Here u can find the information, how u can identify the unknown Bacteria .Different biochemicals test for identification of bacteria

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