BACTERIAL

IDENTIFICATION
 Derivatize to f
methyl este

Table 12.2 Some phenotypic characteristics of taxonomic value

Gas chromatog
Category Characteristics
Morphology Colony morphology; Gram reaction; cell size
and shape; pattern of flagellation; presence of
spores, inclusion bodies (e.g., PHB,a glycogen,
or polyphosphate granules, gas vesicles, (b)
magnetosomes); capsules, S-layers, or slime layers;
stalks or appendages; fruiting-body formation Figure 12.30
identification. (a)
Motility Nonmotile; gliding motility; swimming (flagellar)
motility; swarming; motile by gas vesicles each class, but in
bacterial sources.
Metabolism Mechanism of energy conservation (phototroph, on the carboxylic
chemoorganotroph, chemolithotroph); utilization
the gas chromato
of individual carbon, nitrogen, or sulfur
compounds; fermentation of sugars; nitrogen
height is proportio
fixation; growth factor requirements
Physiology Temperature, pH, and salt ranges for growth;
response to oxygen (aerobic, facultative,
anaerobic); presence of catalase or oxidase;
analyses, fatty
production of extracellular enzymes grown under
Cell lipid chemistry Fatty acids;b polar lipids; respiratory quinones and analyzed
the types and
Cell wall chemistry Presence or absence of peptidoglycan; amino acid
composition of cross-links; presence or absence rium is then c
of cross-link interbridge profiles of tho
Other traits Pigments; luminescence; antibiotic sensitivity; conditions.
serotype; production of unique compounds, for Fatty acid p
example, antibiotics traits, can var
a
PHB, poly-β-hydroxybutyric acid ( Section 2.14).
nential versus
b
Figure 12.30 results, it is ne
Gram Staining

An important differential-staining
procedure used in microbiology
is the Gram stain.
On the basis of their reaction in
the Gram stain, Bacteria can be
divided into two major groups:
gram-positive and gram-
negative
Gram Staining
 BACTERIAL
IDENTIFICATION
General approach for bacterial identification
General approach for bacterial
identification
4 steps : Step 1 : Sampling

Step 2 : Culturing
on isolation media

Step 3 : Performing an
identification technique

Step 4 : Results
= name of bacteria
General approach for bacterial
identification
Different
identification
techniques

Physical methods Genetical methods Biochemical methods

à Based on the à Based on the à Based on the
characterization of characterization of characterization of
proteome of the specifics genes of metabolic pathways
bacteria the bacteria of the bacteria

To identify unknown bacteria , results are

compared to databases
 BACTERIAL
IDENTIFICATION
Metabolism of the bacteria and biochemical
bacterial identification
Metabolism of the bacteria

Bacteria are living cells

- consume nutrients (carbohydrates, proteins…)
- reject metabolic waste.

Bacteria

Enzymes
Nutrients Metabolic
waste

Biochemical techniques for

identifying bacteria are based
on the characterization of
enzymes and metabolic waste
Metabolism of the bacteria

The main metabolic pathways :

Acidic
Carbohydrates
Bacteria molecules

Enzymes

Proteins, aminoacids Ammonia,

For some
These particular
waste produced acidic
are frequently CO2, amino…
molecules, detection requires some
or alkaline
special reagents
à Possibility of detection with a
colorimetric pH indicator
Application to the identification
Example : research the ability to use glucose
Bacteria
inoculation
incubation
Medium + glucose + pH indicator :
-green color for pH = 7
-yellow color for acid pH (pH <7)
Yellow color :
àAcid pH
àProduction of acidic
waste by bacteria
àProves the presence of
enzymes which allow the
use of glucose as nutrient
by bacteria
àBacteria « glucose + »
Application to the identification

How to differentiate bacteria ???

Examples with two bacteria :

-E.coli can use as nutrient glucose, mannose, and arabinose but

not amylose
àProfile = GLU + MAN + ARA + AMY –

-E.tarda can use as nutrient glucose, but non mannose, arabinose

and amylose
àProfile = GLU + MAN - ARA – AMY –

Each bacteria has a specific biochemical profile

Application to the identification
How to identify an unknown bacteria with a
biochemical method ??

4 steps :

1-Incubate bacteria to test in media with different nutrient

(usually 5 to 20)
à one medium for one nutrient

2-After incubation, for each medium, determine the results

positive or negative for each nutrient

3-Write the complete biochemical profile (+ - + + + - - + …)

4-Compare the profile with a database to identify your bacteria

 BACTERIAL
IDENTIFICATION
Identification media
Identification media

Different packaging of identification media

Tubes media

Agar plate Multi-test

media Classics miniaturized
tubes systems
 BACTERIAL
IDENTIFICATION
Multi-test miniaturized system :
The API system
The API system

The most used API system…

API Strep
à Identification of
Streptococcus species

API Staph
à Identification of
Staphylococcus species

API 20NE
à Identification of Non
Enterobacteria
(Pseudomonas for example)

API 20E
à Identification of
Enterobacteria
The API system : introduction

With an unknown bacteria to identify, which API system use ?

… before use API system, it’s necessary to perform

preliminary tests on bacteria to identify

• Preliminary test 1 : Gram stain + microscopic observation

à Distinguishes bacteria according to their form (cocci, rods) and

their response to color (purple = Gram + bacteria, pink = Gram
– bacteria)

• Preliminary test 2 : Bacteria respiratory enzymes test

à Distinguishes bacteria into groups according to the existence of

two enzymes, « oxidase » enzyme, or « catalase » enzyme
 The API system

With an unknown bacteria to identify, which API system use ?

API NH API Listeria

for Neisseria for Listeria

Cocci
Rods Gram+ API 50CH
Gram- for Bacillus
Results
for the
Gram
Catalase+ stain
API Staph Cocci Oxidase +
for Staphylococcus Gram+ Rods Gram-
API 20NE
for no
Enterobacteria
Oxidase-
Catalase-
API 20E
API Strep for Enterobacteria
for Streptococcus
The API system

Introducing the API 20E system for Enterobacteria

identification…

1 bacterial
colony

Fill microtubes gallery

5 mL of sterile water with the bacterial
suspension
The API system

View of API 20E just after seeding…

Nitrogen metabolisms Metabolism of carbohydrates

and specific enzymes

24h / 37°C…..
The API system

API 20 E after incubation…Positive results for all tests :

API 20 E after incubation…Negative results for all tests :

The API system

Example of results for bacteria to test :

1-Reading results :

2-Entering results in the database software:

The API system

Example of results for bacteria to test :

3-Expression of results by software :

Name of the In this example we

identified have a very good
Quality identification of
bacteria identification
bacteria
Salmonella spp
The API system

From the time when bacteria was

collected and the results…à two
days

… there are new and more

rapid methods !!