Chapter 4 1

STAINING METHODS
(SIMPLE, GRAM AND ACID-FAST
STAINING)
Micro-organisms are semi-transparent, as the refractive index of microbes is approximately equal to2
the refractive index of the surrounding environment. Hence, bacteria are not easily visible in unstained
state.
Stains are used to-
1- To render microscopic and semitransparent objects visible
2- To study the morphology (size, shape and arrangement of microbes)
3- To observe the presence of various internal and external structures (capsule, spore, flagella etc.)
4- To produce specific physical or chemical reactions.

Colouring agent used for general purposes – dye

Biologic- stain
Stain – it is an organic compound containing both chromophore and auxochrome 3
groups linked to benzene ring.
Chromophore- imparts colour to the stain.
Any substance which possesses only chromophore group is not a stain. To behave as
stain it must have the affinity to bind with cells or tissues.
The group that imparts ability to binding tissue is auxochrome.
 4

Depending upon positive or negative electrical charge present on chromophore, stains are grouped as acidic
stains and basic stains.
A) Acidic Stain (anionic stain) 5
A stain that possesses negatively charged chromophore and therefore, has a strong affinity for
the positive constituents of the cell. Proteins, positively charged cellular components readily
bind to and accept the colour of the negatively charged, anionic chromogen of an acidic stain.
e.g. sodium eosinate, picric acid, nigrosine, congo red, Rose Bengal stain etc.
A) Basic Stain (cationic stain) 6
A stain that possesses positively charged chromophore and therefore, has a strong affinity for
the negative constituents of the cell. Nucleic acids, negatively charged cellular components
readily bind to and accept the colour of the positively charged, cationic chromogen of a basic
stain.
e.g. methylene blue, safranin, crystal violet, malachite green etc.
A number of staining techniques are available for visualization, differentiation and separation of bacteria.
Commonly used staining techniques are as follows- 7

All staining procedures require preparation of smears prior to the execution of any of the specific staining
techniques listed above.
1) Cleaning of glass slides- 8
Cleaning glass slides required for preparation of microbial smears. Grease or oil from slides must be
removed from slides by washing it thoroughly with soap solution. Slides are rinsed with water and
followed by 95% alcohol. After cleaning, dry the slides and the use.
2) Preparation of smear-
A thin dried film of bacterial culture on glass slide prepared for staining is referred to as smear. Good
thin smear is prepared by avoiding thick and dense smears. Those made from broth cultures or
cultures from the solid medium require variation in technique.
a) Broth cultures-
A target circle of approx. 1.5cm diam. is marked on the underside of a slide. Aseptically transfer a
loopful of bacterial suspension on the circled area and spread evenly on area of target circle with the
help of nichrome wire loop. Allow the slide to air dry completely.
b) Cultures from a solid medium- 9
MOs cultured in a solid medium produce thick, dense surface growth. These cultures must be
diluted by placing a loopful of water on the glass slide. Transfer of cell from the culture required
the use of sterile inoculating needle. Only the tip of needle should touch the culture to prevent
the transfer of too many cells. The culture is suspended in water drop on the glass slide and
spreaded evenly on the area of target circle with the help of nichrome wire loop. Allow the smear
to dry completely.
3) Fixation of smear-
Smear fixation causes bacteria to adhere to a slide so that they can be stained and observed.
Generally heat is used for fixation. In heat fixation, bacterial proteins are coagulated and fixed to
the glass surface. Heat fixation is performed by the rapid passage of the air-dried smear two or
three times over the flame of the bunsen burner.
10
SIMPLE STAINING
11
Aim: To study the bacterial morphology by simple staining
Requirements:
Cultures- Bacillus subtilis or staphylococcus aureus, Escherichia coli (E. coli)
stains- methylene blue or crystal violet or carbol fuschin
Apparatus- Staining tray, glass slides, Bunsen burner, inoculating loop, lens paper, glass
marker
Equipments- Microscope
Principle:
The use of single stain to colour the bacteria is commonly
known as monochrome staining.
Unstained bacterial cell (Bacterial cell- ) (Na+ )
Methylene blue stain (MB+ ) (Cl- )
(Bacterial cell- ) (Na+ ) + (MB+ ) (Cl- ) → (Bacterial cell- ) (MB+ ) + (Na+Cl- )
Basic dyes are commonly used for this staining. These dyes are available as salt of acids. Eg.
Methylene blue chloride. When methylene blue rehydrates, it ionizes to form methylene blue and12
chloride ions. The +ve charge ion have the colouring property.
MB.Cl→ionization→(MB+ ) (Cl- )
The structure of the bacterial cell has negative acidic characteristics because of large amount of
carboxyl groups located on the cell surface due to acidic amino acids.
Therefore, when ionization of carboxyl groups takes place it imparts negative charge to the cell
surface.
(Bacterial cell- COOH) →ionization→ (Bacterial cell- COO-) + H+
In nature, H+ is replaced by positive ion (Eg. Na+, K+) and H+ ion bonds with oxygen to form water.
Thus, the surface of an unstained bacterial cell wall is represented as
(Bacterial cell-- ) (Na+ ) + (MB+ ) (Cl- ) → (Bacterial cell--) (MB+ ) + (Na+Cl- )
 -On addition of methylene blue for staining, exchange of MB+ with Na+ on the bacterial 13
cell surface takes place, resulting into ionic bond formation between MB+ and cell surface.
-Thus, when colouring agent forms ionic bond with cell or cell components, it results into
the staining of cell.
-The most commonly used basic stains are-
methylene blue (2-3min.) ; Crystal violet (1-2 min.); Carbol fuschin (15-30 secs)
Procedure:
Prepare separate bacterial smears of each MOs and heat fix it.
Place the slide on the staining tray and flood the smear with one of the indicated stains
using the appro. exposure time.
Pour off the staining solutions. Wash the slide in running tap water. Dry the slide between
blotting papers and examine the stained smear under microscope using oil immersion
objective
Observation and Results:
14
Bacterial sp. are seen blue, violet or red depending upon the stain used against the
colourless background. Note the shape, size and arrangement of cells. S. Aureus is ovoid
or spherical in shape, arranged in clusters. E. Coli and B. subtilis are small, rod shaped
bacteria.
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S. aureus E.coli
15
GRAM STAINING
16
Aim: To study the bacterial morphology by Gram’s staining
Requirements:
Cultures- Nutrient broth 24 hr. cultures of A) Escherichia coli (E. coli) B) Bacillus cereus or (C)
staphylococcus aureus.
stains- crystal violet and safranin
Reagents- Grams iodine, ethyl alcohol (95%)
Apparatus- Staining tray, glass slides, Bunsen burner, inoculating loop, lens paper, glass marker
Equipments- Microscope
Principle:
Gram’s staining is one of the most imp. Differential staining. This technique divides bacteria into 2
groups.
1. Gram positive- Those which primary stain like crystal violet and appear deep violet in colour.
2. Gram negative- Those which lose the primary stain on application of decolourizer and take the colour of the counter stain like
safranin or basic fuchsin. They appear red in colour
17
Difference between gram positive and gram negative bacteria:-
COMPONENT GRAM POSITIVE GRAM NEGATIVE
Thickness of cell wall 20-25nm and thick 10-15nm and thin
Peptidoglycan More in number Less in number

Teichoic acid Present absent

Polysaccharide Present absent
Lipid Less /absent More/present
Cell wall Simple complex
Outer membrane Absent Present
Effect of lyzosome Easily destroy resistant
Type of amino acid Few in number Several in number

Four types of reagents are used in Gram staining

o Primary stain-
The crystal violet stain is used first and stain all cell deep violet in colour

o Mordant (Gram’s iodine)

May define as any substance that forms an insoluble compound with stain and serves to fix the colour to bacterial cell. It
should be noted that mordant is not stain. It leads to formation of crystal violet iodine- magnesium ribonucleated [CV-I-
Mg ribonucleate] complex in gram +ve bacteria. The complex (Mg-ribonucleate) is absent in cell wall of
Gram –ve bacteria. Hence only CV-I complex is formed in gram negative bacteria.
o Decolourising agent (ethyl alcohol, 95%)
Cell wall of gram positive cell contain less lipid. On application of decolourising agent like alcohol, acetone, 18
shrinkage of cell wall take place due to dehydration and decreases the permeability for CV-I– Mg ribonucleate
complex . Thus, complex is retain in the cell and hence cell is stain deep violet in colour. On other hand the treatment
of decolourising agent extracts lipid from cell wall of gram negative cell and increases the permeability property of
cell wall. Due to this, CV-I complex extracted and cell gets decolourised (lose violet colour)
o Counter stain (safranin)-
Final reagent to stain red, those cells which have been previously decolourised. Since, only gram –ve cells are
decolourized, they may now absorb the counter stain. Gram +ve cells retain the violet colour of the primary stain.

Procedure:
Prepare a smear from bacterial suspension on clean grease free slide.
Allow the smears to air dry and then heat fix in the usual manner.
Cover the smear by crystal violet (primary stain) and keep for 1 min.
Wash the smear with water and cover it with Gram’s iodine (mordant) for 1 min.
 19
Wash the smear with water and decolourize
with 95% ethyl alcohol vey carefully till
washing does not contain violet colour in it.
For normal smear 10-15 sec. are enough.
Rinse the smear with water and counterstain
with safranin/dil. Carbol fuchsin for 1 min.
Wash the smear with water and allow it to
air dry.
Put a drop oil on smear and examine under
oil
immersion objective.
Observation and Results:
Smear prepared from suspension marked A shows red coloured rod shaped bacteria i.e. Gram –ve bacilli 20
Smear prepared from suspension marked B shows violet coloured rod shaped bacteria i.e. Gram +ve bacilli
Smear prepared from suspension marked C shows deep violet coloured cocci arranged in clusters which means that the organism
may be Gram +ve staphylococci.

Escherichia coli Bacillus cereus Staphylococcus aureus

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21
22
ACID-FAST STAINING
23
Aim: To study the bacterial morphology by acid fast staining/Ziehl-Neelsen staining
Requirements:
Cultures- (A)Nutrient broth (72 hr.) cultures of Mycobacterium tuberculosis,
(B) Broth culture (24hr.) of staphylococcus aureus
stains- Carbol fuchsin, methylene blue, malachite green
Reagents- Acid-alcohol (3% HCl+95% ethanol), 20% H2SO4
Apparatus- Staining tray, glass slides, Bunsen burner, inoculating loop, lens paper, glass marker
Equipments- Microscope
Principle:
Acid fast staining is another type of the most imp. differential staining. The bacteria’s which resist
decolourization with acid and alcohol are called as acid fast organisms. Acid +alcohol (3%
HCl+95% ethanol) is very intensive decolourizer. This technique divides bacteria into 2 groups.
1. Acid-fast
2. Non-acid fast 24
This process is mainly used in the diagnosis of M. tuberculosis and M. leprae.
Acid fastness property in mycobacteria and some species of Nocardia is correlated with their
high lipid content. Due to high lipid content of cell wall, acid-fast cells have relatively low
permeability to dye and hence it is difficult to stain.
To stain these bacteria penetration of primary dye is facilitated with the use of 5% aq. Phenol
which acts as a chemical intensifier. Heat is also used which acts as a physical intensifier.
Three types of reagents are used in acid-fast staining
o Primary stain (carbol fuchsin)
It is a phenolic stain that is soluble in the lipoidal material which constitute the major
portion of the mycobacterial cell wall allows penetration and retention of red stain.
Penetration is increased by application of heat which drives the carbol fuchsin through the
lipoidal wall and into the cytoplasm. All cells appear red in colour after primary stain.
o Decolourising agent (acid-alcohol, 20% H2SO4)

Smear is cooled before decolourization which allows the waxy cell substances to harden. On application of 25
decolourising agent, acid-fast cells show resistant to decolourization. Primary stain is more soluble in the cellular
waxes than decolourising agent. Hence, primary stain is retained by mycobacteria species. But this is not the case with
non-acid fast bacteria that lack cellular waxes. Primary stain is easily removed during decolourization and non acid
fast cells are observed as colourless.
o Counter stain (methylene blue/malachite green)-
Non-acid fast bacteria absorb the counter stain and appear as blue or green colour while acid-fast cells retain the red colour
of the primary stain.
Procedure:
Prepare a smear from bacterial suspension on clean grease free slide.
Allow the smears to air dry and then heat fix in the usual manner.
Flood the smear with carbol fuchsin and heat the slide from below till steam rises for 5 min.
Do not boil the stain and ensure that the stain does not dry out.
Allow the slide to cool down for 5 min. to prevent the breakage of slide in the subsequent step. Wash with tap water.
Decolourize the slide by using acid-alcohol or 20% sulphuric acid until carbol fuchsin fails to wash from smear.
Wash with water and counter stain with 1% aq. solution of malachite green or methylene blue for 1 to 2 min.
Wash the smear with tap water, dry and examine under oil immersion objective.
Observation and Results:
26
Smear (A) shows thin bright red, slightly curved bacilli with beaded appearance showing
palisade arrangement. Thin, bright red, slightly curved, breaded appearance are the features
of M. tuberculosis i.e. ACID-FAST bacteria.
Smear from suspension B shows blue coloured cocci arranged in clusters which means that
the organism may be staphylococci i.e. non acid fast bacteria.
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Mycobacterium tuberculosis (acid fast) Staphylococcus aureus (non-acid fast)