Staining techniques are used in microbiology to increase the visibility and contrast of bacterial structures under a light microscope. There are several types of staining including simple stains that color all bacteria the same hue, differential stains that color different types of bacteria differently, and special stains like acid-fast stains. Gram staining is a common differential technique that divides bacteria into Gram-positive or Gram-negative based on differences in cell wall structure. Acid-fast staining identifies bacteria with high mycolic acid in their cell walls like Mycobacterium tuberculosis. Proper fixation of smears is also important to preserve cell structure before staining.

1. Staining Techniques
Ankur vashishtha

2. Staining Techniques
• Structural details of bacteria cannot be seen under light
microscope due to lack of contrast.
• Hence, it is necessary to use staining methods to produce color
contrast and thereby increase the visibility.
• Before staining, the fixation of the smear to the slide is done.

3. Fixation
• Fixation is the process by which the internal and external
structures of cells are preserved and fixed in position.
• It Kills and fixes the cells on to the slide.
• There are two types of fixation as follows
1. Heat Fixation
2. Chemical Fixation

4. Heat Fixation
• It is usually done for Bacterial
smears by gently flame heating
an air-dried film of bacteria.
• This adequately preserves
overall morphology but not
structures within the cells

5. Chemical Fixation
• It can be done using ethanol, acetic acid, mercuric chloride,
Formaldehyde, methanol and glutaraldehyde.
• They are used to protect the fine internal structure of the cells.
• This is useful for examination of blood smears.

6. Staining Techniques in Microbiology
1. Simple stain
2. Negative stain
3. Impregnation methods
4. Differential stain

8. Simple stain
• Use basic dyes, Such as methylene blue or basic fuchsine are
used as simple stains.
• They provide the color contrast, but impart the same color to
all the bacteria in a smear.

9. Negative stain
• A drop of bacteria suspension is mixed with dyes, such as
India ink or nigrosin.
• The background gets stained black where as unstained
bacterial/ yeast capsule stand out in contrast.
• This is very useful in the demonstration of bacterial/yeast
capsules which do not take up simple stains.

10. Impregnation methods
• Bacterial cells and structures that are too thin to be seen under
the light microscope, are thickened by impregnation of silver
salts on their surface to make them visible,
• e.g. for demonstration of bacterial flagella and spirochetes.

11. Differential stain
• Here, two stains are used which impart different colors to different bacteria
or bacterial structures, which help in differentiating bacteria.
• The most commonly employed differential stains are:-
 Gram stain:- It differentiates bacteria into gram-positive and gram-
negative group.
 Acid-fast stain:- It differentiates bacteria into acid-fast and non acid-
fast group.
 Albert stain:- It differentiates bacteria having metachromatic granules
from other bacteria that do not have them.

12. GramStain
• This staining technique was originally
developed by Hans Christian Gram
(1884).

13. STAINS
Crystal violet (Primary stain)
Gram’s iodine (Mordant)
Decolorizer (Decolorization)
Carbol fuchsin/ safranin
(Counter stain)

14. procedure
• Fixation:- The smear
made on a slide from
bacterial culture or
specimen, is air dried and
then heat fixed.

17. Interpretation of gram stain
• Smear is examined under oil immersion objective:-
▫ Gram-positive bacteria resist decolorization and retain the color of
primary stain i.e. violet
▫ Gram-negative bacteria are decolorized and, therefore, take counter
stain and appear pink.

19. Principle of gram staining
• The following theories have been put forward.
▫ pH theory
▫ Cell wall theory

21. Cell wall theory
• This is believed to be the most important postulate to describe
the mechanism of Gram stain.
▫ Gram-positive cell wall has a thick peptidoglycan layer. (50-100
layers thick).

22. pH theory
▫ Cytoplasm of gram-positive bacteria is more acidic, hence ,
can retain the basic dye (e.g. crystal violet) for longer time.
▫ Iodine serves as mordant, i.e. it combines with the primary
stain to from a dye-iodine complex which gets retained
inside the cell.

23. ACID-FAST STAIn
• The acid-fast stain was discovered by Paul Ehrlich and subsequently
modified by Ziehl and Neelsen.
• This staining is done to identify acid-fast organisms, such as
Mycobacterium tuberculosis and others.
• Acid-fastness is due to presence of mycolic acid in the cell wall.

25. Smear Preparation
• Smear measuring 2x3 cm in size is prepared in a new clean
grease free scratch free slide from the yellow purulent portion
of the sputum .
• The smear should neither be too thick nor too thin.
• Smear preparation should be done near a flame,

26. STAINS & REAGENTS
1. Carbol fuchsin 1% (Primary stain)
2. 25% sulfuric acid (Decolorization)
3. 0.1% Methylene blue (Counter
staining)

27. Procedure
5 Minutes 2-4 minutes 30 Second

28. Interpretation
• Mycobacterium tuberculosis
appears as long slender straight
or slightly curved & beaded,
red colored acid-fast bacillus.

29. ALBERT STAIN
• Albert stain is used to demonstrate the metachromatic granules of
corynebacterium diphtheriae.

30. Procedure
• Fixation:- the smear is heat fixed
• Smear is covered with Albert-I (Albert’s stain) for 5 Minutes
• Then washed in water, and blotted dry
• Albert – II (Iodine solution) is added for 1 minute.
• Slide is washed in water, blotted dry and examined under oil
immersion field.

32. Interpretation
• corynebacterium diphtheriae
appears as green colored bacilli
arranged in chines latter or
cuniform pattern, with bluish
black metachromatic granules at
polar ends.

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