ID EN TIFIC ATIO N A N D

C U LTIV ATIO N O F
M IC R O R G A N IS M S
 Identification of organisms is needed to give
the diagnosis of a disease.
For identification :
COLLECTION OF SAMPLE;
Sample should be appropriate, this requires
understanding of pathogenesis of the infection.
Avoid the collection of normal flora.
Container should be sterile
Transportation should be proper and immediate
& storing it correctly if delay in transport
 PROCESSING OF SPECIMEN
1. Staining of specimen after preparing the
smear and examined under oil immersion
lens.
2. Obtaining a pure culture of the organisms by
inoculating in to bacteriological medium.
3. Growing the organisms on selective media
4. Biochemical reactions
5. Sp antibody reaction by serological reactions
6. Molecular methods.
 Processes Involved

Staining
Microscopy
Microbial Culture
Colony Morphology
Biochemical Characteristics
Immunological Methods
Ultrastructure Studies
Staining
Used to increase visibility of microorganisms
being studied
Used to identify the shape of bacteria
Used to determine the morphological features
of microorganisms
Used to detect contamination
Used to differentiate and classify
microorganisms (differential stains)
Used to detect bacterial parts such as capsule,
spores, flagella or inclusion bodies (special
stains)
 STAINING
Direct smear of sputum, pus, body fluids.but not
required in urine , blood e.t.c.
Bacteria have clear cytoplasm so Refractive index of
medium in which bacteria are growing and that of
bacteria are same, so bacteria can not be visualised.
Staining is must for their recognition .
Methods : Simple staining using: methyl violet,
carbol fuchsin,
Differential staining: some organisms and their
morphological features , bring out special characters
with differential stains e.g flagellar stain, capsular
stain, metachromatic granules.
 MAKING OF SMEARS: fluid material e.g broth culture,
urine, sputum, pus, e.t.c one loopful is taken up with
inoculating wire and spread in an area of 15-20 m.m thinly
on the slide .solid culture is first emulsified in normal
saline then smear is prepared.
Smear is then dried and kept over a flame.
Fixation
For killing of vegetative bacteria
Bacteria stick to the surface of slide and are preserved
from undergoing autolytic changes.
Fixation renders the bacteria permeable to stain .
Methanol fixation is better than heat fixation as:
Greater control of decolourization
Liquid specimens are prevented from washing off.
Preserves rbc,s
Back ground is clear
Fixation

The process by which the internal and

external structures of microorganisms are
preserved and fixed in place.
Heat fixation fixation by means of
application of heat
The prepared smear of microorganisms is gently
heated and air-dried.
Chemical fixation involves the use of
chemicals such as ethanol and
formaldehyde.
Sim ple Staining Technique

Only one stain is used

Crystal Violet, Carbol Fuchsin, and
Methylene Blue are examples of
basic dyes used in simple staining
technique.
D iff
erentialStaining

Used to classify bacteria into separate

groups based on their distinct staining
properties
Makes use of a primary stain, a
decolorizer, and a counterstain
Gram Stain and Acid-Fast Stain are
examples
 DIFFERENTIAL STAINS:
Grams staining
ZN staining
Flourescent dyes
Positive staining methods
Negative staining methods
Supravital staining
Vital staining
G ram Stain
Differentiates bacteria into two groups: Gram
positive and Gram negative
Stain mechanism is generally related to the
thickness of the cell wall, pore size and
permeability properties of intact cell envelope
Makes use of a primary stain (Crystal Violet), a
mordant (Grams Iodine) a decolorizer (Alcohol or
Acetone) and a counterstain (Safranin).
Gram positive organisms stain blue while gram
negative organisms stain red.
Most cocci are gram positive; a third of all cocci,
half of the bacilli, and all the spiral bacteria are
gram negative.
Exam ples ofG ram Positive Bacteria

Staphylococcus aureus

Streptococcus pyogenes

Clostridium perfringens

Listeria monocytogenes
Exam ples ofG ram N egative Bacteria

Escherichia coli

Haemophilus influenzae

Vibrio cholerae

Neisseria meningitidis
GRAM STAINING:
Principal: some bacteria when treated with
basic P-rosaniline dye e.g methyl violet,
gentian violet, take purple color with addition
of iodine, fixed , decolorization with acetone,
some organisms are decolorised which then
take counter staining and are called gram
negative, others retain the first dye crystal
violet .
Iodine acts as mordant causes di iodo complex
which can not be removed after decolorisation.
 First Gram staining done by Christian
Gram(1884)
Genetian violet as 1st stain, Gm +ve: dark
purple
Lugols iodine as mordant Gm ve :red
Bismarck brown as counter stain yeast cells :dark
purpl
Presently Grams staining nuclei of pus
cells:
Crystal violet as 1st stain, pink
Grams iodine as mordant
Acetone as decoloriser
Dilute carbol fuchsin as counter stain
 Modifications :
Preston Morell modification
Jensen modification
Kopeloffs & Beerman modification
Disadvantages :
Gram positive appear Gram negative or vice versa:
Smears from old culture
If patient on antibiotics , cell wall of organisms
damaged
Bacteria lose their osmotic integrity by mechanical shakers
e.t.c
Over or improper decolorisation with acetone
Thick smear where decolorisation is not proper
USES:
Number of microrganisms: rare , scanty, moderate, many.
Morphology: cocci, spiral, rod shaped, comma shaped
Arrangement :clumps, chains, pairs,
Gram positive bacilli, chinese letter pattern , smear from throat swab may be
corynaebacteriae diptheriae.
Fungi ,yeasts gram positive
Why some organisms take gram stain
oIntegrity of cellwall in gram positive not permeable to 1ST stain becouse thick
peptidoglycan in cellwall.
o Normally basic stains used because protoplasm is acidic and takes basic stains easily.
oIsoelectric point of cell wall or gram positive organisms is 2.3,basic dyes in gram positive
more easily taken up .Gram negative 4-5 isoelectric point.
oIodine uptake better in gram positive forming diiodine complex ,absent in gram
negative .
oGram positive have mucoproteins in cell wall when stain added , mucoprotein pores
constrict dye retained .In gram negative cellwall, lipopolysacharide is present. Acetone
dissolves lipid, pore size increases 1st dye moves out .
O rganism s w hich are not stained by gram
staining

Mycobacyteria : mycolic acid in the

cellwall
Spirocaetes : Very thin
Mycoplasma : deficient cellwall
Chlamydia : I/ cellular
Rickettsia : I/ cellular
ZN STAIN IN G /ACID FAST STAIN IN G `

Devised by Ehrlich in 1882 modified by Ziehl and

Neelsen.
Distinguishes the tubercle bacilli which are acid fast
from others.
Normal aniline dye solutions do not readily
penetrate the substance of tubercle bacilli and are
therefore unsuitable staining. However with the
use of powerful staining soln. that contains phenol
and by application of heat the dye can be made to
penetrate the bacillus. Once stained the bacilli will
stand the action of powerful decolorizing agents
and retain the stain while all other structures the
smear are decolorized.
Acid-Fast Stain
Used to differentiate Mycobacteria (M.
tuberculosis) from other bacteria
Mycobacteria are lipophilic or difficult to stain.
Once stained, Mycobacteria are resistant to
counterstain.
Nocardia are partially acid fast
Makes use of a primary stain (Kinyoun Carbol
Fuchsin), decolorizer (Acid Alcohol), and
counterstain (Methylene Blue).
Acid fast bacteria appear as bright red bacilli
against a light blue background.
Reagents used:
Strong carbol fuchsin(alcohol
&phenol)
Decolorizing agents -20% HSO
Counter stain-methylene blue
Method
AFB-dark pink rest all blue
Factors responsible for acid fastness

High lipid content of cellwall also called as Mycolic acid , a waxy

substance.

Integrity of cell wall which gives a selective permeability to the

movement of dye and decolorising agent.

Acid fastness can be destroyed if organisms are exposed to anti

tubercular drugs like Isonized there fore cell wall is destroyed.

The lipids are tightly bound in tubercle bacilli . lipid soluble chemicals
make organisms non A.F.

Some organisms like Nocardia, parasitic oocysts like cryptosporidium,

cyclospora are also acid fast but not as much as T. bacilli hence in
these decolorisation is done with 5% HSO instead of 20% HSO
also called as modified A.F staining.
 Modifications
Kinyon stain
Flourescent stain
Gabbets stain
SpecialStaining Techniques

Negative Staining using India Ink or

Negrosine is used to reveal the
presence of capsule
Schaefer-Fulton using Malachite
Green and Safranin is used to stain
endospores
The Leifson Method (pararosalanine),
and Gray Method (basic fuchsin) are
used to stain flagella.
M icrobialCulture

Makes use of a growth environment

called culture medium
Microorganisms form culture when
inoculated in or on the medium.
May be purely chemical (defined or
synthetic), may contain organic
materials, or may consist of living
organisms.
Culture m ethods

These provide additional information for

the identification of bacteria. Most
bacteria grow when culture medium
contains required ingredients in the
correct amount at correct pH.
Incubation is done in an atmosphere
and temperature best suited to there
metabolism aerobic and anaerobic .
Culture is done on solid medium &liquid
medium .
Types ofCulture M edium

Nutrient broth and Nutrient agar used

to cultivate bacteria. Both contain
proteins, salts, and growth enhancers.
Selective Medium enhances the growth
of a desired organism while retards the
growth of unwanted organisms.
Differential Medium does not retard
growth but provides environment where
different bacteria can be differentiated
from each other.
Types ofCulture M edium

Nutrient broth and Nutrient agar used

to cultivate bacteria. Both contain
proteins, salts, and growth enhancers.
Selective Medium enhances the growth
of a desired organism while retards the
growth of unwanted organisms.
Differential Medium does not retard
growth but provides environment where
different bacteria can be differentiated
from each other.
Selective M edium

MacConkey Agar retards the growth of Gram + bacteria

and enhances the growth of Gram - bacteria
Selective M edium

Thayer-Martin Medium favors the growth of Neisseria

gonorrhea only
Selective M edium

Lowenstein-Jensen medium is
used for the detection of M.
tuberculosis
D iff
erentialM edium

BLOOD AGAR PLATE (BAP)

D iff
erentialM edium

MacConkey Agar is used to differentiate lactose

fermenters from non-lactose fermenters
CU LTURAL CH ARACTERS

Provide additional information for the identification of

the bacterium. While studying colonies on solid
medium , following features are noted.:
Shape :circular , irregular ,
Size
Elevation : convex , concave , umbonate , umblicate
Margins : bevelled
surface: : smooth , wavy, rough , granular, papillate ,
Edges : entire, undulate, crenate, fimbriate ,
Colour
Structure : opaque, transulucent or transparent ,
Colony M orphology
S. Pneumoniae in
P. aeroginosa
BAP
Colony M orphology
 Liquid medium shows turbidity or
depoisits or pellicle.
Degree of growth scanty moderate ,
or abundant can be known.
Biochemicals
To see the productions of certain end products like
indole , HS, nitrite when grown in suitable medium.
Certain enzymes are produced by bacteria like
oxidase, catalase, urease, collagenase, lecithinase,
gelatinase.
Analysis of end products by thin layer
chromatography.
Precautions
Inoculum from pure culture.
Controls
Sterility testing of each batch
 CARDOHYDRATE FERMENTATION TEST
(Sugar Fermentation Test)
Aim: To determine the ability of microbes to ferment carbohydrates with the production of
an acid and/or gas.

Principle:
Sugars are metabolized through different metabolic pathways depending on types of
microbial species and aerobic or anaerobic environment .If fermenting bacteria are grown in a
liquid culture medium containing the carbohydrate, they may produce organic acids as by-
products of the fermentation. These acids are released into the medium and so lower pH of
medium. If a pH indicator such as phenol red or bromocresol blue is included in the medium,
the acid production will change the medium from its original color to yellow.
Gases produced during the fermentation process can be detected by using a small,
inverted tube, called a Durham tube, within the liquid culture medium. If gas is produced, the
liquid medium inside the Durham tube will be replaced; by the gas in the form of a bubble.

Interpretation:
If the medium changes from colorless to yellow and gas bubble is found in Durhams tube
then it indicates acid and gas production. In some cases gas may not be evolved during the
process. If no change observed in the colour of medium then sugar is not degraded by the
organism.
CARBOHYDRATE FERMENTATION
TEST (DURHAM TUBES)

Nutrient Broth + Respective Sugar

From left to right: uninoculated
positive, and negative.
 TSI (TRIPLE SUGAR IRON) AGAR TEST
Aim: To differentiate among and between the members of
Enterobacteraceae family.
Principle:
Study and identify the organisms belonging to Enterobacteraceae
family.
It is also used to distinguish the Enterobacteriaceae from other gram-
negative intestinal bacilli (by their ability to catabolize glucose, lactose,
or sucrose, and to liberate sulfides from ferrous ammonium sulfate or
sodium thiosulfate. )
TSI agar slants contain a 1% concentration of lactose and sucrose, and
0.1% glucose.
The pH indicator, phenol red, is also incorporated into the medium to
detect acid production from carbohydrate fermentation.
The uninoculated medium is red in colour due to presence of phenol
red dye.
 TSI (TRIPLE SUGAR IRON) AGAR TEST
Aim: To differentiate among and between the members of
Enterobacteraceae family.
Principle:
Study and identify the organisms belonging to Enterobacteraceae
family.
It is also used to distinguish the Enterobacteriaceae from other gram-
negative intestinal bacilli (by their ability to catabolize glucose, lactose,
or sucrose, and to liberate sulfides from ferrous ammonium sulfate or
sodium thiosulfate. )
TSI agar slants contain a 1% concentration of lactose and sucrose, and
0.1% glucose.
The pH indicator, phenol red, is also incorporated into the medium to
detect acid production from carbohydrate fermentation.
The uninoculated medium is red in colour due to presence of phenol
red dye.
The indicator is pink at alkaline pH and yellow at acidic pH, at neutral
pH it remains red.
The following reactions may occur in the TSI tube:
Yellow butt (A) and red slant (K) due to the fermentation of glucose
(phenol red indicator turns yellow due to the persisting acid formation in
the butt). The slant remains red (alkaline) (K) because of the limited
glucose in the medium and, therefore, limited acid formation, which does
not persist.
A yellow butt (A) and slant (A) due to the fermentation of lactose
and/or sucrose (yellow slant and butt due to the high concentration of
these sugars) leading to excessive acid formation in the entire medium.
Gas formation noted by splitting of the agar.
Gas formation (H2S) seen by blackening of the agar.
Red butt (K) and slant (K) indicates that none of the sugars were
fermented and neither gas nor H2S were produced.
The indicator is pink at alkaline pH and yellow at acidic pH, at neutral
pH it remains red.
The following reactions may occur in the TSI tube:
Yellow butt (A) and red slant (K) due to the fermentation of glucose
(phenol red indicator turns yellow due to the persisting acid formation in
the butt). The slant remains red (alkaline) (K) because of the limited
glucose in the medium and, therefore, limited acid formation, which does
not persist.
A yellow butt (A) and slant (A) due to the fermentation of lactose
and/or sucrose (yellow slant and butt due to the high concentration of
these sugars) leading to excessive acid formation in the entire medium.
Gas formation noted by splitting of the agar.
Gas formation (H2S) seen by blackening of the agar.
Red butt (K) and slant (K) indicates that none of the sugars were
fermented and neither gas nor H2S were produced.
TSI AGAR TEST
 OXIDASE TEST
Aim: To determine the ability of microbes to produce Oxidase enzyme
Principle:
Oxidase enzyme plays a key role in Electron Transport Chain during
aerobic respiration.
Cytochrome Oxidase catalyzes the oxidation of reduced Cytochrome
by molecular oxygen (O2), resulting in the formation of H2O and H2O2.
Aerobic as well as some facultative anaerobes and microaerophillic
bacteria shows oxidase activity.

Purple
 CATALASE TEST
Aim: To determine the ability of an organism to produce catalase.
Principle:
Certain organisms produce hydrogen peroxide during aerobic
respiration and sometimes extremely toxic superoxide
radicals.
These are extremely toxic because they are powerful
oxidizing agents and destroy cellular constituents very rapidly.
A bacterium must be able to protect itself against such O2
products or it will be killed.
Many bacteria possess enzymes that afford protection against
toxic O2 products.
 CATALASE TEST
Aim: To determine the ability of an organism to produce catalase.
Principle:
Certain organisms produce hydrogen peroxide during aerobic
respiration and sometimes extremely toxic superoxide
radicals.
These are extremely toxic because they are powerful
oxidizing agents and destroy cellular constituents very rapidly.
A bacterium must be able to protect itself against such O2
products or it will be killed.
Many bacteria possess enzymes that afford protection against
toxic O2 products.
 Catalase Test on Slants

Tryptic soy agar slants

(a) Staphylococcus aureus,
catalase positive. Notice the bubbles of oxygen (tube on the left).
(b) Enterococcus faecalis,
catalase negative; note the absence of bubbles (tube on the right).
CATALASE TEST
PURPOSE

To differentiate members of the family Microco-

coccaceae (including Staphylococcus) which are
catalase positive from Streptococcus species
which are catalase negative.

To differentiate Listeria monocytogenes and

corynebacteria(catalase positive) from other gram
positive, non-sporeforming bacilli.
PRINCIPLE
The enzyme catalase catalyzes the release of
water and oxygen from hydrogen peroxide.

catalase
2 H202 -------------- 2 H20 + O2
bubbles or
effervescence
INTERPRETATION

Positive rapid and sustained appearance of

bubbles or effervescence

Negative lack of bubble formation 30 seconds

later
 A B

Catalase test

A. Positive Staphylococcus aureus.

B. Negative Streptococcus pyogenes
COAGULASE TEST
PURPOSE

To determine the ability of the organism to produce

coagulase which clots plasma.

To distinguish the pathogenic coagulase positive

staphylococcus from the nonpathogenic coagulase
negative staphylococcus.
PRINCIPLE
Coagulase is an enzyme that converts soluble
fibrinogen into soluble fibrin.

Two forms of coagulase

bound coagulase (clumping factor) detected in the
coagulase slide test

can directly convert fibrinogen to insoluble fibrin

and causes the staphylococci to clump together
PURPOSE

To determine the ability of the organism to produce

coagulase which clots plasma.

To distinguish the pathogenic coagulase positive

staphylococcus from the nonpathogenic coagulase
negative staphylococcus.
PRINCIPLE
Coagulase is an enzyme that converts soluble
fibrinogen into soluble fibrin.

Two forms of coagulase

bound coagulase (clumping factor) detected in the
coagulase slide test

can directly convert fibrinogen to insoluble fibrin

and causes the staphylococci to clump together
 free coagulase detected in the coagulase tube test

reacts with a globulin plasma factor(coagulase

reacting factor-CRF) to form a thrombinlike factor,
staphylothrombin--- catalyzes the conversion
of fibrinogen to insoluble fibrin
INTERPRETATION
Slide Coagulase test

Positive white fibrin clots in plasma

Negative smooth suspension

Tube Coagulase test

Positive formation of fibrin clot

Negative no clot is formed
 A B

Slide coagulase test

A.Negative Staphylococcus epidermidis
B.Positive Staphylococcus aureus
 Open tube Closed tube Metabolism

Acid(yellow) alkaline(green) oxidative

Acid(yellow) acid(yellow) fermentation

Alkaline(green) alkaline(green) nonsaccharolytic

(nonutilizer)

Oxidative-Fermentation Medium of Hugh and Leifson

CARBOHYDRATE FERMENTATION

IN TRIPLE SUGAR IRON AGAR

CARBOHYDRATE FERMENTATION

IN TRIPLE SUGAR IRON AGAR

PURPOSE
1. As an initial step in the identification of
Enterobacteriaceae

PRINCIPLE:
2. The action of many species of microorganisms on a
carbohydrate substrate results in the acidification of
the medium with or without gas formation.
2. Iron salts(ferrous sulfate and ferric ammonium
citrate) reacts with H2S to produce an insoluble black
precipitate(ferrous sulfide).
 COMPOSITION
Protein sources beef extract, peptone, yeast
extract, proteose peptone
Sugars(lactose, sucrose, glucose)
Indicators
a. phenol red carbohydrate fermentation
b. ferrous sulfate hydrogen sulfide production
Sodium thiosulfate source of sulfur atoms
Sodium chloride osmotic stabilizer
 BIOCHEMICAL REACTIONS
carbohydrate fermentation
acid production
yellow deep glucose fermented
yellow slant lactose and/ or sucrose fermented
gas formation
bubble formation
cracking or splitting of the agar
upward displacement of the agar
pulling away of the medium from the walls of test tube

H2S production
blackening of the butt(FeS black precipitate)
 K/@H2S+
alkaline slant; acid butt; with gas formation
with H2S

glucose fermented; lactose and

or/sucrose not fermented; with gas
formation and black precipitate

Salmonella
Proteus
Citrobacter
K/A H2S( )
alkaline slant; acid butt; no gas; no H2S

glucose is fermented; lactose

and/or sucrose not fermented;
no gas formation; no black
precipitate

Shigella
Providencia
Serratia

anaerogenic Escherichia coli

 K/KH2S(-)
alkaline slant; alkaline butt; no gas;
no H2S

no sugars fermented; no gas;

no black precipitate in the butt

Pseudomonas
Alcaligenes
A/@H2S+
acid slant;acid butt; with gas; with H2S

all sugars fermented; with gas formation;

with black precipitate in the butt

Citrobacter freundii
METHYL RED-VOGES

PROSKAUER TEST
PURPOSE:

To identify the lactose fermenting Enterobacte-

riaceae such as Escherichia coli (MR positive
and VP negative) whereas most members of
the Klebsiella-Enterobacter-Serratia-Hafnia
group are VP positive.
 Metabolism of glucose using MR and VP pathways

Glucose

Acetoin Pyruvic acid Mixed acid fermentation

KOH + air pH less than 4.4(red)

Diacetyl

Napthol + creatine
pink red complex
Positive VP
PRINCIPLE METHYL RED TEST

In the first pathway, mixed acid products (lactic,

acetic, formic and succinic) result, leading to
a decrease in the pH of the medium and a
positive MR test.
The pH must drop to 4.4 or less for the MR indicator
to take on its acidic red color.
PRINCIPLE VOGES PROSKAUER TEST
In the second pathway, acetylmethyl carbinol
acetoin is an intermediate product to butylene
glycol.
It is the neutral product detected in the
VP reaction.
In the presence of oxygen and 40% potassium
hydroxide, acetoin is converted to the diacetyl form,
which results in a red color in the presence of
alpha-napthol.
INTERPRETATION

Methyl red test

Positive distinct red color at surface of the medium
Negative yellow color at the surface of the medium

Voges Proskauer test

Positive pink red color at surface of the medium
Negative yellow color at surface of the medium
 A B

Methyl Red test

A.Positive Escherichia coli

B.Negative Klebsiella pneumoniae
 A B

Voges Proskauer test

A.Positive Klebsiella pneumoniae

B.Negative Escherichia coli
AMINO ACID DEGRADATION
PURPOSE:
To determine the production of decarboxylase
by bacteria(Enterobacteriaceae).
Composition Moeller decarboxylase medium

1. Glucose
2. Amino acid substrate(1% lysine, 1% arginine
1% ornithine)
3. pH indicator
a. bromcresol purple
1. alkaline pH- purple
2. acid ph-yellow
b. phenol red
1. alkaline pH red
2. acid pH-yellow
PRINCIPLE
Decarboxylase enzyme - removes carboxyl groups
from the amino acids lysine and ornithine.
Dihydrolase enzyme - removes a carboxyl group
group from arginine.
Glucose base without the amino acid and tubes
containing glucose plus the amino acid substrates
are inoculated.
Decarboxylation and dihydrolation are anaerobic
reactions so overlay the inoculated tubes with
mineral oil to exclude air.
pecific amine products
Lysine ----------------- cadaverine

Ornithine-------------- putrescine

Arginine--------------- citrulline----------- ornithine

dihydrolase
reaction decarboxylation

putrescine
 Early incubation both tubes yellow due to
acidification of the indicator (bromcresol purple)
by the acid end products of glucose fermentation.

If amino acid is decarboxylated, alkaline amines

are formed and cause the indicator to revert to
an alkaline pH.
INTERPRETATION
Control tube yellow- glucose fermentation;
viable organisms; pH of the medium has been
lowered sufficient to activate the decarboxylase
enzyme
Positive test purple decarboxylation;
formation of the amino acids from the
decarboxylation
 A B

Moeller decarboxylase medium

A.Positive purple; decarboxylation
B. Negative yellow; no decarboxylation;
only glucose fermentation
 A B C D

carboxylase-dihydrolase reactions Enterobacter clo

.Control without amino acid C. lysine-negative

.Arginine positive D. ornithine-positive
 Enterobacter cloacae Klebsiella pneumoniae

Arginine +(purple)alkaline -(yellow) acid

Lysine -(yellow)acid +(purple)alkaline

Ornithine +(purple)alkaline -(yellow)acid

DEAMINASE REACTIONS
PURPOSE
To determine the deaminase activity using the
amino acids phenylalanine or tryptophan.

Only Proteus, Providencia and Morganella species

possess the deaminase enzyme.
PRINCIPLE
Deamination of the amino acid results in a colored
compound with the addition of 10% ferric chloride

Phenylalanine ----------------PPA + 10% FeCl3

Phenylalanine deaminase green

Tryptophan-------------indole-pyruvic acid +10% FeCl3

Tryptophan deaminase brown
INTERPRETATION
Positive deamination for phenylalanine intense
green color
Positive deamination for tryptophan brown color
Negative slant retains its original color after the
addition of ferric chloride
 A. Negative Escherichia coli
B. Positive Proteus vulgaris

A B

enylalanine deamination test

SINGLE SUBSTRATE

UTILIZATION
SINGLE SUBSTRATE

UTILIZATION
PURPOSE:
To determine if a member of the Enterobacteriaceae
is capable of utilizing citrate as the sole source
of carbon.

Useful in the identification of the lactose fermenting

Enterobacteriaceae: Escherichia coli is citrate
negative; Enterobacter and Klebsiella are positive
PRINCIPLE
Sodium citrate is the only carbon source in
Simmons citrate agar.
If the organism can utilize citrate, the sodium
citrate is converted to ammonia, which is then
converted to ammonium hydroxide.
The alkalinity of the compound formed raises
the pH of the medium, and the bromthymol blue
indicator takes on its alkaline color which is blue.
INTERPRETATION

Positive growth with an intense blue color on the

slant or solely the presence of growth

Negative absence of growth and no color change

in the medium (remains green)
 A B

Citrate Utilization test

A.Positive - Klebsiella pneumoniae

B.Negative - Escherichia coli
N ew er approaches

G&C ratio DNAcomposition.

Difference in metabolic products.
Cellwall composition
Amino acid composition
Infra red spectrometry
Mass spectrometry
Immuno detection of bacterial antigens by ppt
reactions , counter immuno electrophoresis and
immunoblotting.typing of monoclonal abs ELISA
RIA
 Identification of microrg is a
elaborate and time consuming
process and should be done by
considering sum total of
characteristics and not by single
character.