Order of Biochemical Tests

I) Test for metabolism of carbohydrates and

related products
II) Test for specific break down products
III) Test to show ability to utilize a specific
substance
IV) Test for metabolism of protein and amino
acids
V) Test for enzymes
 Test for Enzymes
• Catalase test
• Oxidase test
• Urease test
• ONPG (ß- galactosidase)
• Nitrate reduction
• Lecithinase test (phospholipase-c)
 Oxidase Test
Principle
Determine the presence of bacterial
cytochrome enzyme oxidase which participates
in the electron transport and in the nitrate
metabolic pathway
Demonstration
Using the substrate tetra methyl-p-
phenylenediamide dihydrochloride to
indophenol a dark purple colored end product
 Oxidase Test….
Methods
Moist filter paper method
Dry disc method
Direct plate method
Quality controls
Positive control- ATCC Pseudomonas spp
Negative control – ATCC E.coli
 Oxidase Test…..
• Cytochromes are iron containg hemoprotiens
and in aerobic respiration they transfer
electrons(H) to oxygen to form water
• The reagent used is a dye p-
phenylenediamide dihydrochloride acts as an
artificial electron acceptor substituting the
oxygen. In the reduced stage dye is colorless ,
but in the presence of enzyme cytochrome
oxidase dye is oxidised to indophenol blue
 Oxidase Test…..
Oxidase reagent
• 1% Kovacs reagent(tetra methyl -p-
phenylenediamide dihydrochloride )
• 1% Gordans & Mc leods reagent (Dimethly - p-
phenylenediamide dihydrochloride )
Modified Oxidase
• Reagent in Dimethly sulfoxide (DMSO) 6% solution
used for micrococci spp (positive in 30 secs)
 Oxidase Test…..
Positive Negative
• Neisseria spp • Enterobacteriaceae
• Haemophilus spp
• Pseudomonas spp(except
• Acinetobacter spp
P.maltophilia) • Brucella canis
• Aeromonas spp • Bordetella parapertusis
• Alcaligenes
• Vibrio • Franscisella tularensis
• Campylobacter • Gardenella vaginalis
• Plesiomonas shigelloides
• Micrococcus
• Moraxella
 Oxidase Test…..
Precautions
• Use platinum wire or wooden stick
• Should be read within 10 secs
• Do not perform the test from colonies growing in
medium having glucose as its fermentation will inhibit
the oxidase enzyme activity
• Reagent should be freshly prepared
• To be stored in dark bottle
• Do not use pigmented colonies or colonies from mac
conkey agar
(1 gm dye in 100ml distilled water)
 Catalase test
Principle
Demonstrate the presence of enzyme catalase
Biochemistry
Enzyme that decomposes hydrogen peroxide
into water and oxygen. Chemically it is a
hemoprotien structure similar to hemoglobin
2H2o2 catalase 2H2o + o2 (gas bubbles)
 Catalase test….
• Catalase is present in most cytochrome containing
aerobic and facultative anaerobic bacteria (except
streptococcus spp). Anaerobic bacteria eg clostridium
spp possess the peroxidase enzyme in lieu of catalase
• Hydrogen peroxide forms as one of the oxidative end
product of aerobic carbohydrate metabolism. If this is
allowed to acculmulate in the bacterial cells it
becomes lethal to the bacteria
• Catalase thus helps in converting H2o2 to H2o and o2
• Optimal PH for catalase action is 7
 Catalase test….
Reagents
3% hydrogen peroxide stored in dark brown
bottle under refrigeration
18 to 24 hrs culture of the organism to be tested
Quality control
Positive control - Staphylococcus aureus
Negative control – Streptococcus spp
Blood agar plate (+ve) & Chocolate agar (-ve)
 Catalase test….
Methods
• Slide method
• Tube method
• Direct plate method
 Catalase test….
Precautions
• Avoid colonies from blood agar
18 to 24 hrs colony only should be used
• Reagent to be fairly fresh as it is very unstable
and easily breaks down on exposure to light
• Reagent to be stored in brown or amber
colored bottle in refrigerator at 4°c
 Catalase test….
Positive Negative
• Micrococcus • Streptococcus
• Staphylococcus • Clostridium
• Bacillus • Erysipleothrix
• Listeria monocytogenes
• Corynebacterium (except
C.pyogenes & C.haemolyticum)
• Moraxella spp (except Kingella
kingae)
• Enterobacteriaceae
• Gonococcus & Meningococcus
• Vibrio cholerae
• Pseudomonas
• Aeromonas
• Plesiomonas
 Urease Test
Principle
To determine the ability of the organism to
split urea forming 2 molecules of ammonia by
the action of the enzyme Urease with
resulting alkalinity
Quality control
Positive – Proteus spp & Klebsiella spp(weak positive)
Negative – E.coli
 Urease Test….
Stuarts Urea broth (pH 6.8)
• Phenol red
• Monopotassium phosphate
• Disodium phosphate
• Urea
• Phenol red indicator
• Distilled water
Christensens urea agar (pH 6.8)
• Peptone
• Glucose
• Sodium chloride
• Monopotassium phosphate
• Urea
• Phenol red indicator
• Agar
• Distilled water
 Urease Test….
Procedure
The surface of the agar slant is streaked with the test
organism and incubated at 35°c for 18-24 hrs
Interpretation
Rapid hydrolysers gives +ve results in 1-2 hrs less
active spps require 3 or more days
Stuarts broth – Positive red color through out the broth
Christensens agar – Rapid urea splitters red through out
the medium, slow urea splitters red color initialy in
slant only & then gradually entire tube. Negative
yellow or straw color
 Urease Test….
Alternate tests
• Reagent impregnated urease strip test (2hrs)
• Key urease tablets test
• Urea discs
• Ewings Urea R broth (pH 6.9)
 Urease Test….
Precaution
• Both the Urease test medium depend upon the
demonstration of alkalinity i.e. not specific for
Urease i.e. even protein hydrolysis may result in
alkalinity hence false positive may be seen in
Pseudomonas . To eliminate such false positivity
control test using the same medium without urea
should be tested.
Rapid Urease splitters – Proteus spp
Delayed Urease splitters – Enterobacteriaceae
 Urease Test….
Positive Negative
• Klebsiella pneumoniae • Escherichia
• Proteus • Providencia
• Bacillus lentus • S.typhi
• Enterobacter • Edwarseilla
• Cryptococcus • Aeromonas
• Helicobacter pylori
• Bordetella
bronchoseptica
• Actinobacillus spp
 Urease Test….
Species differentiation

Positive
• Bordetella parapertusis &
P.brochiseptica
• Moraxella phenlypyruvica
• Pasteurella pneumotrpica &
P.urea
• Yersenia
pseudotuberculosis &
Y.enterocolitica
• Brucella suis
Negative
• B.pertusis
• Moraxella spp (others)
• P.multocida & P.hemolytica
• Y.pestis
• Other Brucella spp
 ß Galactosidase (Onpg and Pnpg)
Principle
To demonstrate the presence or absence of the
enzyme ß Galactosidase
Biochemistry
O – Nitrophenyl – ß – D – galactopyranoside is
structurally similar to lactose except that
orthonitrophenyl has been substituted for glucose
Onpg (colorless) H 2o Galactose+ Orthonitrophenol(yellow)
galactosidase
Lactose H20 Galactose + Glucose
galactosidase
 Lactose(ß-galactoside)
• Disaccharide compound composed of glucose and galactose
connected thru an oxygen linkage known as galactoside bond
• Lactose fermentation depends upon the 2 enzymes ß-galactoside
permease and ß-galactosidase
• ß-galactoside permease permits the transport of ß-galactoside such
as lactose into the bacterial cell wall
• ß-galactosidase cleave the ß-galactoside bond after it enters into
the bacterial cell liberating glucose and galactose
• Non lactose fermenting bacteria are devoid of both the enzymes
• Late lactose fermenters lack the enzyme permease but have ß-
galactosidase activity and so the permeases activity occurs
sluggishly and lactose fermentation is delayed by 2-8 days. in these
instances a +ve ONPG test may provide a rapid identification of
delayed lactose fermentors
 ß Galactosidase….
Media and reagents
pH 7.0
Sodium phosphate buffer
O-Nitrophenyl- ß-D-galactopyranoside(ONP)
Toulene
Physiologic saline
Quality control
Positive – E.coli
Negative – Proteus spp
 ß Galactosidase….
Procedure
• Bacteria grown in medium like TSI or KIA gives optimal results
• Loopful of bacterial growth is emulsified in 0.5 ml of physiologic
saline to produce heavy suspension
• One drop of toulene is added to the suspension and mixed for few
seconds to release the enzyme for the bacterial cells
• Equal volume of buffered ONPG solution is added to the suspension
and the mixture is placed at 37°c in water bath
Interpretation
Yellow color within 20 mins to 24 hrs- positive
Colorless after 24 hrs – Negative
Rapid test
Reagent impregnated ONPG test strip or discs
 Nitrate reduction
Principle
To determine the ability of the organism to
reduce nitrate to nitrites or fee nitrogen gas
Purpose
Aid in the spp deferentiation of i)H.duceryi(-) and
H.vaginalis(-) from other Haemophillus spp.
ii)Branhamella catarrhalis(+) and Neisseria
mucosa(+) from other Neisseria spp
Aid in the identification of Enterobacteriaceae(+)
 Nitrate reduction….
• In order to determine if a bacteria can reduce nitrate, the
test organism is inoculated into nitrate reduction broth, an
undefined medium that contains large amounts of nitrate
(KNO3). After incubation, alpha-napthylamine and sulfanilic
acid are added . These two compounds react with nitrite
and turn red in color, indicating a positive nitrate reduction
test
• If there is no color change at this step, nitrite is absent. If
the nitrate is unreduced and still in its original form, this
would be a negative nitrate reduction result. However, it is
possible that the nitrate was reduced to nitrite but has
been further reduced to ammonia or nitrogen gas. This
would be recorded as a positive nitrate reduction result
 Nitrate reduction….
• To distinguish between these two reactions, zinc
dust must be added. Zinc reduces nitrate to
nitrite. If the test organism did not reduce the
nitrate to nitrite, the zinc will change the nitrate
to nitrite. The tube will turn red because alpha-
napthylamine and sulfanilic acid are already
present in the tube
• Thus a red color after the zinc is added indicates
the zinc found the nitrate unchanged(-ve)
 Nitrate reduction….

Neg

Posi Posi
Neg
 Test for Break down products
• Indole
• MR
• VP
 INDOLE
Principle
To determine the ability of the organism to split
Indole from the tryptophan molecule
Biochemistry
Indole is one of the metabolic degradation
product of the amino acid tryptophan
Bacteria that possess the enzyme tryptophanase
are capable of hydrolyzing and deaminating
tryptophan with the production of Indole, Pyruvic
acid and ammonia.
 INDOLE…
Media & Reagents
Media: Tryptophan 1% or peptone broth
Reagents a) Ehrlichs
b) Kovacs
Ehrlichs : p - dimethyl aminobenzaldehyde
Ethyl alcohol & HCL
Kovacs : p - dimethyl aminobenzaldehyde
Pure amyl or Iso amyl alcohol & HCL
 Indole…
Other media which can be used for indole production
• Sulphide indole motility agar
• Motility indole ornithine agar

Quality Control
Positive control : E.coli
Negative control : Klebsiella pneumoniae
 Indole….
Procedure
Inoculate peptone broth with the test
organism and incubate for 18 to 24 hrs at
35°c
Add 15 drops of Indole reagent down the
inner wall of the tube
If Ehrlich reagent is used it should be
preceded by addition of 1 ml of xylene this
is not necessary for Kovac reagent
Intrepretation
Development of bright fuchsia red color at
the interface of the interface of the reagent
and the broth within seconds after adding
the reagent is indicative of the presence of
Indole and is a positive test
 Indole….
Indole rapid tests
Indole spot test(filter paper)
Indole spot test for anaerobic bacteria
Indole microtechnique(indole test strip)
Precautions
Medium containing glucose should not be used
 Indole….
Positive Neggative
• E.coli • Salmonella
• Proteus vulgaris • Klebsiella
• K.oxytoca • Enterobacter
• K.ornitholytica • Proteus mirablis
• Citrobacter diversus
• Citrobacter amalonaticus
• E.tarda
• Morganella morgagni
 Methyl Red
Principle
To test the ability of the organism to produce
and maintain stable acid end products from
glucose fermentation and to overcome the
buffering capacity of the system
This is a qualitative test for acid production
 Methyl Red…
Biochemistry
• Methyl red is a pH indicator with a range
between 6(Y) and 4.4(R)
• The pH at which the MR detects acid is
considerably lower than the pH of other
indicators
• Thus to produce a color change the test
organism must produce a large quantity of
acid from the substrate being used
 Methyl Red….
Media & Reagents
MR/VP Broth : Polypeptone, Glucose, Di
potassium phosphate &
Distilled water
MR pH indicator : MR 0.1 g in 300ml of 95%
Ethanol & Distilled water
200ml
Quality Control
Positive : E.coli
Negative : E. aerogenes
 Methyl Red….
Procedure
Innoculate the MR/VP broth with a pure
culture of the test organism and incubate at
35° for 48 to 72 hrs
Add 5 drops of MR reagent to the broth
Interpretation
Positive : Culture sufficiently acid to allow
the MR reagent to remain a distinct red
color(pH4.4) at the surface of the medium
Negative : Yellow color (pH 6.0) at the
surface of the medium
 Methyl Red ….
Positive Negative
• E.coli • Enterobacter aerogenes
• Yersenia spp • Enterobacter cloacae
• Listeria monocytogenes • Klebsiella
• Gram negative non
enteric bacilli
 Voges Proskauer Test
(acetoin production)
Principle
To determine the ability of the organisms to
produce neutral end product acetyl methyl
carbinol (acetoin) from glucose fermentation
Quality control
Positive : Enterobacter aerogenes
Negative : E.coli
 Voges Proskauer Test…
Media
MR/VP Broth : pH 6.9
Polypeptone
Glucose
Di potassium phosphate
Distilled water
Reagents
VP (A) : Alpha naphthol 5% (color intensifier)
Absolute ethylalcohol
VP (B) : 40% potassium hydroxide (oxidising agent)
Distilled water
 Voges Proskauer Test…
Procedure
Innoculate pure culture of the test organism into
MR/VP broth and incubate for 24 hrs at 35°c
Aliquot 1 ml of the broth to a sterile test tube and
add 0.6ml of VP(A) followed by 0.2ml of VP(B)
Shake the tube gently to expose the medium to
atmospheric oxygen and allow the tube to remain
undisturbed for 10 to 15 mins
Intrepretation
Positive : Pinkish red color at the surface of the
medium
Negative : Yellow color at the surface of the
medium
 Voges Proskauer Test…
Alternate Tests
• Gas liquid chromatography measure of diacetyl
• Electron capture gas liquid chromatography
• Gas chromatography – chemical ionization mass
spectrography
• Calorimetric method of measurement of diacetyl
Rapid test
• Reagent impregnated VP strip
 Voges Proskauer Test…

Precautions
• Organisms which are MR +ve is VP – ve or vice
versa is true for most of the organisms belonging
to Enterobacteriacea
• Organism like Hafnia alvei & Proteus mirabilis
may give both MR & VP positive results although
VP reaction is delayed
• Excess KOH may mask a weak VP positive
reactions
 Voges Proskauer Test…

Positive Negative
• Klebseilla pneumoniae • E.coli
• Enterobacter • Micrococcus
• Staphylococcus • K.ozanae
• Hafinia alvei(25°+ve) • K.rhinoscleromatis
Hafinia alvei(37°variable) • Y.enterocolitica 37°
• Yersinia enterocolitica 25°
• Listeria monocytogenes
 Test for metabolism of carbohydrates and related
products

• OF
• 1% sugar fermentation
 Oxidation fermentation Test
(Huge & Liefson)
Principle
To determine the oxidative or fermentative
metabolism of a carbohydrate or its non utilization
Biochemistry
Fermentation is a anaerobic process and bacterial
fermenters of carbohydrates are usually facultative
anaerobes
Oxidation is a aerobic process and bacterial oxidisers
are usually strict aerobes
 Oxidation fermentation Test
•The method described, sometimes referred to as the Hugh and
Leifson test employs a semi-solid medium in tubes containing the
carbohydrate under test (usually glucose) and a pH indicator
•Two tubes are inoculated and one is immediately sealed to
produce anaerobic conditions
•Oxidising organisms, eg Pseudomonas species, produce an acid
reaction in the open tube only
•Fermenting organisms, eg Enterobacteriaceae, produce an acid
reaction throughout the medium in both tubes
•Organisms that cannot break down the carbohydrate aerobically or
anaerobically, eg Alcaligenes faecalis, produce an alkaline
reaction in the open tube and no change in the covered tube
• Hugh and Leifson’s medium can also be used for recording gas
production and motility
•Staphylococci and micrococci are tested with the Baird-Parker
modification of the medium
 Coagulase Test
Principle
Ability of an organism to clot plasma by the
action of the enzyme coagulase
Used for the differentiation of Staphylococcus
aureus (+) from S. epidermis and S. saprophyticus
 Biochemistry:
Staphylocoagulase is an extracellular enzyme produced by
Staphylococcus aureus and is protein. It has a prothrombin like activity and can
convert fibrinogen into fibrin

The enzyme coagulase is found in two forms bound coagulase and free coagulase
 Bound coagulase (Slide test):
• Also known as clumping factor is attached to
the bacterial cell wall and is not present in the
culture filtrates
• Fibrin strands are formed between the
bacterial cells when suspended in the plasma
(fibrinogen) causing them to clump into visible
aggregates
 Free coagulase:
• Free coagulase is a thrombin like substance
present in culture filtrates.
• When a suspension of coagulase producing
organism is prepared in plasma in a test tube
the enzyme coagulase, reacting with a serum
substance coagulase reacting factor form a
complex which reacts with the fibrinogen to
produce the fibrin clot
 Quality controls:

Positive control : ATCC Staphylococcus aureus

Negative control: ATCC Staphylococcus epidermidis
 Slide test (Bound coagulase):
• Place saline or distilled water in a slide,
emulsify heavy suspension of staphylococcus
(18-24 hr pure broth culture) in the drop of
saline or distilled water.
• Add fresh plasma into the suspension and mix
it homogenously.
• Observe for the formation agglutinates which
usually occurs within 5 to 20 secs.
 Slide test (Bound coagulase): …..
• If no agglutination the test is negative for
bound coagulase.
• Controls should also be prepared in the same
slide by emulsifying the bacterium in the saline
without adding plasma to rule out auto
agglutination.
• Slide coagulase is only a presumptive and all
negative results or delayed results must be
confirmed with tube coagulase test.
Tube coagulase (Free Coagulase): ...
• To 0.5 ml of fresh plasma add 0.5 ml of
overnight broth pure culture of Staphylococcus
and incubate the tubes in water bath at 37° c
• Every 30 mins tilt the tube and look for the clot
formation till 4hrs
• If the clot is formed then the test is positive for
enzyme free coagulase and negative if the clot
has not formed till 4hrs
Tube coagulase (Free Coagulase):
• Simultaneously positive controls (ATCC
Staphylococcus aureus), negative controls
(ATCC Staphylococcus epidermidis) and
plasma controls are also performed to rule
out false positive
• Rapid test :
Staph strips - Strips impregnated with lyophilized
plasma with ETDA premeasured on strip.
• Alternate tests
• Pour plate coagulase test
• Tube coagulase – Thermo nuclease test
• Coagulase - mannitol agar plate test
• Latex agglutination test
• Passive Haemagglutination test
 Precautions
• Citrated plasma should not be used as false
positive results may arise from those organisms
that capable of utilizing citrate
• If citrate plasma has to used, it has to be treated
with heparin before employing for the test
• Rabbit plasma is preferred over human plasma
as its clots is firmer and coagulation is faster
•  
• Tube coagulase positive organisms –
Staphylococcus aureus , Staphylococcus
intermedius, Yersinia pestis, Erysiplothrix etc.
• Slide coagulase positive organisms -
Staphylococcus aureus , Staphylococcus
hyicus, Staphylococcus lugdunensis.
• Coagulase negative organisms- Staphylococcus
epidermidis, Staphylococcus saprophyticus
 Bile esculin test
• Principle: To determine the ability of the
organisms to hydrolyze the glycoside esculin to
esculetin in the presence of bile (10 to 40%)
• Esculetin reacts with an iron salt to form a dark
brown or black complex
• Ferric citrate is incorporated in the bile esculin
medium in a 0.05% concentration as an
indicator of esculin hydrolysis and resulting
esculetin formation
 Bile esculin test….

• It helps in differentiating group D streptococci

from other streptococci
• It aids in differentiation of Listeria
monocytogenes(+)
• Helps in selectively isolate and presumptively
separate Bacteroides fragilis group from other
anaerobic gram negative bacilli
 Bile esculin test…
• Positive : Presence of black or dark brown
color on the slant, half or more of medium
should be blackened in any interval of time.
• Negative : No blackening of the medium or
blackening of less than half of the tube after
72 hrs of incubation.
• Rapid tests – Bile esculin strip test, Esculin
spot test.
 Bile esculin test….
Precautions : Bile esculin test cannot be used
for the identification of group D Enterococcus,
but a positive reaction can be used in
combination with other tests to identify group
D Enterococcus. Other streptococci such as
some viridian species like S.sanguis, S.mutans
and S.anginosus also hydrolyze bile esculin
 Bile Solubility test
• Principle : To test the ability of bacterial cells to lyse in the
presence of bile salts within a specific time and temperature
• Helps in differentiating bile soluble Streptococcus pneumoniae
and other bile insoluble alpha hemolytic Streptococci
• Species differentiation of bile soluble Haemophillus influenzae
and H.aegyptius from other bile insoluble Haemophillus
species
• Mechanism : S.pnuemoniae produces an intracellular
autolytic enzyme autolysin which cause the organism to
undergo rapid autolysis when grow in a artificial medium.
Autolysin enzyme gets accelerated and activated in the
presence of bile acids
 Bile Solubility test..
• Direct plate method : Add a drop 10% sodium
deoxycholate or sodium taurocholate directly over the
colony to be tested in the plate and incubate the plate
and look for the disappearance of the colonly after
some time if it is bile soluble
• Tube method : Take a 1ml overnight
todd-hewitt broth culture of the test
organism and to that add 0.5ml of
sodium deoxycholate or sodium
taurocholate and incubate the tubes
for 3hr and check the tube every half
an hr for any clearing. If the tubes are
clearing within 3 hrs then the test
organism is bile soluble
 Phenylalanine deaminase test

• Principle: To determine the ability of an

organism to deaminate phenylalanine to
phenylpyruvic acid by enzymatic activity with
resulting acidity
 Phenylalanine deaminase test…
• Biochemistry: The aromatic amino acid phenylalanine
undergoes oxidative deamination, catalyzed by an amino acid
oxidase enzyme (Phenylalanine deaminase), a flavoprotien to
yield the keto acid phenyl pyruvic acid

Fecl3 acts as a chelating agent and chelates phenyl pyruvic acid to form green color.
Positive control: Proteus spp
Negative control: Escherichia coli
 Phenylalanine deaminase test…
Interpretation
• Positive test: A light to deep green color
formation on the slant and in the
syneresis fluid
• Negative test: No color change remains
yellow due to the color of the fecl3
reagent
• Rapid tests -Reagent impregnated
phenylalanine strip test
• Precautions - A positive test results to
be interpretated immediately after
addition of Fecl3 reagent because the
green color fades quickly
 Phenylalanine deaminase test…
Positive organisms
Proteae genera
- Morganella
- Proteus
- Providencia
Moraxella phenylpyruvica (other moraxella spp negative)
Agrobacterium radiobacter
Ochrobactrum
Oligella ureolytica
Actinobacillus ureae