Himanshu Khandelwal, Nikhil Sharma

1. #-Identification of Bacteria using
Staining Techniques:-
• Staining Techniques:-
• Staining, in microbiology, can be defined as a technique
which is used to enhance contrast a biological specimen at the
microscopic level. Stains and dyes are used to highlight the specimen
at the microscopic level to study it at higher
Table 1: Different stains for different bacteria
Reagent Gram-Positive Gram-Negative
Crystal violet Purple or blue Purple or blue
Gram’s iodine Purple or blue Purple or blue
Acetone or Alcohol Purple or blue Colorless
Staining, in microbiology, can be defined as a technique
which is used to enhance and contrast a biological
specimen at the microscopic level. Stains and dyes are
used to highlight the specimen at the microscopic level to
study it at higher magnification for histopathological
studies and diagnostic purposes

3. Simple Staining:-Simple staining involves directly staining the bacterial cell with a
positively charged dye in order to see bacterial detail, in contrast to
negative staining where the bacteria remain unstained against a
dark background.
Simple staining is carried out to visualize bacteria and to
compare morphological shapes and arrangements of the
bacterial cell. In this technique, the bacterial smear is stained
with a single basic dye such as crystal violet, safranin, methylene
blue, etc
Principle:- Simple staining uses single basic dyes such as crystal violet which is
dissolved in a solvent and applied to the microorganisms. The
microorganisms give the colour characteristics of the staining
solution. Because of which shape and size of microorganisms can
be determined.
Procedure:- 1. Take a grease-free slide and mark the circle with the help of a wax
pencil or glass marker pen.

4. 2. Make a smear with the help of inoculating loop by using bacterial culture.
3. Once a uniform smear is prepared subject it to drying.
4. The smear must be held above the Busen burner flame once or twice. Excess heating is
strictly avoided.
5. Add a few drops of crystal violet over the smear which covers the circle area for 1-2 min.
6. Remove the stain down from the slide and wash with a gentle stream of running water.
7. The slide must be held at the inclined position and then placed on a piece of blotting paper
8. Dry the slide and completely wipe off the smear.
9. Add a drop of immersion oil on the smear and observe under the immersion lens.
10. Check the different shapes of microorganisms and analyze the bacterial cell.

6. Observation:- Examine the microbial preparations under an oil immersion lens. Note the
observation and write the description of the organisms, including their
shape, colour and arrangements. Draw the shape of the organism.

7. Result:- A bacterial strain is a violet or deep blue. Spherical-shaped bacteria are presently
single, in pairs, tetrads, in short-chain, or in clusters
Application:- i) Simple staining is useful to study the shape of the organism.
ii) Simple staining provides contrasting characters between object and
background so objects can be easily studied.
iii) With the help of Simple staining size of an object can be studied.
iv) Only one type of dye is required in simple staining so it is also
referred to as Monochrome staining.
Differential Staining:- Differential staining is a procedure where more than one dye is used
to differentiate between different types of microorganisms on a slide
It differentiates between the physical and chemical properties of
two different groups of organisms based on cell-wall
characteristics.
It is further classified into two types as;
Gram Staining.
Acid Fast Staining.

8. Gram Staining:- This separates bacterias into two major groups,
Gram +ve Bacteria.
Gram -ve Bacteria.
This technique was invented by Danish Dr Hans Christian Gram in 1884.
Principle:- The Gram stain technique is based on the differential structure of the
cellular membranes and cell walls of Gram +ve and Gram -ve bacterias.
Gram +ve Bacteria:- Gram-positive organisms contain a highly cross-linked layer of
peptidoglycan that retains the primary dye, crystal violet (CV),
following the application of the mordant, iodine (I).
The iodine and crystal violet form a complex (CV-I) within the
peptidoglycan.
When decolorizer is applied to the cells, the CV-I complex remains
within the cell, making it appear dark purple to blue.

9. # Gram -ve Bacteria:-
The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan,
which is loosely distributed.
Following the application of the crystal violet and iodine, the CV-I complexes are not trapped
within the peptidoglycan.
Application of the acid-alcohol decolorizer dehydrates the outer cellular membrane, leaving
holes in the membrane and effectively washing or removing the CV-I complex from the cells.
The cells appear colorless.
To make the colorless cells visible, a secondary stain, safranin, is applied, making the gram-
negative cells pink.
Gram Stain Reagents:- Primary stain: Crystal Violet.
Gram’s iodine: (Potassium Iodide +Iodine).
Decolorizer: 50 mL acetone and 50 mL ethanol.
Counterstain: Safranin.

10. Procedure: 1-Prepare and fix the specimen to the microscope slide before staining.
2-Cover the smear with crystal violet, the primary stain, for 20 seconds.
3-Gently rinse off the stain with water.
4-Cover the smear with Gram’s iodine for 1 minute.
5-Pour off the excess Gram’s iodine.
6-Run the decolorizer over the smear until the solution appears clear.
7-Gently rinse with water.
8-Cover the smear with safranin, the secondary or counterstain, for 20 seconds.
9-Gently rinse the stain with water.
10-Blot dry with highly absorbent paper and observe under a microscope.

12. Observation:- Gram-positive: Blue/Purple Color
Gram-Negative: Red/Pink Color
Examples:-Gram-Positive: Streptococcus,
Staphylococcus, Corynebacterium,
Listeria, Bacillus, Clostridium, etc.
Gram-Negative: E. coli, Salmonella Typhi,
Shigella spp, Pseudomonas aeruginosa,
Neisseria gonorrhoeae, Chlamydia
trachomatis, Yersinia pestis, etc.
Acid Fast Staining:- Acid-fast staining was first introduced by a scientist Paul Ehrlich in
the year 1882.
Later, this was modified by Ziehl and Neelson in the year 1883.
Thus, acid-fast staining is sometimes called Ziehl and Neelson
staining.
It is a type of differential staining method, which is used to
differentiate between the acid-fast and non-acid fast bacteria.

13. Objective:-
To differentiate Mycobacterium species from other species of bacteria.
Differentiate between the acid-fast and non-acid fast bacteria
Principle:-Mycobacterium does not bind readily to simple stains and therefore the use of heat
along with carbol-fuchsin and phenol allows penetration through the bacterial cell
wall for visualization.
Mycobacterium cell wall contains high lipid content made up of mycolic acid on its
cell wall making it waxy, hydrophobic, and impermeable.
These are ß-hydroxycarboxylic acids made up of 90 carbon atoms that define the
acid-fastness of the bacteria.
Use of Carbol-fuchsin which is basic strongly binds to the negative components of the
bacteria which include the mycolic acid and the lipid cell wall. The addition of acid
alcohol along with the application of heat forms a strong complex that can not be
easily washed off with solvents.
The acid-fast bacilli take up the red color of the primary dye, carbol-fuschin.
While non-acid-fast bacteria easily decolorize on the addition of the acid-alcohol and
take up the counterstain dye of methylene blue and appear blue
.
This technique has been used in the identification of Mycobacterium tuberculosis
and Mycobacterium leprae
Reagents:-Primary Stain: Carbol-fuchsin.
Decolorizer: acid-alcohol or 20% Sulphuric acid.
Counterstain: Methylene Blue or Malachite green

14. Procedure :-
1-On a clean sterile slide, make the smear of the sample culture and heat fix the smear over
blue heat.
2-Over the smear, pour and flood the smear with carbol fuchsin and heat gently until it
produces fumes.
3-Allow it to stand for 5 minutes and wash it off with gently flowing tap water.
4-Add decolorizer and leave it for 1-2 minutes. Repeat this step until the smear appears pink
in color.
5-Wash off the acid with water.
6-Flood the smear with methylene blue dye and leave it for 2-3 minutes and wash with water.
7-Air dry and examine the stain under the oil immersion lens.

15. Results:-1-Acid-fast bacteria retain the primary dye, carbol-fuschin, and stain pink.
2-Non-acid fast bacteria take up the counterstain methylene blue dye and appear blue.
Applications:-1-Used for examination and
identification of Mycobacterium species.
2-Used to differentiate between acid-fast and
non-acid fast bacilli
3-Used for the identification of some fungal
species such as Cryptosporidium.
Biochemical tests (imvic):- The IMViC tests are a group of individual tests used in microbiology
lab testing to identify an organism in the coliform group. A coliform
is a gram negative, aerobic, or facultative anaerobic rod, which
produces gas from lactose within 48 hours. The presence of some
coliforms indicate fecal contamination.
-Indole test
-Methyl red (MR) test
-Voges-Proskauer (VP) test
-Citrate utilization test
#Indole test:- Some microorganisms can metabolize tryptophan by the tryptophanase. It is
based on the principle of the formation of a hydrolytic product from an amino
acid tryptophan.
Media:- Sulfide Indole Motility (SIM)
-Tryptophan is used as substrate.

16. Reagent:- Kovac’s reagent (Consist of isoamyl alcohol, para-dimethyl amino benzaldehyde, and
concentrated hydrochloric acid). It is used to determine the ability of the organism to split
indole from the amino acid tryptophan.
Colour:- Pink or red indicates a positive test.
Tryptophan → Tryptophanaseindole + Acid pyruvate + NH3 → Kovac’s Reagent
The upper organic layer forms red colour (positive for indole); black (production of H2S2) and
motility (turbidity forms) of bacteria.
Example:- Proteus mirabilis can differentiate from Proteus Vulgaris by this test. P. Vulgaris
shows a positive test (red or pink).
# Methyl Red (MR) test:-This test is utilized to detect presence of acids during fermentation of
glucose.
Principle:-In acid fermentation, three acids (acetic, lactic and succinic) are formed in significant
amounts per mol of glucose fermented.
These large amounts of acid result in a significant decrease in the pH of the medium below
4.4.
This is visualized by using a pH indicator, methyl red, which is yellow above pH 5.1 and red at
pH 4.4.
Procedure:- Glucose Phosphate Broth is used in this test.
-Inoculate two tubes containing Broth with a pure culture of the microorganisms under
investigation.
Incubate at 35 °C for up to 4 days.

17. -Add about 5 drops of the methyl red indicator solution to the tube.
-A positive reaction is indicated if the color of the medium changes to red within a few minutes.
Result:-
The development of a stable red color on the surface of the medium indicates sufficient acid
production to lower the pH to 4.4 and constitutes a positive test.0000
# Voges-Proskauer Test:-
Principle:-Pyruvic Acid the major metabolite of glucose
fermentation is further metabolized by the bacterias to produce
acetoin (acetyl methyl carbinol), a neutral-reacting end product.
-In the presence of atmospheric oxygen and 40% potassium
hydroxide, acetoin is converted to diacetyl, and alpha-naphthol
serves as a catalyst to bring out a red complex.
Procedure:- 1-Inoculate a tube of Glucose phosphate broth
with a pure culture of the test organism.
2-Incubate for 24 hours at 35°C
3-At the end of this time, aliquot 1 mL of broth to
clean the test tube.
4-Add 0.6mL of 5% α-naphthol, followed by 0.2 mL
of 40% KOH. (Note: It is essential that the reagents
be added in this order.)
5-Shake the tube gently to expose the medium to
atmospheric oxygen and allow the tube to remain

18. Results:--A positive test is represented by the development of a red color 15 minutes or more
after the addition of the reagents indicating the presence of diacetyl, the oxidation product of
acetoin.
-The test should not be read after standing for over 1 hour because negative Voges-Proskauer
cultures may produce a copper like color, potentially resulting in a false positive interpretation.
# Citrate utilization test:-
-Citrate utilization test is used to determine the ability of
bacteria to utilize sodium citrate.
-When an organic acid such as citrate is used as a carbon and
energy source, alkaline carbonates and bicarbonates are
produced ultimately.
-The carbon dioxide that is released will subsequently react
with water and the sodium ion in the medium to produce
sodium carbonate, an alkaline compound that will raise the
pH.
-Growth usually results in the bromothymol blue indicator,
turning from green to blue. The bromothymol blue pH
indicator is a deep forest green at neutral pH. With an
increase in medium pH to above 7.6, bromothymol blue
changes to blue.

19. Result:-Citrate positive: growth will be visible on the slant surface and the medium will be an
intense blue.
-The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise
the pH of the medium to above 7.6, causing the bromothymol blue to change from the original
green color to blue.