as terminal or sub
terminal. The background is stained as dark by the Negrosin stain.
 

Acid Fast Stain:

 
Ziehl Neelsen Method
 
The Ziehl Neelsen Method is used for staining the Mycobacterium in clinical specimens. The thick outer
waxy covering (mycolic acid) of the Mycobacterium cell walls act as a barrier  and does not allow all
the stains to enter into the cell. In order to visualize these cells higher concentrations of the staining
solution is needed and once this stain enters the cell, it is too difficult to remove the stain using a
decolorizer. When the clinical specimen is stained with basic dyes such as carbolfuchsin (primary
stain) with the continuous application of heat, softens the waxy lipid outer covering of the cell wall and
the stain readily enters the cell and stains the cell cytoplasm. When decolorizing agents such as acid-
alcohol is added over the primary stain, some bacterial cells cannot be easily decolorized and such
bacterial cells are called as acid fast bacteria. The bacteria with high concentration of lipid are easily
decolorized by the decolorizing agent and are said to be non-acid fast bacteria. Finally, the addition of
the counter stain, Methylene blue, dyes the colorless non acid fast cells as blue thus differentiating
them from the pink acid fast bacteria which are unaffected by the Methylene blue.
 

Capsule Stain:
 
Capsules are the gelatinous outer layer of the bacterial cells and these structures cannot retain the
color of the staining agents. The capsules can be visualized by means of two methods.
 
 Positive Capsule Staining
 
Since capsule is water soluble in nature, it is too difficult to stain the capsule with normal staining
methods. The positive capsule staining method (Anthony Method) uses two reagents to stain the
capsular material. The primary stain Crystal violet is applied over a non heat fixed bacterial smear so
that both the bacterial cells and capsular material take up the color of the primary stain. The ionic
nature of the bacterial cell binds the crystal violet stain more strongly while the non-ionic nature of the
capsule get adhere with the crystal violet stain. When the decolorizing agent copper sulfate is added
over the bacterial smear, the loosely adhered crystal violet stain is washed off from the capsular
material without removing the tightly bound crystal violet from the cell wall. The capsular material
absorbs the light blue color of the copper sulfate in contrast to the purple bacterial cell.
 
Negative Capsule Staining
 
Another simple method to visualize the bacterial capsules is by using negative staining Technique.
During staining the non heat fixed bacterial smear with the acidic stains such as Nigrosin will not
penetrate the bacterial cells (since both acidic stain and bacterial surface has negative charge).
Instead the acidic stain deposits around the bacterial cells and create a dark back ground and the
bacteria appear as unstained with a clear area around them, capsule.

Note: If you heat fix the bacterial smear for capsule staining, the cells will shrink creating a hallow
zone around the bacterial cell and will be mistaken for the capsule.
 

Metachromatic Granule Staining: Albert’s

Staining
 
Albert’s staining is specially demonstrates the presence or absence of the metachromatic granules, a
characteristic feature of Cornybacterium diphtheriae. During gram staining if a smear appears as
purple rods with straight or slightly curved with clubs at the end, with a characteristic V shape then it
is suspected as Cornybacterium diphtheriae. The further confirmation can be done by Albert’s staining
technique. This techniques employ two stains Albert Stain A( combination of Toluidine blue, Malachite
green, Glacial acetic acid, alcohol and distilled water) and Albert Stain B(Iodine, potassium iodide and
distilled water). After the staining process the metachromatic granules appear as bluish black where
as the cell appears as green color.
 

Flagella Staining
 
Bacteria are motile by means of flagella. The flagella are too thin to be seen in ordinary stains, special
stains and techniques needed to visualize the flagellum enough stain to obtain a visible thickness.
Specialized stains are usually found in microbiology laboratories to detect the presence or absence of
flagella to indicate the nature of that bacterium (motile/non-motile).
 
 
Liefson Stain
 
When a bacterial culture is stained with Liefson stain, the tannic acid component of the stain produce
a colloidal precipitate which can be taken up by the bacterial flagella will become colorized which can
be easily visualized using microscopy. The concentration of the tannic acid and dye is important in
staining the bacterial flagella while the alcohol concentration in the Liefsons stains helps in maintaining
the solubility of the components. On microscopic observation, the bacterial cells and flagella will stain
red and the flagellar arrangement can be visualized easily. The age of the bacterial culture, condition
of staining solutions, concentrations of the staining solution etc can also affect the staining reaction.
 

1) Primary stain used for staining the spores of Bacillus

   

2) Dorner’s method is used to visualize which of the following structures of bacteria?

 Capsule

 Metachromatic granules
   
 Endospores

 Flagella

3) The characteristic feature of Cornybacterium diphtheria is

Chinese characters or palisades

Drum like arrangement

   

Cluster like arrangement

None of the above

4) Mycobacterium tuberculosis cannot be easily decolorized with acid alcohol due to

 Mycolic acid

 Peptidoglycan
   
 Lipid

 All the above

 5) The gelatinous outer layer of the bacterial cells
 Cell wall

 Capsule
   
 Flagella

 Cell membrane

6) The decolorizing agent in positive capsule staining

 Sulphuric acid

 Water
   
 Acid alcohol

 Copper sulfate

7) Example of an endospore producing organism

 Staphylococcus aureus

 Streptococcus pneumonia
   
 Bacillus subtilis

 All the above

8) Example of capsulated organism

Bacillus megaterium

Streptococcus pyogenes

   
Streptococcus pneumoniae

Streptococcus agalactiae

All the above