Identification of Bacteria

Dr K Ravi Kiran
Meningitis
 Sample collection
Laborato
ry Transport
diagnosis
Process for
identification
 CONVENTIONAL

Identificatio AUTOMATION
n Methods

MOLECULAR
 CONVENTIONAL

• Staining
• Culture of bacteria
• Biochemical reactions
Identificatio
n Methods
AUTOMATION

MOLECULAR
Conventional
method
Direct microscopy by Gram staining

GPC GNB
Direct microscopy (gram staining)

Selection of culture media

Colony morphology & gram staining

Biochemical reactions

Antimicrobial Susceptibility Test

Colony morphology
• Shape - circular, irregular
• Size - in millimetres
• Elevation - flat, elevated
• Surface and Margins - smooth, irregular
• Colour & Pigmentation
Pigmentatio
n
Biochemical
Reactions/Tests
Sugar Sugar media is used
fermentation and checked for acid
test production
Durham’s tube is used
for gas collection
 • When a drop of hydrogen peroxide (3% H2O2 ) is added to a
Catalase colony bacteria - effervescence or bubbles appear in positive
reaction
Test • Differentiate - Staphylococcus (positive) from Streptococcus
(negative).
18
Oxidase Test

• Detects the presence of

cytochrome oxidase enzyme
in bacteria
• Oxidase positive organisms
produce - deep purple)
• Eg: Pseudomonas ,
Vibrio, Neisseria,
Bacillus

19
Indole Test
• Indole positive - Red colored ring
is formed near the surface of the
broth.
• Eg: Escherichia coli, Proteus
vulgaris, Vibrio cholerae
• Indole negative - Yellow colored
ring is formed near the surface of
the broth,
• Eg: Klebsiella pneumoniae,
Proteus mirabilis, Pseudomonas,
Salmonella
20
Citrate Utilization Test

• Citrate test - positive for Klebsiella

pneumoniae, Citrobacter,
Enterobacter, etc.
• Negative for Escherichia coli,
Shigella, etc.
21
 Urea Hydrolysis Test
• Urease test is positive for -
Klebsiella pneumoniae, Proteus
species, Helicobacter pylori,
Brucella, etc.
• Urease test is negative for -
Escherichia coli, Shigella,
Salmonella, etc.

22
Triple Contains three sugars - glucose,
sucrose and lactose in the ratio of
Sugar 1:10:10 parts.

Iron Uninoculated TSI medium - red in

color
(TSI)
Agar After inoculation - the medium is
incubated at 37°C for 18–24
Test hours.
23
 •Interpretation: detects three properties of
Triple bacteria, such as
Sugar • Fermentation of sugars to produce acid
Iron • Fermentation of sugars to produce gas
(TSI)
• Production of H2S (hydrogen Sulphide)
Agar Test

24
Triple
Sugar
Iron
(TSI)
Agar Test

25
Reactions in TSI Examples
Acidic slant/acidic butt ≥2 sugars fermented - (1) glucose, (2) lactose
or/and sucrose
A/A, gas produced, no H2S Escherichia coli, Klebsiella pneumoniae

Alkaline slant/acidic butt Only glucose-fermenter group

K/A, no gas, no H2S Shigella
K/A, no gas, H2S produced (small amount) Salmonella Typhi

K/A, no gas, H2S produced (abundant) Proteus vulgaris

K/A, gas produced, H2S produced (abundant) Salmonella Paratyphi B

K/A, gas produced, no H2S Salmonella Paratyphi A

Alkaline slant/alkaline butt Non-fermenters group
K/K, no gas, no H2S Pseudomonas, Acinetobacter
Interpretation (Analysis)
Series of tests
& observation
will lead to
definitive
identification of
the bacteria
Review
• Describe briefly how you proceed with laboratory diagnosis
in suspected bacterial infections ?
• Name some of the biochemical tests that you know & their
use ?
• Name the sugars present in TSI test ?
• What are the applications of Gram Staining ?
Thank you