dye for microbial identification

1. Bacterial Staining
Methods
Dr. Nusrat Mannan
AssistantProfessor
Dept. of Microbiology
US BANGLAMEDICALCOLLEGE

4. According to nature of stain, it can be classified into:
1. Acidic Dyes: It is a dye that has a negative charge so they bind to positively charged cell
structures like some proteins. Acidic dyes are not very often used in the Microbiology lab.
except to provide background staining like Capsule staining. Examples: Nigrosine, Picric
acid, Eosin, Acid fuschin, India ink etc.
2. Basic Dyes: This dye has a positive charge & binds to negatively charged molecules
(nucleic acid, -COOH -OH). Since the surface of bacterial cells is negatively charged (due to
Teichoic acid), basic dyes are most commonly used in bacteriology. Examples: Crystal
Violet, Methylene Blue, Safranin, basic fuschin.
3. Neutral Dyes: They are usually formed from precipitation in which are produced when
aqueous acidic & basic stains are combined. Neutral dyes stain nucleic acids, & cytoplasm. Eg;
Eosinate of Methylene blue, Giesma stain.

6. BACTERIAL SMEAR PREPARATION:
Smear - is a distribution of bacterial cells on a slide for the purpose of viewing them under
the microscope.
Method:
-Aseptically a small sample of the culture is spread over a slide surface.
-This is then allowed to air dry.
-The next step is heat fixation to help the cells adhere to the slide surface.
-The smear is now ready for staining.

10. STAINING METHODS:
1. POSITIVE STAINING: - where the actual cells are themselves colored and
appear in a clear background.
(a) Simple staining: A stain that provides color contrast but gives the same color
to all bacteria and cells. Ex: Loeffler’s methylene blue, Polychrome methylene blue,
Diluted carbol fuchsin.
(b) Differential Staining: A stain that imparts different colors to different
bacteria is called differential stain(which contains more than one stain). Ex: Gram’s
stain , Acid fast staining, Special stains.
2. NEGATIVE STAINING: where the cells remain clear (uncolored) and the
background is colored to create a contrast to aid in the better visualization of the
image. (a) Indian ink (b) Nigrosin .

13. Differential Stains
Differential Stains use two or more stains and
allow the cells to be categorized into various
groups or types.
Both techniques allow the observation of cell
morphology or shape, but differential staining
usually provides more information about the
characteristics of the cell wall (Thickness).

15. Gram staining
Principles
Gram staining is used to determine gram status to classify bacteria broadly. It is based on the
composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a
mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important
in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to
some antibiotics.
Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with
peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in
Gram-negative bacteria

21. ACID FAST STAINING

22. Mycobacterium are Acid Fast Bacilli
Mycobacterium are Gram-resistant (waxy cell walls), non-motile, pleomorphic
rods, related to the Actinomyces. Most Mycobacteria are found in habitats such
as water or soil. However, a few are intracellular pathogens of animals and
humans. Mycobacterium tuberculosis, along with M. bovis, M. africanum, and
M. microti all cause the disease known as tuberculosis (TB) and are members of
the tuberculosis species complex. Each member of the TB complex is
pathogenic, but M. tuberculosis is pathogenic for humans while M. bovis is
usually pathogenic for animals.

23. Acid-Fast Stain:
Acid-fast cells contain a large
amount of lipids and waxes in
their cell walls, primarily
mycolic acid. Acid fast bacteria
are usually members of the
genus Mycobacterium or
Nocardia. Therefore, this stain
is important to identify
Mycobacterium or Nocardia

25. Ziehl-Neelsen stain
Ziehl-Neelsen staining is used to stain species of Mycobacterium tuberculosis that do not
stain with the standard laboratory staining procedures like Gram staining. The stains used are
the red-colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene
blue or Malachite green.
Acid-Fast Organisms
Primary stain binds cell wall mycolic acids. Intense decolorization does not release primary
stain from the cell wall of AFB. Color of AFB based on the primary stain. Counterstain
provides contrasting background.

26. Acid fastness of acid-fast bacilli is attributed to the
presence of large quantities of the unsaponifiable
wax fraction called mycolic acid in their cell wall
and also the intactness of the cell wall.
The degree of acid fastness varies in
different bacteria. In this staining method,
application of heat helps the dye to penetrate the
tubercle bacillus.
Once stained, the stain cannot be easily
removed. The tubercle bacilli resist the decolorizing
action of acid-alcohol which confers acid fastness to
the bacteria. The other microorganisms, which are
easily decolorized by acid-alcohol, are considered
non-acid fast.
The non-acid fast bacilli readily absorb the
colour of the counter stain appearing blue, while the
acid-fast cells retain the red colour of primary stain.

30. DIFFERENT MODIFICATION OF ACID-FAST
STAIN
1) 5% Sulphuric acid is used as a decolourizing agent for staining Mycobacterium
leprae.
2) 1% Sulphuric acid is used as a decolourizing agent for staining Nocardia species,
Cryptosporidium and Isospora oocysts (Kinyoun’s modification of acid fast stain).
3) 0.25% Sulphuric acid is used as a decolourizing agent for staining spores.