Identification of Bacteria using:

Staining Techniques (Based on

bacterial cell structure)
&
Biochemical Tests (Based on
Biochemical activities of the
bacterial cells)
2024
Introduction to Microbiology
- Size (1cm=10mm, 1mm=1000µm)-0.2 to 2 µm in size.
- Microscopy (Simple, Compound & Electron).
- Procaryotic = Before nuclei (Bacteria & Blu-green algae) and
Eucaryotic = True nuclei (Fungi, algae, protozoa, plants &
animals)organisms.
 Staining Techniques:
• Every bacterial cell is individually colorless, hence needs a stain to be
observed under microscope for enhanced visualization.
• Staining techniques in microbiology use dyes and stains to enhance the
contrast and structure of biological specimens at a microscopic level.
• They are basically of 2 types: Simple staining and Differential staining
techniques.
• Simple staining: Stains all the bacterial cells without differentiating between
them.
• Differential staining: Used for identifying various stains of bacteria.
Various differential staining techniques are-
- Gram staining
- Acid fast staining
- Endospore staining
- Capsule staining
- Flagella staining
- Ziehl-Neelsen/Acid-fast staining
- Periodic Acid-Schiff staining etc.,.
Types of stains/dyes used:
 The chemicals that are used in various staining techniques are
basically of three types:
 Acidic stains – These are negatively charged acid radicals and
they combine with the positive charges present in the bacterial
cells to impart colors.
Examples: Eosin, Acid fuchsine, Malachite green & Indian ink.
 Basic stains - These are positively charged basic radicals that
combine with the negatively charged particles in the cytoplasm
of the bacteria to impart colors. Generally used for simple
staining.
Examples: Methylene blue & Crystal violet.
 Neutral stains - Both positively charged and negatively charged
radicals are present in the stain and they impart different colors
to different components of the bacterial cell.
Examples: Giemsa's stain & Leishman’s stain.
Positive staining and Negative
staining
Positive staining:
In positive staining, the actual cells (to be observed) are colored and they
appear in a clear background.
Examples: Simple staining techniques & Differential staining techniques.
 Negative staining:
In negative staining, the cells remain clear/uncolored and the background is
colored to create a contrast that aids in better visualization of the image.
Examples: Nigrosine & Indian ink.
Preparation of a bacterial smear/slide
before staining:
 Smear preparation: Distribution of bacterial cells on a slide for the purpose of
viewing them under a microscope.
 Clone (colony formed from a single cell)& Strain (usually a pathogen obtained
from another living organism)
Simple staining technique:
 In simple staining technique, all the bacterial cells are stained with the dye. So,
only the presence or absence of bacterial cells can be determined using this
technique.
 The principle of simple staining is to use a single dye to stain a sample so that cells
are more visible under a microscope.
 Examples: (Any basic dyes) Safranin, Malachite green, Loeffler’s Methylene Blue,
Dilute Carbol Fuchsin (made by diluting Ziehl-Neelsen’s stain 10-20 times with
water).
 Procedure:
- Make a thin smear on a slide.
- Heat fix the smear by passing the slide 2-3 times gently over the Bunsen flame
with the smear side up.
- Pour Loeffler’s methylene blue over the smear and allow it to stand for 3 minutes.
- Wash the stained smear with water and air-dry it.
- Observe the smear first under low power (10x) objective and then under oil-
immersion (100x) objective.
- Observe the presence of micro-organisms.
 Safety: Dyes used for bacteriological staining are usually aniline dyes, which can be
carcinogenic. Avoid contact, inhalation, or ingestion of the dye.
Gram Staining technique:
 A differential staining technique that uses different stains to identify
bacteria as either Gram-positive or Gram-negative. This technique was
developed by Danish bacteriologist Hans Christian Gram in 1882.
 This staining procedure is used to identify bacteria based on their cell
wall composition. There are two types of Gram’s staining, and the
bacteria can be divided into gram positive or gram negative bacteria.
 It uses:
- primary stain - crystal violet for the staining of cell walls,
- mordant - iodine (Mordants are chemical agents that are used along
with dye to make the specimen stainable, which otherwise is unstainable,
- decolorizing agent - ethanol/acetone/mixture of both – for rapid
decolorization and
- counterstain - safranin or fuchsin.
Difference between gram +ve and gram –
ve bacteria:
The peptidoglycan is the major constituent of the cell wall of gram +ve
bacteria (50-90%) whereas in the gram –ve cell wall its presence is only 5-
10%.
Peptidoglycan layer in gram positive bacterial cell wall:
EXAMPLES:
Gram-positive bacteria Examples include:
- Bacillus: A rod-shaped bacteria that can be free-living or parasitic.
- Corynebacterium: A rod-shaped or club-shaped bacteria that can be a pathogen in
humans, animals, or plants.
- Listeria: A rod-shaped bacteria that can be an opportunistic pathogen in humans and
animals.
- Staphylococcus aureus: A bacteria found in the nose, respiratory system, and on the
skin.

Gram-negative bacteria Examples include:

- E. coli: A rod-shaped bacteria that is part of the natural flora of animals and humans.
- Pseudomonas aeruginosa: A rod-shaped bacteria that can survive in a variety of
environmental conditions, such as water and soil.
- Enterobacter: A rod-shaped bacteria that can thrive in both aerobic and anaerobic
environments.
- Klebsiella: A bacteria that can inhabit both humans and animals and can survive in
water and on inanimate objects.
The outer phospholipid membrane of the gram –ve bacteria protects it from
penicillin and lysozymes
Acid-fast staining:
 This technique is used to stain acid-fast bacteria belonging to the
genus Mycobacterium especially Mycobacterium tuberculosis,
Mycobacterium leprae and also for Nocardia (pulmonary infection)
that do not stain with Gram’s staining.
 It is a modification of Ehrlich’s method (1882) and is also known as
Ziehl-Neelsen stain.
 Acid-fastness of the acid-fast bacilli is attributed to the presence of
unsaponifiable wax fraction called mycolic acid in their cell-walls.
 In this technique:
- Carbol fuchsin is used as a stain,
- (Application of) heat is used as mordant and
- Methylene blue is used as a counterstain.
Mycolic acids are long-chain fatty acids that are a major
component of the cell walls of certain bacteria, including the
tubercle bacillus Mycobacterium tuberculosis.
Acid-fast staining technique:
It's a rapid and inexpensive method that uses a
primary stain, carbol-fuchsin, to stain acid-fast
organisms bright red. The background of the sample
stains blue.
Slide after acid-fast staining:
Biochemical Tests: I M ndole ethyl red V
oges-Proskauer iC
itrate Tests
 Biochemical tests are used for identification of bacteria
based on the differences in the biochemical activities of
different bacteria.
 Bacterial physiology differs from one type of organism to
another and that is the basis for this test.
 The ability of bacteria to form organic compounds by
metabolizing certain carbohydrates and related compounds
is a widely used method for the identification of micro-
organisms.
 IMViC Tests are a group of 4 individual tests that are used in
the lab to identify an organism in the coliform group (A
coliform is a gram –ve, aerobic or facultative anerobic rod
that produces gas from lactose within 48 hours. The
presence of some coliforms indicates fecal contamination).
Indole (Production) Test
 Demonstrates the ability of certain bacteria to decompose the amino acid
tryptophan present in peptone water (culture medium) to indole.
 Indole is then tested for by adding few drops of Kovac’s reagent which gives a pink
ring in the presence of indole.
 Procedure: The organism is inoculated in peptone water and after incubation at 37
degree Celsius for 24 hours, Kovac’s reagent is added.
Tryptophan Tryptophanse Indole + Pyruvic acid + Ammonia
HCl,alcohol
p-dimethylaminobenzaldehyde + Indole Quinoidal red-violet
compound
 Interpretation:
If a pink ring is produced, the organism is indole +Ve (E. coli).
If a yellow ring is produced, the organism is indole –Ve (Klebsiella).
Methyl Red Test:
 Demonstrates the ability of microbes to oxidize glucose with production and
stabilization of high content of acid end-product.
Voges-Proskauer Test:
 Some bacteria ferment glucose with production of acetyl methyl carbinol.
 Bacteria is grown in glucose phosphate peptone water for 48 hours.
 Then KOH is added to test for acetyl methyl carbinol (acetoin) formation.
 Interpretation: If an eosin pink color is produced, VP reaction is +Ve.
Citrate Utilization Test
 Bacteria are inoculated on a medium containing sodium citrate and a pH
indicator such as bromothymol blue.
 The medium also contains inorganic ammonium salts, which are utilized as sole
source of nitrogen.
 Use of citrate involves the enzyme citrase, which breaks down citrate to
oxaloacetate and acetate.
 Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO2).
 Production of sodium bicarbonate (NaHCO3) as well as ammonia (NH3) from the
use of sodium citrate and ammonium salts results in alkaline pH.
 This results in a change of the medium’s color from green to blue.