Bacterial identification

BACTERIAL IDENTIFICATION
Chota alex
Biomedical science

IDENTIFICATION METHODS
The most important task of a bacteriologist is to
identify the pathogens from the clinical sample so
that appropriate treatment can be instituted.

IDENTIFICATION METHODS
The methods fall into three categories :
Phenotypic - morphology (micro and macroscopic)
 Immunological - serological analysis
Genetic techniques

PHENOTYPIC METHODS
Phenotypic Methods : MICROSOPIC ,MACROSCOPIC

PHENOTYPIC METHODS- stages
First stage tests will identify the genus of an unknown bacterium
Or at least, will narrow it down to two closely related genera
Second stage tests will identify the species of an unknown
bacterium
Third stage tests will further differentiate the species into sub-
species or sub-types
All the tests require pure cultures

First stage
 Colony morphology and Gram stain (reaction and cell shape)
 Acid fast stain
 Spores
 Motility
 Catalase
 Oxidase
 It may not be necessary to perform all the first stage tests, depending on the Gram
stain result

Second stage
The choice of second stage tests depends on the genus of the
bacterium
These tests are used to identify most species of clinically relevant
bacteria (pathogens and normal flora) with as few tests as
possible
Common second stage tests include:
Carbohydrate fermentation
Haemolysis
Growth in the presence of inhibitors – high salt, bile
Species-specific tests – e.g., coagulase for S. aureus

PHENOTYPIC METHODS
Macroscopic morphology are traits that can be accessed with
the naked eye such as:
Appearance of colony-size, shape, color.
Pigment
Speed of growth

COLONY
MORPHOLOGY
Form- shape of colon
color, surface, texture and
size
Elevation-side view
Edge- margin

Phenotypic Methods -microscopic appearance
Microscopic Morphology include a combination of
Cell shape
Size
Gram stain
Acid fast reaction
Special structures e.g. Endospores, Granules and
Capsules can be used to give an initial putative
identification.

Gram stain-Differential stain
distinguishing between gram-positive and
gram-negative bacteria
 Narrows possible identities
of an organism
 Excludes many possibilities
 Generally insufficient alone
for diagnosis
e.g., E. coli and Salmonella gram
stains look alike

Gram stain
Sometimes highly suggestive of a
particular microorganism
e.g., Gram-negative rods in ♀ urine E.
coli UTI
e.g., Gram-positive encapsulated
diplococci and numerous white blood
cells in sputum Streptococcus
pneumoniae
Sometimes enough for complete
diagnosis
e.g., Gram-negative diplococci clustered
in white blood cells of male urethral
secretions Neisseria gonorrhoeae

Special stains
Some microbes have unique
characteristics that can be detected with
special staining procedures
e.g., Mycobacterium species possess cell
walls with a high lipid content
Acid-fast stain on sputum is diagnostic for
tuberculosis

Phenotypic Methods- Biochemical Tests
The microbe is cultured in a media with a special substrate
and tested for an end product.
Prominent biochemical tests include:
Enzymes
Carbohydrate fermentation
Acid production
Gas production
Sensitivity to drugs

Phenotypic Methods- Biochemicals….
Enzymes
Catalase test
Oxidase test
Urease test
Coagulase test

Enzymes
 CATALASE TEST
 Catalase is present in most aerobic and facultative anaerobic bacteria (except streptococcus
spp).
 Hydrogen peroxide forms as one of the oxidative end product of aerobic carbohydrate
metabolism.
 If this is allowed to accumulate in the bacterial cells it becomes lethal to the bacteria
 Catalase thus helps in converting H2o2 to H2o and o2
 The presence of the enzyme in a bacterial isolate is evident when a small inoculum is
introduced into hydrogen peroxide and the rapid effervescence of O2 bubbles occurs.

Catalase test….
Positive Negative
Micrococcus Streptococcus sp
Staphylococcus Clostridium
Bacillus
Listeria monocytogenes
Enterobacteriacae
Gonococcus & Meningococcus
Vibriocholerae
Pseudo/Aero/Plesiomonas

Enzymes
OXIDASE TEST
This test depends on the presence of cytochrome oxidase in
bacteria
Procedure- Place a piece of filter paper in petri dish and
add 3 drops of freshly prepared oxidase reagent (1%
solution of tetramethyl-p-phenylene diamine)
Using a sterile glass rod or toothpick, remove a colony of
test organisms from a culture plate and smear it on the
filter paper
Oxidase positive organisms give blue/ dark purple color
within 5-10 seconds, and in oxidase negative organisms,
color does not change.

Oxidase test…..
Positive Negative
Pseudomonas spp. Enterobacteriaceae
Aeromonas spp. Acenitobactoer spp.
Vibrio spp.
Alcaligenes spp.
Neisseria spp.
Haemophilus sps

EnzymesCoagulase test
 This test is used to differentiate Staphylococcus aureus (positive) from
coagulase negative Staphylococci
 When a bacterial suspension is mixed with plasma, this enzyme causes
alteration in fibrinogen.
 leading to precipitation on the staphylococcal cells, causing the cells to
clump.
 Slide test: Positive when there is Macroscopic clumping in 10 seconds or
less in a plasma drop and no clumping in a saline or water drop.
 Tube test: Positive when there is a Clot of any size

pictures
Coagulase Positive : Staphylococcus aureus
Coagulase negative: Staphylococcus epidermidis
Tube test:
-Positive: Clot of any size (a)
-Negative: No clot (b)

Enzymes
Urease test
Some bacteria produce urease an enzyme that hydrolyzes urea
into ammonia and carbon dioxide.
The test for urease production relies on the fact that the ammonia
produced upon hydroysis is alkaline.
The test organism is inoculated into a urea broth that contains
phenol red, a pH indicator, and has a pH of 6.8. At this pH phenol
red is salmon color. However, when the pH rises above 8.1 phenol
red turns a cerise (hot pink) color.
The urease test is useful for differentiating Salmonella which is
urease negative, from Proteus which is urease positive.

Fermentation of sugars and Gas production
Triple Sugar Iron Agar (TSI) test
 TSI agar is used to determine whether a gram negative rod utilizes glucose
and lactose or sucrose fermentatively and forms hydrogen sulphide (H2S).
 TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and
peptone. Phenol red and ferrous sulphate serves as indicators of
acidification and H2S formation, respectively.
 The formation of CO2 and H2 is indicated by the presence of bubbles or
cracks in the agar or by separation of the agar from the sides or bottom of
the tube.
 The production of H2S requires an acidic environment and is indicated by
blackening of the butt of the medium in the tube.

Results interpretation:
Alkaline slant/no change in the butt (K/NC) = Glucose, lactose
and sucrose non-utilizer (alkaline slant/alkaline butt)
Alkaline slant/acid butt (K/A) = Glucose fermentation only.
Acid slant/acid butt (A/A), with gas production = Glucose,
sucrose, and/or lactose fermenter.
Alkaline slant/acid butt (K/A), H2S production = Glucose
fermentation only.
E.g:
A/A, with gas: E. coli
K/A, H2S: Salmonella typhi
K/NC: Pseudomonas aeruginosa

TSI results
A/A, with gas: E. coli
K/A, H2S: Salmonella typhi
K/NC: Pseudomonas aeruginosa

SIM- SULFIDE,INDOLE, MOTILITY
 This is a differential medium. It tests the ability of an organism to do several things:
reduce sulfur, produce indole and swim through the agar (be motile). SIM is
commonly used to differentiate members of Enterobacteriaceae.
 If sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for
H2S production.
 Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to
indole.
 Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color
(indole +).
 Escherichia coli is indole positive.
 SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is
motile then the growth will radiate from the stab mark and make the entire tube appear
turbid.
 Pseudomonas aeruginosa and Proteus mirabilis are motile.

MOTILITY
 If bacteria is motile, there will be growth
going out away from the stab line, and test
is positive.
 If bacteria is not motile, there will only be
growth along the stab line.
A colored indicator can be used to make the
results easier to see.

LIA- Lysine Iron Agar
Lysine Iron Agar was developed to detect lactose fermenting
Salmonellae which are known to decarboxylate lysine rapidly and
produce large amounts of hydrogen sulfide.
This medium is a sensitive medium for the detection of Lactose
fermenting and lactose non-fermenting Salmonella species.
Many strains of this group, ferment Iactose very rapidly thus
suppressing H2S production on Triple Sugar Iron Agar
 It is recommended to use LIA and TSI together for better
discrimination between coliform organisms e.g. Escherichia and
Shigella.

CITRATE
 The citrate test is commonly employed as part of a group of tests distinguish between members of
the Enterobacteriaceae family based on their metabolic by-products.
 The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate
as its sole carbon and energy source.
 Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6.
 Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline
pH's (around 7.6).
 Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color.
 Growth of bacteria in the media leads to development of a blue color (positive citrate).
 Enterobacter and Klebsiella are citrate positive while E.coli is negative.

OTHERS
 BILE SOLUBILITY
 CAMP
 MSA
 HEMOLYSIS
 CHOLERA RED REACTION
 BACITRACIN
 SEROLOGY FOR STREP GROUPS

Immunological Methods
Immunological methods involve the interaction of a microbial
antigen with an antibody (produced by the host immune system).
Testing for microbial antigen or the production of antibodies is
often easier than test for the microbe itself.
Lab kits based on this technique are available for the identification
of many microorganisms

Genotypic Methods
Genotypic methods involve examining the genetic material of
the organisms and has revolutionised bacterial identification
and classification.
Genotypic methods include PCR (RT-PCR, RAPD-PCR), use of
nucleic acid probes, RFLP and plasmid fingerprinting.
Genotypic techniques are becoming the sole means of
identifying many microorganisms because of their speed and
accuracy.

Challenges in Bacterial Identification
Traditional methods of bacterial identification rely on
phenotypic identification of the causative organism
These methods of bacterial identification suffer from two
major drawbacks.
They can be used only for organisms that can be
cultivated in vitro.
Second, some strains exhibit unique biochemical
characteristics that do not fit into patterns that have been
used as a characteristic of any known genus and species.

In the past decade or so, molecular
techniques have proven beneficial in
overcoming some limitations of traditional
phenotypic procedures for the detection and
characterization of bacterial phenotypes

Bacterial identification

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