The document discusses techniques for classification and identification of microorganisms, focusing on methods such as morphological, physiological, biochemical, immunological, and molecular approaches. It details various staining techniques, including simple and differential staining, and specific biochemical tests that help identify bacterial species based on metabolic needs. Additionally, it covers molecular methods like ribotyping and PCR for microbial DNA evaluation and provides references for further reading.

1. .
Submitted by – Ankur Kumar
M.Sc. Microbiology
Central University Of Haryana

2.  Classification and identification?
 Classification- Placing an organism in groups of
related species. List of characteristics of known
organisms.
 Identification- Matching characteristics of an
unknown to list of known organisms.

3.  Identification and characterization
 Morphological approach
 Physiological and Biochemical approach
 Immunologial approach
 Molecular approach

5.  Staining:-
 Biochemical technique of coloring specimen.
 Use to increase visibility of microorganisms being
studied and to identify the shape of bacteria.
 Use to determine the morphological features of
microorganisms.
 Use to differentiate and classify
microorganisms.
 Use to detect bacterial parts such as capsule,
spores, flagella or inclusion bodies.

6.  Types of staining
 Simple Staining Technique:- Only one stain is
used. Crystal Violet, and Methylene Blue are
examples of basic dyes used in simple staining
technique.
 Differential Staining:- Used to classify microbes
into separate groups based on their distinct
staining properties.
 Use of two contrasting stains seperated by
decolurizing agents
 Examples- Gram Stain, Acid-Fast Stain and Spore-
Staining .

7.  Gram staining
 G+ve retained the crystal violet so appear Purple
 G-ve did not retain crystal violet so appear Pink

9.  Acid-Fast Stain
 Acid-fast stain is a differential stain used to identify
acid fast organisms.
 First introduced by Dr. Paul Ehrlich.
 Also known as the Ziehl-Neelson’s staining.
 Acid fast bacteria appear as bright red bacilli against a
light blue background.
 Used to differentiate Mycobacteria (M. tuberculosis)
from other bacteria.
 EX- Corynebacterium ,Nocardia spp.,
Rhodococcus spp.

10.  Acid fast bacteria do not decolourize on applying
acid alcohol so appear Red
 Non acid fast bacteria appear blue after applying
methylene blue. (counter stain)

11.  Special Staining
 Negative Staining - India Ink or Nigrosine
is used to stain capsule.
 Schaefer-Fulton- Malachite Green and
Safranin is used to stain endospores.

12.  Hanging Drop Method
.
 To examine the cell motility and morphology by
taking the living microorganism from the media.

13.  Observations
 Inside edge of drop
motile cell move
independently .
 All other cells seem to
show Brownian
movement.
Applications –
o Cytological changes.
o Motilty test- Ex- Vibrio cholerae.

14.  Cultural charateristics
Characteristics of growth in broth.
• Clear – no growth
• surface
• subsurface
• sediments
• Amount of Growth

15.  Characteristics of colonies on plates
 size, color, form, elevation, and margin

16. Characteristics of growth on slants
 amount of growth
 colour
 Opacity
 form

17.  Biochemical Tests
 Each species of bacteria has specific metabolic
needs and relies on different enzymes to fuel those
unique needs.
 The presence of catalase, gelatinase, oxidase,
urease can be used to identify the species of
bacteria.
 Biochemical characterization relies on the
characteristic patterns of substrate utilization,
metabolic product formation and sugar formation,
etc.

18. .
1.Fermentation tests- whether the microbe is
capable to carry out various fermentation reactions or
not.
Ex., Mixed-acid fermentation (methyl red test)
2.Hydrolytic Reactions:- starch , casein, fat,
tryptophan, urea, phenylalanine.

19. Starch Hydrolysis
Positive test: A clear zone
around the line of growth
indicates that the organism has
hydrolyzed starch.
Negative test: A blue, purple, or
black coloration of the medium
(depending on the concentration of
iodine).

20. .
3.oxidative tests- for respiratory metabolism.
 Eg., Catalase, and Nitrate tests.
The presence of the enzyme in a bacterial isolate is evident when
a small inoculum is introduced into hydrogen peroxide,
and the rapid elaboration of oxygen bubbles occurs.

21.  IMViC Tests
 IMViC is group of four different tests can be used to
differentiate organisms.
 I: indole test – detects ability of bacteria to produce indole.
 M: methyl red test – for mixed acid production.
 V: Voges-Proskauer test-to metabolize pyruvate into
acetoin.
 C: citrate test- to utilize citrate as a sole source of energy.

22. .
Name of
Bacteria
Indole Test MR Test VP Test Citrate Test
E. coli + ve + ve – ve – ve
Klebsiella
pneumoniae
– ve – ve + ve + ve
Klebsiella
oxytoca
+ ve – ve + ve + ve
Pseudomona
s aeruginosa
– ve – ve – ve + ve
Salmonella
Paratyphi A
– ve + ve – ve – ve
Salmonella
Typhimurium
– ve + ve – ve + ve

23.  Molecular approaches
Ribotyping- rRna sequencing.
 Polymerase Chain Reaction (PCR)- can be
used to amplify a small amount of microbial DNA in
a sample.
o The presence or identification of an organism is
indicated by amplified DNA

24.  Nucleic acid hybridization
 Single strands of DNA or RNA, from related organisms
will hydrogen bond to form a double stranded molecule;
this bonding is called nucleic acid hybridization.

25. Taxonomical
characterization
of newly
isolated strains.

26.  Referance:
 Microbiology, a loboratory manual ,12th Edition
James G. Cappuccino
 Prescott's Microbiology 9th Edition, by Willey.
 https://www.sciencedirect.com
 https://www.ncbi.nlm.nih.gov/.
 https://microbenotes.com