MICROBIOLOGY LAB

BIOCHEMICAL TEST IN MICROBIOLOGY

LAB
RAHAF ALDLBH
LABORATORY TECHNOLOGIST
 ACETAMIDE UTILIZATION TEST:
Acetamide test is used to test an organism’s ability to utilize acetamide
by deamidation. It is used in the qualitative procedures to differentiate
non-fermentative, gram negative bacteria, especially Pseudomonas
aeruginosa, on the basis of acetamide utilization.

Objective
To differentiate microorganisms based on the ability to use acetamide as the
sole source of carbon.

Uses
The test is recommended for differentiating Pseudomonas aeruginosa
from other non-glucose-fermenting, Gram-negative rods.
Expected Results
Positive: De-amination of the acetamide, resulting in a blue color.
Negative: No color change or development of a yellow color.
 ACETATE UTILIZATION TEST:
Acetate utilization test is employed in the qualitative procedures to differentiate
species of Shigella from Escherichia coli and non-fermentative gram negative
bacteria.

Objective
To differentiate organisms based on ability to use acetate as the sole source of
carbon.
Uses
Generally used to differentiate Shigella spp. from Escherichia coli.

Expected Results
Positive: Medium becomes alkalinized (blue) as a result of the growth and use
of acetate.
Negative: No growth or growth with no indicator change to blue.
 AMES TEST:
Ames test it is a biological assay to assess the mutagenic potential of
chemical compounds. It utilizes bacteria to test whether a given chemical can
cause mutations in the DNA of the test organism. The test was developed by
Bruce N. Ames in 1970s to determine if a chemical at hand is a mutagen.

Objective
To determine the mutagenic activity of chemicals by observing whether
they cause mutations in sample bacteria
Uses
Ames test is used to identify the revert mutations which are present in
strains, it can also be used to detect the mutagenicity of environmental
samples such as drugs, dyes, reagents, cosmetics, waste water,
pesticides and other substances which are easily solubilized in a liquid
suspension.
Result Interpretation
The mutagenicity of chemicals is proportional to number of colonies observed.
If there is a large number of colonies on the test plate in comparison to control,
then such chemical are said to be mutagens.
Very few numbers of colonies can be seen on control plate also. This may be
due to spontaneous point mutation on hisidine encoding gene.
 AMINO ACID DECARBOXYLASE TEST:
Amino acids are metabolized variably by gram negative aerobic and facultatively
anaerobic bacteria as well as gram positive cocci.
These amino acids are decarboxylated, hydrolysed or deaminated depending on
the organism and the amino acid in question. In decarboxylation, the enzymes
break the bond holding the carboxylic (-COOH) group to the rest of the amino
acid.
Objective
To differentiate decarboxylase producing Enterobacteriaceae from other
gram negative rods.
Uses
Arginine decarboxylase test aids in differentiating enteric bacteria with
closely related physiological characteristics.
Lysine decarboxylase test assist in the identification of Salmonellae (+ve)
and Shigellae (-ve).
Expected Results
Positive: Alkaline (purple) color change compared with the control tube
Negative: No color change or acid (yellow) color in test and control tube.
Growth in the control tube.
 API (ANALYTICAL PROFILE INDEX) 20E TEST:
API identification products are test kits for identification of Gram positive and
Gram negative bacteria and yeast.
API strips give accurate identifications based on extensive databases and are
standardized, easy-to-use test systems.
The kits include strips that contain up to 20 miniature biochemical tests
which are all quick, safe and easy to perform.
API (Analytical Profile Index) 20E is a biochemical panel for identification and
differentiation of members of the family Enterobacteriaceae.
It is hence a well-established method for manual microorganism identification
to the species level.
Objective
To identify and differentiate members of family Enterobacteriaceae.
Uses
API offers manual identification of microorganisms for:
Infectious disease diagnosis
Identification of important industrial microorganisms
Result Interpretation
1. For some of the compartments, the color change can be read straightway after
24 hours but for some reagents must be added to them before interpretation.
2. Add following reagents to these specific compartments:
TDA: Put one drop of Ferric Chloride
IND: Put one drop of Kovacs reagent
VP: Put one drop of 40 % KOH (VP reagent 1) & One drop of VP Reagent 2 (α-
Naphthol)
3. Get the API Reading Scale (color chart) by marking each test as positive or
negative on the lid of the tray. The wells are marked off into triplets by black
triangles, for which scores are allocated.
4. Add up the scores for the positive wells only in each triplet.
5. Three test reactions are added together at a time to give a 7-digit number,
which can then be looked up in the codebook. The highest score possible for a
triplet is 7 (the sum of 1, 2 and 4) and the lowest is 0.
6. Identify the organism by using API catalog or apiweb (online)
 BACITRACIN SUSCEPTIBILITY TEST:
Bacitracin is a polypeptide antibiotic produced by organisms of the licheniformis
group of Bacillus subtilis var Tracy.
It is used to determine its effect of a small amount (0.04 IU or 0.05 IU not higher)
on different microorganisms. The presumptive identification of beta hemolytic
Group A streptococci from beta hemolytic non-Group A Streptococci is usually
done by testing for sensitivity to bacitracin.
Many laboratories use this test as the sole test for diagnosing GAS infections
due to the difficulty in performing sero-grouping and high cost of antisera.
Uses
The test is used for presumptive identification and differentiation of beta-
hemolytic group A streptococci (Streptococcus pyogenes– susceptible) from
other beta-hemolytic streptococci.
It is also used to distinguish staphylococci species (resistant) from
micrococci (susceptible).
Expected Results
Positive: Any zone of inhibition greater than 10 mm; susceptible.
Negative: No zone of inhibition; resistant
 BETA LACTAMASE TEST:
Different bacteria produce an important class of enzymes called beta-
lactamases, which may be mediated by genes on plasmids or chromosomes.
These enzymes confer resistance to a number of penicillin antibiotics by
cleaving the beta-lactam ring of penicillins and cephalosporin antibiotics,
resulting in inactivation of these drugs.
They are capable of inactivating “penicillinase-labile-penicillins”, such as
amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin.
β-lactamases thus play a key role in bacterial resistance to beta-lactam
agents, and detection of their presence can provide useful information.
Various assays are available to detect β-lactamases, such as the iodometric
method, the acidometric method, and by the use of chromogenic substrates
Objective
To detect the enzyme beta-lactamase, which confers penicillin resistance to
various bacterial organisms.
Uses
Useful applications include detection of:
N. gonorrhoeae resistance to penicillin
H. influenzae resistance to ampicillin
Staphylococcal resistance to penicillin

Expected Results
Positive: Yellow to pink-red color change on the area where the culture is
applied.
Negative: No color change.
 BILE ESCULIN TEST :
Esculin is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-
coumarin). The two moieties of the molecule (glucose and 7-
hydroxycoumarin) are linked together by an ester bond through oxygen.
Many bacteria can hydrolyze esculin, but few can do so in the presence of
bile.
Thus the bile esculin test is based on the ability of certain bacteria, notably
the group D streptococci and Enterococcus species, to hydrolyze esculin in
the presence of bile (4% bile salts or 40% bile).
Bacteria that are bile-esculin positive are able to grow in the presence of bile
salts and the hydrolysis of the esculin in the medium results in the formation
of glucose and a compound called esculetin.
Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium
component ferric citrate) to form a black diffusible complex.
Uses
This test is used for the presumptive identification of enterococci and
organisms in the Streptococcus bovis group.
The test differentiates enterococci and group D streptococci from non–group D
viridans streptococci.

Expected Results
Positive: Growth and blackening of the agar slant
Negative: Growth and no blackening of medium ; No growth
 BILE SOLUBILITY TEST:
Bile Solubility Test is the test which differentiate Streptococcus pneumoniae
(positive- soluble) from alpha-hemolytic streptococci (negative- insoluble).
Streptococcus pneumoniaeis bile soluble whereas all other alpha-hemolytic
streptococci are bile resistant.
Expected Results
Tube Method :
Positive Result: Suspension clears in tube labelled test and remains turbid in
control tube.
Negative Result: Suspension remains turbid.
Note: Partial clearing (partial solubility) is not considered positive for S.
pneumoniae identification.
Plate Method:
Positive Result: Colony disintegration or flattening of the colony within 30 minutes,
leaving an alpha-hemolytic where colony may be located.
Negative Result: No change i.e. colonies remain intact.

Modified Bile Solubility Test:

Positive Result: No cocci are seen in the test smear and control smear shows
intact bacteria.
Negative Result: Cocci are seen in both test smear and control smear.
 BUTYRATE DISK TEST:
hydrolysis of bromo-chloro-indolyl butyrate.
The Test Disc is used for rapid detection and qualitative procedures of
butyrate esterase for presumptive identification of Moraxella catarrhalis.
Moraxella catarrhalis is recognized as a significant pathogen whose isolation
from routine sputum cultures is associated with clinical infection.
The rapid identification of this organism is important, since most strains
produce beta-lactamase and are resistant to penicillin and ampicillin.
Conventional methods of identification may take up to 48 hours to perform
while the detection of an enzyme, butyrate esterase, is a rapid means of
identifying Moraxella catarrhalis, which is performed using butyrate discs.

Objectives
To detect the enzyme butyrate esteraste in microorganisms
Uses
Aid in the identification of Moraxella (Branhamella) catarrhalis.
Helps to differentiate Neisseria gonorrhoeae(negative) and Moraxella
catarrhalis(positive), which are both Oxidase positive, Gram negative diplococci

Expected Results
Positive: Development of a blue color during the 5-minute incubation period.
Negative: No color change.
 CAMP TEST:
CAMP test is used to distinguish the species Streptococcus agalactiae from
other species of beta-hemolytic Streptococcus. S. agalactiae, a member of
the Lancefield Group B streptococci, is one of the causative agents of
mastitis in cows. CAMP is an acronym for the authors of this test (Christie,
Atkinson, Munch, and Peterson) which was identified in 1944.
Uses
It is used to distinguish the species Streptococcus agalactiae from other
species of beta-hemolytic Streptococcus.
It is used to identify Listeria monocytogenes which also produces a positive
CAMP reaction.

Result Interpretation

Positive: Enhanced hemolysis is indicated by an arrow head-shaped zone of

beta-hemolysis at the junction of the two organisms.
Negative: No enhancement of hemolysis.
 CASEIN HYDROLYSIS TEST:
Casein, the major milk protein, is a macromolecule composed of amino acid
subunits linked together by peptide bonds (CO—NH). It makes around 85% of the
protein found in milk as well as the white color of milk.
Casein is way too large to enter the cell membrane. Before their assimilation into
the cell, proteins must undergo step-by-step degradation into peptones,
polypeptides, dipeptides, and ultimately into their building blocks, amino acids.
These are mediated by extracellular enzymes called proteases. The function of
these proteases is to cleave the peptide bond CO–NH by introducing water into
the molecule. The reaction then liberates the amino acids which are low-
molecular in weight which can be transported through the cell membrane for use
in the synthesis of structural and functional cellular proteins.
Objectives
To determine the ability of the organism to degrade the casein protein.
To differentiate the organism on the basis of production of exoenzyme
proteinase (caseinase).
Uses
Helpful in identifying bacteria that grow in milk
Differentiating among Enterobacteriaceae, Bacillaceae, and several other
families.
For the differentiation of aerobic actinomycetes based on casein proteolysis.
Identification of organisms that can hydrolyse casein, such as Streptomyces,
Pseudomonas , and Actinomadura
Result Interpretation
Positive Test: Clearing is observed around and/or beneath colony growth
(hydrolysis).
Negative Test: No clearing is observed around and/or beneath the inoculum
 CATALASE TEST:
This test demonstrate the presence of catalase, an enzyme that catalyses the
release of oxygen from hydrogen peroxide (H2O2). It is used to differentiate
those bacteria that produces an enzyme catalase, such as staphylococci, from
non-catalase producing bacteria such as streptococci. Normally 3% H2O2 is used
for the routine culture while 15% H2O2 is used for detection of catalase in
anaerobes.
Uses
The morphologically similar Enterococcusor Streptococcus (catalase negative)
and Staphylococcus (catalase positive) can be differentiated using the catalase
test.
Also valuable in differentiating aerobic and obligate anaerobic bacteria.
Semiquantitative catalase test is used for the identification of Mycobacterium
tuberculosis.
It is used to differentiate aerotolerant strains of Clostridium, which are catalase
negative, from Bacillus species, which are positive.
Catalase test can be used as an aid to the identification of Enterobacteriaceae.

Result Interpretation
Positive: Copious bubbles produced, active bubbling
Examples: Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae,
Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli,
Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia),
Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and
Rhodococcus equi.

Negative: No or very few bubbles produced.

Examples: Streptococcus and Enterococcus spp
 CETRIMIDE TEST:
Cetrimide is a quaternary ammonium salt, which acts as a selective agent and
inhibits most bacteria by acting as a cationic detergent (Cetyltrimethylammonium
bromide).
Cetrimide when added into a culture medium results into a selective solid
medium recommended for use in qualitative procedures for selective isolation
and presumptive identification of Pseudomonas aeruginosa and other
nonfermentative, gram-negative bacilli.
Objective
To isolate and purify Pseudomonas aeruginosa from contaminated
specimens.
Uses
Used for the isolation and identification of Pseudomonas aeruginosafrom
clinical and non-clinical specimens.
It may also be used for determining the ability of an organism to produce
fluorescein and pyocyanin.
The test can also be used for microbial limit testing for non-sterile products.

Expected Results
Positive: Growth, variation in color of colonies. Visual examination may also reveal
the typical yellow-green to blue color which indicates the production of pyocyanin.
Negative: No growth
Pyocyanin Production:
Positive Test – Blue-green pigmentation surrounding growth
Negative Test – No color development
Fluorescein Production (requires the use of ultraviolet light):
Positive Test – Yellow-green fluorescence
Negative Test – No fluorescence
 CITRATE UTILIZATION TEST:
This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer,
and Citrate) that are used to differentiate among the Gram-Negative bacilli in the
family Enterobacteriaceae.

Result Interpretation
Positive Reaction: Growth with color change from green to intense blue
along the slant.
Examples: Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter,
Serratia, Providencia, etc.
Negative Reaction: No growth and No color change; Slant remains green.
Examples: Escherichia, Shigella, Morganella, Yersinia etc.

COAGULASE TEST:
Coagulase test is used to differentiate Staphylococcus aureus (positive) which
produce the enzyme coagulase, from S. epidermis and S. saprophyticus
(negative) which do not produce coagulase. i.e Coagulase Negative
Staphylococcus (CONS).

Result Interpretation
Fibrin Clot of any size- Positive
No Clot- Negative
 DEOXYRIBONUCLEASE (DNASE) TEST:
Deoxyribonucleic acid (DNA) is a large polymer of nucleotides that is way too large to
enter the cell membrane. In order to utilize external DNA, bacteria cells secrete
exoenzymes (DNases) outside of the cell that hydrolyze DNA into nucleotides.
The nucleotides can then move across the cell membrane via transport proteins to be
utilized. The cell can use nucleotides to make nucleic acids and to use as a source of
nitrogen, phosphate and carbon.
Extracellular DNases have been reported in a relatively small subset of prokaryotes,
including a number of human pathogens. DNA hydrolysis is tested by growing an
organism on a DNase Test Agar plate (providing nutrients and DNA) and then checking
the plate for hydrolysis.
DNA Hydrolysis test or Deoxyribonuclease (DNase) test is thus used to determine the
ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy
for growth. It helps in the detection of deoxyribonuclease activity of bacteria and fungi,
and especially for identification of pathogenic Staphylococci.

Objective
To differentiate organisms based on the production of deoxyribonuclease.
Uses
Used to determine the ability of an organism to hydrolyze deoxyribonucleic acid.
Used to differentiate Staphylococcus aureuswhich produces the enzyme
deoxyribonuclease from other Staphylococci which do not produce DNase.
Particularly useful if plasma is not available to perform coagulase test or when the
result of coagulase tests are difficult to interpret.
It is also used to distinguish Serratia (positive) from Enterobacter sp.
Moraxella catarrhalis (positive) from Neisseria

Expected Results
Positive: Medium is colorless around the test organism.
Negative: If no degradation of DNA occurs, the medium remains green
 FERMENTATION TEST:
The ability of bacteria to form organic compounds by metabolizing certain
carbohydrates and related compounds is a widely used method for the
identification of microorganisms. Different fermentation media are used to
differentiate organisms based on their ability to ferment carbohydrates
incorporated into the basal medium.
Purple Broth is used for studying carbohydrate fermentation reactions,
particularly in the identification of gram-negative enteric bacteria with desired
carbohydrates added. Purple media were originally formulated by Vera. This
medium is recommended by FDA for fermentation studies of sugars.

Objective
To determine the fermentation reactions of pure cultures of microorganisms
using purple broth

Uses
It is recommended for the determination of fermentation reactions of
microorganisms, especially enteric bacilli and Enterococcus
They are used primarily for the differentiation and presumptive identification
of gram-negative enteric bacilli based on patterns of carbohydrate
fermentation.

Result Interpretation
Positive: The development of a yellow color in the medium is indicative of a
positive carbohydrate fermentation reaction.
Negative: Lack of yellow color development is indicative of a negative
carbohydrate fermentation reaction.
Gas formation is indicated by the appearance of gas bubbles in the Durham
tube.
 GELATIN HYDROLYSIS TEST:
Gelatin is a protein derived from the connective tissues of vertebrates, that is, collagen.
It is produced when collagen is boiled in water. Gelatin hydrolysis detects the presence
of gelatinases. Gelatinases are proteases secreted extracellularly by some bacteria
which hydrolyze or digest gelatin. The production of gelatinases is used as a
presumptive test for the identification of various organisms, including Staphylococcus
sp., Enterobacteriaceae, and some gram-positive bacilli.

Uses
This test is used to determine the ability of an organism that produce gelatinases.
This test is helpful in identifying and differentiating species of Serratia, Proteus,
Bacillus, Clostridium, Pseudomonas and Flavobacterium.
This test differentiates pathogenic Staphylococcus aureus which is gelatinase-
positive from non-pathogenic epidermidis which is gelatinase negative.
This test can be used to differentiate Serratia and Proteus species which are gelatin
positive from other members of Enterobacteriaceae family.
Bacillus anthracis, B. cereus and several other members of the genus are
gelatinase-positive, as are Clostridium tetani and perfringens.

Expected Results
Positive: Partial or total liquefaction of the inoculated tube (the control tube
must be completely solidified) at 4°C within 14 days. On plates, gelatin
hydrolysis is indicated by clear zones around gelatinase-positive colonies.
Negative: Complete solidification of the tube at 4°C. On plates, no clear zones
around colonies are observed.

Gelatin hydrolysis. A, Positive; note liquefaction at

top of tube. B, Uninoculated tube.

Gelatin hydrolysis.
A, Positive B, Negative
 GROWTH AT 42 TEST:
Microbes can be roughly classified according to the range of temperature at which they can
grow. The growth rates are the highest at the optimum growth temperature for the organism.
The lowest temperature at which the organism can survive and replicate is its minimum growth
temperature. The highest temperature at which growth can occur is its maximum growth
temperature. However, growth at both extreme cold and hot temperatures require evolutionary
adjustments to macromolecules and biological processes.
Only few organisms grow well in such temperature ranges. Thus the effect of temperature on
organisms can be observed to differentiate organisms

Objective
To differentiate species of the fluorescent pseudomonas group
To differentiate other non-fermentative bacteria.

Uses
Growth at 42oC is advocated to differentiate species of the fluorescent
pseudomonas group as well as to differentiate other non-fermentative bacteria.
This test is mostly used to differentiate a pyocyanogenic pseudomonads from other
Pseudomonas.

Expected Results
Positive: Good growth at both 35°and 42°C
Negative: No growth at 42°C, but good growth at 35°C.

Growth at 42°C. A, Positive; good growth.

B, Negative; no growth.
 HIPPURATE HYDROLYSIS TEST:
Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyse
substrate hippurate into glycine and benzoic acid by action of hippuricase
enzyme present in bacteria.
Hippuricase is a constitutive enzyme that hydrolyzes the substrate hippurate to
produce the amino acid glycine. Glycine is detected by oxidation with Ninhydrin
reagent, which results in the production of a deep purple color.
Hippurate hydrolysis test is used in the presumptive identification of Gardnerella
vaginalis, Campylobacter jejuni, Listeria monocytogenes and group B
streptococci by detecting the ability of the organism to hydrolyze hippurate.
Objective
To detect the production of the enzyme hippuricase for the presumptive
identification of a variety of microorganisms.
Uses
The hippurate test is most frequently used in the identification of Gardnerella
vaginalis, Streptococcus agalactiae, Campylobacter jejuni, and Listeria
monocytogenes.
Used in the differentiation of β-hemolytic Streptococcus agalactiae from other β-
hemolytic streptococci.
Aids in the separation of Campylobacter jejuni and Campylobacter coli strains.

Expected Results
Positive: A positive test is indicated by the appearance of a deep blue/violet
color in 30 minutes.
Negative: Colorless or slightly yellow pink color
 HYDROGEN SULFIDE TEST:
Some microorganisms have an ability to reduce sulfur (Sulphur) containing
compounds to hydrogen sulfide during metabolism which is commonly
employed as a test measure for their identification in laboratories.
Numerous methods are used to detect H2S production by micro-organisms
which vary with the source of sulfur and the metal salts used to indicate
H2S formation. SIM is more sensitive in the detection of H2S than either TSI
or KIA, because of its semisolid nature, its lack of interfering carbohydrates,
and the use of peptonized iron as an indicator. However, Lead acetate paper
is 10 times more sensitive than other media.

Objective
To determine whether the microbe reduces sulfur-containing compounds to
sulfides to produce hydrogen sulfide gas.

Uses
It is used mainly to assist in the identification of members of family
Enterobacteriaceae and occasionally to differentiate other bacteria such as
Bacteroidessps and Brucella sps.
The test aids in the identification and differentiation of members of
Enterobacteriaceae (enterics) from other Gram- bacilli.
It is especially helpful in identifying Salmonella, Francisella, and Proteus species.

Results
Positive result:Blackening on the medium
Negative result:No blackening on the medium
 INDOLE TEST:
This test demonstrate the ability of certain bacteria to decompose the amino acid tryptophane
to indole, which accumulates in the medium. Indole production test is important in the
identification of Enterobacteria. Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani and
Providencia species break down the amino acid tryptophan with the release of indole. This is
performed by a chain of a number of different intracellular enzymes, a system generally
referred to as “tryptophanase.” It is used as part of the IMViC procedures,a tests designed to
distinguish among members of the family Enterobacteriaceae.

Uses
To differentiate Proteus mirabilis (indole negative) from all other Proteus species
(indole positive).
To differentiate Klebssiella pneumoniae (indole negative) from Klebsiella
oxytoca(indole positive).
To differentiate Citrobacter freundii (indole negative) from Citrobacter koseri (indole
positive).

Result Interpretation
Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top
of the medium within seconds of adding the reagent.
Examples: Aeromonas hydrophila, Aeromonas punctata, Bacillus alvei,Edwardsiella sp.,
Escherichia coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella oxytoca, Proteus sp.
(not P. mirabilis and P. penneri), Plesiomonas shigelloides,Pasteurella multocida, Pasteurella
pneumotropica, Enterococcus faecalis, and Vibrio sp.
Negative: No color change even after the addition of appropriate reagent.
Examples: Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp., most
Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most
Haemophilus sp., most Klebsiellasp., Neisseria sp., Pasteurella haemolytica,
Pasteurella ureae, Proteus mirabilis, P. penneri, Pseudomonas sp.,Salmonella sp.,
Serratia sp., Yersinia sp.

Positive: A positive reaction is denoted by the appearance of a blue to

blue-green color change on the bacterial smear within 2-3 minutes.
Negative: Negative reactions remain colorless or light pink.
Note: Positive reaction is Red-violet in the case of Providencia alcalifaciens
 KLIGLER’S IRON AGAR TEST:
The Kligler’s Iron Agar test employs a medium for the identification of
Enterobacteriaceae, based on double sugar fermentation and hydrogen sulphide
production. In 1918, Kligler described a medium for detection of H2S and
differentiation of Salmonella spp. Bailey and Lacey further modified the medium
by substituting phenol red indicator for Andrade indicator. This medium became
known as KIA. It is recommended for determination of H2S production by enteric
gram-negative bacilli and for detection of H2S produced by some strains of
Pseudomonas.
Objective
To differentiate organisms by demonstrating hydrogen sulfide production and the
fermentation of dextrose and lactose.
Uses
The test is recommended for the differential identification of gram-negative enteric
bacilli from clinical and non clinical samples on the basis of the fermentation of
dextrose, lactose and H2S production.
It is used as a differentiation medium for typhoid, dysentery and allied bacilli.
It differentiates Salmonella Typhi from other Salmonellae and also Salmonella
Paratyphi A from Salmonella Scottmuelleri and Salmonella Enteritidis.
Kligler Iron Agar test differentiates lactose fermenters from the nonfermenters.

Expected Results
Carbohydrate Fermentation: KIA Color Reactions:
Positive Test for Slant Reaction – Yellow (acid) Red slant/ yellow butt – dextrose (+), lactose (-)
Negative Test for Slant Reaction – Red (alkaline) Yellow slant/ yellow butt – dextrose (+), lactose (+)
Positive Test for Butt Reaction – Yellow (acid) Red slant/ red butt – dextrose (-), lactose (-)
Negative Test for Butt Reaction – Red (alkaline.

Hydrogen Sulfide Production: GAS Production:

Positive Test – Black color throughout medium, a black Positive Test – Bubbles in the medium, cracking and
ring at the juncture of the slant and butt, or a black displacement of the medium, or separation of the
precipitate in the butt medium from the side and bottom of the tube
Negative Test – No black color development Negative Test – No bubbles and no separation or
displacement of the medium.
 LECITHINASE TEST:
Phosphatidylcholine or simply, lecithin is a substance widely distributed in
animal tissues, egg yolk, and some higher plants, consisting of phospholipids
linked to choline.
Some microorganisms possess lecithinase, also called phospholipase C, which
is an enzyme that splits the phospholipid lecithin. Such lecithinase activity is
used to characterize several gram positive and gram negative bacteria.

Objectives
To determine the ability of microorganisms to produce the enzyme lecithinase.
To identify the bacteria on the basis of its lecithinase activity.

Uses
Bacterial lecithinases are of special interest because of the possible role of these
enzymes in pathogenicity.
In identification of Clostridium perfringens, Staphylococcus aureus, Pseudomonas
aeruginosa or Listeria monocytogenes .
The lecithinase activity of S. aureus is used in detection of coagulase-positive
strains, because of high link between lecithinase activity and coagulase activity.
Can be used to differentiate between certain species within the genus Bacillus

Result Interpretation
Positive test:Appearance of a white, opaque, diffuse zone that extends into the
medium surrounding the colonies.
Negative test:Absence of a white, opaque zone extending from the edge of the colony.
 LEUCINE AMINO PEPTIDASE (LAP) TEST:
Leucine amino peptidase (LAP) test is a rapid test for detection of enzyme
leucine aminopeptidase. Leucine- β- napthalamide impregnated disk serves
as a substrate for the detection of leucine aminopeptidase. This test is
usually used, in conjunction with other tests, for the identification of
streptococci and other catalase negative gram positive cocci.
Objective
To perform the preliminary characterization of catalase negative, gram-
positive cocci.

Uses
The LAP test is often used in conjunction with PYR and other biochemical tests to
help differentiate between catalase, gram-positive cocci.
In general, Streptococcus pneumoniae and Streptococcus pyogenes, Pediococcus,
Lactococcus, and Enterococcus species are all LAP positive, while other beta-
hemolytic Streptococci, Aerococcus and Leuconostoc species are LAP negative
which help in their identification.
Aminopeptidase Test has been used to perform a Gram analysis of bacteria isolated
from Scleroderma citrinum mycorrhizae, the mycorrhizosphere and bulk soil.

Result Interpretation
Positive: Development of a red/pink color
Negative: No change or a slight yellow color
 LIPASE TEST:
Lipid is the general term used to describe all types of fats. Fats are formed by ester
linkage between three molecules of fatty acids and one molecule of glycerol. Simple
fats are known as triglycerides or triacylglycerols.
Triglycerides are composed of glycerol and three long chain fatty acids. Enzymes that
break simple fat into its component fatty acid and glycerol are known as lipase.
Some microorganisms possess the enzyme lipase. Thus they can be classified based
on their ability to produce and secrete lipases. A variety of simple fats can be used for
this determination, depending on the type of organism being tested

Objective
determine the ability of microorganisms to produce the enzyme lipase.
To identify the bacteria on the basis of its lipase activity

Uses
The lipase test is used to detect and enumerate lipolytic bacteria, especially in high-
fat dairy products.
A variety of other lipid substrates, including corn oil, olive oil, and soybean oil, are
used to detect differential characteristics among members of Enterobacteriaceae,
Clostridium, Staphylococcus, and Neisseria.
Several fungal species also demonstrate lipolytic ability.

Result Interpretation
Positive test: A positive lipase test is noted by the appearance of an iridescent
sheen (oil on water) immediately around colonies that can be seen when the plate
is held at an angle to a light source.
Negative test: A negative lipase test is indicated by the absence of an iridescent
sheen.
Clostridium sporogenes. Lipase positive; iridescent sheen on agar surface when
plate is held at an angle to the light source.
 LIPID HYDROLYSIS TEST:
Lipids are high-molecular-weight compounds possessing large amounts of energy.
Once assimilated into the cell, they are metabolized through aerobic respiration to
produce cellular energy, adenosine triphosphate (ATP). The components may also
enter other metabolic pathways for the synthesis of other cellular protoplasmic
requirements.
However, before their assimilation by bacteria, they need to be degraded. The
degradation of lipids such as triglycerides is accomplished by extracellular
hydrolyzing enzymes, called lipases (esterases), that cleave the ester bonds in this
molecule by the addition of water to form the building blocks glycerol (an alcohol) and
fatty acids
Objective
To determine the ability of the organism to hydrolyse lipid.
To identify bacteria capable of producing the exoenzyme lipase.
Uses
Helpful in identifying bacteria that secrete lipase, including members of
Enterobacteriaceae, Fusobacterium, Propionibacterium, Clostridium,
Pseudomonas, Mycoplasma, Corynebacterium, and Staphylococcus.
For the detection and enumeration of lipolytic microorganisms in food and other
material.

Result Interpretation
Positive test: Formation of a clear zone around the bacterial growth
Negative test:No clear zone around growth.

Tributyrin agar plate. No Lipid hydrolysis on

left; lipid hydrolysis on right.
 LITMUS MILK TEST:
Milk is an excellent medium for the growth of microorganisms because it contains
the milk protein casein, the sugar lactose, vitamins, minerals and water. Litmus milk
is a milk-based medium used to distinguish between different species of bacteria.
The lactose (milk sugar), litmus (pH indicator), and casein (milk protein)
contained within the medium can all be metabolized by different types of
bacteria. The test differentiates microorganisms based on various metabolic
reactions in litmus milk, including reduction, fermentation, clot formation,
digestion, and the formation of gas.

Objective
To determine an organism’s ability to metabolize litmus milk.
To differentiate among microorganisms that enzymatically transforms different milk
substrates into varied metabolic end products.
Uses
The litmus milk test differentiates members of the Enterobacteriacaeae from other
gram-negative bacilli based on the enterics’ ability to reduce litmus.
It is commonly used to differentiate members within the genus Clostridium.
It mainly aids in the identification and differentiation of Enterococcus, and Lactic
acid bacteria. The media may also be used to grow lactic acid bacteria
Result Interpretation
Positive test
Acid pH: pink to red color, Alkaline pH: purplish- blue color, Reduction: white, Acid
curd: hard curd with clear supernatant (whey), Digestion: Dissolution of clot with
clear, grayish, watery fluid and a shrunken, insoluble pink clot, Rennet curd: soft curd
followed by peptonization (alkaline pH, supernatant brown), Gas production: bubbles
in coagulated milk

Negative test:
Color and consistency remain same.
 LYSINE IRON AGAR (LIA) SLANTS TEST:
Lysine iron agar (LIA) slants tests organisms for the ability to deaminate lysine or
decarboxylate lysine. Lysine deamination is an aerobic process which occurs on
the slant of the media. Lysine decarboxylation is an anaerobic process which
occurs in the butt of the media.
Uses
This test is used to differentiate gram-negative bacilli based on decarboxylation or
deamination of lysine and the formation of hydrogen sulfide (H2S).
It employs a sensitive medium for the detection of lactose-fermenting and non
lactose-fermenting salmonellae.
Lysine Iron Agar is specified in standard methods for Salmonella testing.
Result Interpretation
Lysine iron agar. A, Alkaline slant/alkaline butt (K/K). B, Alkaline slant/alkaline butt,
H2S positive (K/K H2S+). C, Alkaline slant/acid butt (K/A). D, Red slant/acid butt (R/A).
E, Uninoculated tube.

Lysine Decarboxylation (detected in butt):

Positive Test: Purple slant/purple butt (alkaline), the butt reaction may be masked
by H2S production
Negative Test:Purple slant/yellow butt (acid), fermentation of glucose only
Lysine Deamination (detected on slant):
Positive Test:Red slant
Negative Test:Slant remains purple
H2S Production:
Positive Test:Black precipitate
Negative Test:No black color development
Gas production:demonstrated by the presence of bubbles or cracks in the medium
 MALONATE TEST:
Malonate test is a colorimetric test of the ability of bacteria to use malonate as a
source of carbon, the endpoint of which is the production of alkaline metabolites
that induce a color change.
Objectives
To test the ability of the organism to utilize malonate as sole source of carbon and
energy for growth.
To differentiate organisms on the basis of malonate utilization.
Uses
The Malonate Test was originally designed to differentiate between Escherichia and
Enterobacter.
Its use as a differential medium has now broadened to include other members of
Enterobacteriaceae.
The malonite reaction can be used todifferentiate among Enterobacteriaceae:
Klebsiella pneumoniae is positive (blue at 24 hours), Escherichia coli is
negative(culture remains green).
The test can also be used to separate Salmonella arizonae (positive) from other
Salmo ella spp(negative).
In differentiating Enterobacteriaceae in food and dairy products.

Result
A positive malonate test is indicated by the development of a blue color in the
medium.
A negative malonate test is indicated by the media remaining green or turning
yellow due to dextrose fermentation.
 METHYL RED (MR) TEST:
The methyl red (MR) test detects the production of sufficient acid during the
fermentation of glucose and the maintenance of conditions such that the pH of
an old culture is sustained below a value of about 4.5, as shown by a change in
the color of the methyl red indicator which is added at the end of the period of
incubation.
Clark and Lubs developed MR-VP Broth which allowed both the MR and VP tests
to be performed from the same inoculated medium by aliquoting portions to
different tubes.

Uses
Originally the paired MR-VP tests were used to distinguish between members of the
family Enterobacteriaceae, but now they are used to characterize other groups of
bacteria including Actinobacteria.

Result Interpretation
Positive Reaction: A distinct red color (A)
Examples: E. coli, Yersinia sps, etc.
Negative Reaction: A yellow color (B)
Examples: Enterobacter aerogenes, Klebsiella pneumoniae, etc.
A weak positive is red-orange. If an orange color is seen, incubate the
remainder of the broth for up to 4 days and repeat the test after further
incubation. In this case it may also be helpful to set up a duplicate broth at 25cْ
 MICRODASE TEST:
Microdase Disk is a reagent-impregnated disk recommended for use in qualitative
procedures to aid in the differentiation of Staphylococcus from Micrococcus by the
detection of the oxidase enzyme.
The oxidase method was originally described by Kovacs in 1956 as a method of
differentiating gram-negative bacilli. However, in 1981, Faller and Schleifer modified
Kovacs’ oxidase reagent by utilizing a tetramethyl-pphenylenediamine (TMPD) in
dimethyl sulfoxide (DMSO).This is referred to as the modified oxidase test.

Objectives
To differentiate gram-positive, catalase-positive cocci

Uses
This test is used to differentiate Micrococci from Staphylococci. (Micrococci should
yield a positive result. Staphylococci should yield a negative result, with the
exception of Staphylococcus sciuri, S. lentus and S. vitulinus

Expected results
Positive: Development of blue to purple-blue color within 2 minutes time
Negative: No color change (white to gray color remains).
 MOTILITY TEST:
Motility is the ability of an organism to move by itself by means of propeller-like flagella
unique to bacteria or by special fibrils that produce a gliding form of motility. Motile
bacteria move using flagella, thread like locomotor appendages extending outward
from the plasma membrane and cell wall either single flagellum or multiple flagella.
Motility has long been recognized as an important taxonomic tool and biological
characteristic of microorganisms. The presence of flagella occurs primarily in bacilli
but there are a few flagellated cocci, thus motility is a very important means of
identification in the family Enterobacteriaceae. From the early days in the field of
microbiology, the ability of bacteria to move has been used as a means of
differentiation and classification of organisms.

Objectives
To determine the motility of bacterium.
To differentiate between motile and non motile bacteria.

Uses
It is used for the differentiation of microorganisms on the basis of motility in a
laboratory setting.
It is performed to assign taxonomic classification to organisms.
Motility tests are important in characterization of pathogens.
The tests are often employed in identification protocols in the family
Enterobacteriaceae
Motility test is also used for the species differentiation of gram positive cocci,
Enterococci. Enterococcus faecium and E. faecalis are non-motile, whereas E.
gallinarum and E. casseliflavus/E. flavescens generally are motile.

Expected Results
Positive: Diffuse, hazy growths that spread throughout the medium rendering it slightly
opaque.
Negative: Growth that is confined to the stab-line, with sharply defined margins and
leaving the surrounding medium clearly transparent.
 MRS BROTH TEST:
The MRS formulation was developed by de Man, Rogosa and Sharpe to replace a
variable product (tomato juice) and, at the same time, provide a medium which would
support good growth of lactobacilli in general. Hence, MRS agar and broth given to
their superiority are commonly employed for culturing and identifying of Lactic acid
bacteria especially the Lactobacillus spp.
“Lactic acid bacteria” includes species of the following genera: Lactobacillus,
Streptococcus, Pediococcus and Leuconostoc. All these species can produce lactic
acid in considerable amounts.
They are Gram-positive, catalase and oxidase negative and are fastidious in their
nutritional requirements. Growth is enhanced considerably by microaerobic conditions.
MRS medium is selective for lactobacilli but some growth of leuconostocs and
pediococci may occur. Selectivity can be altered by pH adjustment. Lactobacilli will
tolerate lower pH levels than streptococci (pH 5.0-6.5) with pediococci and
leuconostocs growing best within this range

Objectives
To determine whether an organism forms gas during glucose fermentation.
Uses
It is used for the identification of some Lactobacillus spp. and Leuconostoc sp.
which produce gas.
The test is a part of the confirmatory tests performed on organisms isolated on MRS
Agar.
Commonly performed to identify Lactobacilli from oral cavity, dairy products, foods,
faeces and other sources.
Result
Positive: Growth with marked turbidity of the broth, gas production indicated by
a bubble in the Durham tube (Leuconostoc spp.) Growth resulting turbidity, no
gas production (Lactobacillus spp)
Negative: No growth (no turbidity) or gas production

MRS broth. A, Positive; gas production by Leuconostoc sp.(Left).

B, Positive: growth, no gas production by Lactobacillus sp.(Right)
 MUG TEST:
has been reported that the enzyme β-glucuronidase is present in most strains of E.
coli(97%).Organisms other than E. coli (e.g., Salmonella, Shigella, Staphylococcus,
Streptococcus, etc.) also possess the enzyme β-glucuronidase. Hence, the detection of
the β-glucuronidase enzyme is commonly employed in laboratories to identify and
differentiate such organisms.
The substrate, 4-methylumbelliferyl-β-D-glucuronide (MUG), is both sensitive and
selective for detection of β-glucuronidase activity. Hence, the MUG test in conjunction
with oxidase, indole, and lactose fermentation can be performed to effectively identify
E. coli and related organisms.
Objectives
To identify glucuronidase activity in orgnasims by fluorescence, under a long-wave
(365nm) UV light source.

Uses
The test is used to presumptively identify various genera of Enterobacteriaceae.
To characterize verotoxin-producing Escherichia coli. (verotoxinproducing strains
of E. coli do not produce MUG, and a negative test result may indicate the presence
of a clinically important strain.)
It aids in the detection of Escherichia colifrom water and food samples
Result
Positive: Electric blue fluorescence
Negative: Lack of fluorescence
 NITRATE REDUCTION TEST:
Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen
(O2). Many gram-negative bacteria use nitrate as the final electron acceptor.
Nitrate reduction test is a test that determines the production of an enzyme called
nitrate reductase, which results in the reduction of nitrate (NO3).
Bacterial species may be differentiated on the basis of their ability to reduce nitrate to
nitrite or nitrogenous gases.

Objectives
To determine the ability of an organism to reduce nitrate to nitrite.
Identify the different ways that nitrate can be reduced by bacteria
Uses
All members of the Enterobacteriaceae family reduce nitrate, but some members
further metabolize nitrite to other compounds. It is thus used to differentiate
members of Enterobacteriaceae that produce enzyme nitrate reductase from Gram
negative bacteria that do not produce the enzyme nitrate reductase.
The reduction of nitrate may be coupled to anaerobic respiration in some species.
It is used in differentiating Mycobacterium
Identifying species of Neisseriaand separating them from Moraxella and Kingella
The nitrate reduction test is a critical test for differentiating between N. gonorrhoeae
and K. denitrificans, particularly when strains of K. denitrificans appear to be gram-
negative diplococci in stained smears.
Facilitating species identification of Corynebacterium
Result
Positive Test:
– Development of a cherry red coloration on addition of reagent A and B
– Absence of a red color development on adding Zn powder
Negative Test:
– A development of red color on addition of Zn powder
 NOVOBIOCIN SUSCEPTIBILITY TEST:
Novobiocin is an aminocoumarin antibiotic, produced by the actinomycete
Streptomyces nivens, with antibacterial property. In 1975, Kloos and Schleifer reported
a simplified scheme for differentiating coagulase-negative Staphylococcus spp. which
included a novobiocin disk test.
Staphylococcus saprophyticus, a gram positive coagulase negative Staphylococci, is
an uropathogenic bacterium that causes acute uncomplicated urinary tract infections,
particularly in young, middle aged female patients. Unlike most other CONS,
saprophyticus is rarely resistant to most antibiotics active against gram-positive
organisms. S. saprophyticus is innately resistant to the antibiotic novobiocin.
Therefore, screening coagulase-negative staphylococci from urine cultures for
novobiocin resistance is a reliable presumptive identification of saprophyticus.
Objectives
To determine the susceptibility pattern of bacterium to antibiotic novobiocin.

Uses
The CoNS have been subdivided into two groups based on their novobiocin
susceptibility pattern. The CoNS group that demonstrates novobiocin susceptibility
includes epidermidis, S. capitis, S. haemolyticus, S. hominis subsp. hominis, S.
lugdunensis, S. saccharolyticus, S. warneri, and other species.
The novobiocin resistant group consists of such species as cohnii, S. kloosii, S.
saprophyticus, and S. xylosus.
It is useful for presumptively distinguishing Staphylococcus saprophyticusfrom
other coagulase-negative staphylococci in clinical specimens.
Expected Results
Positive – A zone of inhibition greater than 16mm indicates that the organism is
sensitive to the antibiotic.
Negative – A zone of inhibition less than or equal to 16mm is indicative of
novobiocin resistance.
 OF (OXIDATION-FERMENTATION) TEST:
Carbohydrates are organic molecules that contain carbon, hydrogen and oxygen
in the ratio (CH2O)n. Organisms use carbohydrate differently depending upon
their enzyme complement.
The pattern of fermentation is characteristics of certain species, genera or
groups of organisms and for this reason this property has been extensively used
as method for biochemical differentiation of microbes.

Objectives
To detect the oxidation or fermentation of carbohydrates by bacteria.

Uses
It aids in the identification of gram-negative bacteria on the basis of their ability to
oxidize or ferment a specific carbohydrate.
It is used to determine whether an organism uses carbohydrate substrates to
produce acid byproducts.
Non fermentative bacteria are routinely tested for their ability to produce acid from
six carbohydrates (glucose, xylose, mannitol, lactose, sucrose, and maltose).

Expected Results
Positive: A positive carbohydrate utilization test is indicated by the
development of a yellow color in the medium.
Oxidative:Development of a yellow coloration in the open tube only.
Fermentative: Development of a yellow coloration in both open and closed
tubes.
Negative: A negative carbohydrate utilization test is indicated by the absence of
a yellow color (media remains green or turns blue).
Non-oxidizer/Non-fermenter
 ONPG TEST:
The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-
galactosidase. Permease allows lactose to enter the bacterial cell wall, where it is then
broken down into glucose and galactose by beta-galactosidase. The glucose and
galactose can then be metabolized by the bacteria. However, some organisms lack
permease and appear as late or non-lactose-fermenters.
The ONPG test is considered to be a very sensitive test for lactose-fermentation. O-
nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, is incorporated
into this test and acts as the substrate for the beta-galactosidase to ascertain the
particular enzyme activity which subsequently aids in the identification and
differentiation of different organisms.

Objectives
To determine the ability of an organism to produce β-galactosidase enzyme.
Uses
The test is used in differentiating members of the Enterobacteriaceae and other
microorganisms based on beta-D-galactosidase activity.
The test distinguishes late lactose fermenters from non–lactose fermenters of
Enterobacteriaceae.
Expected Results
Positive: Development of a yellow coloration (presence of β-galactosidase)
Note: The fluid and disc will turn any shade of yellow if positive for
galactosidase enzyme.
Negative: No color development (absence of enzyme)

ONPG Test Result – o-Nitrophenyl-b-D-

galactopyranoside (OPNG) test. A, Positive. B,
Negative.
 OXIDASE TEST:
The oxidase test detects the presence of a cytochrome oxidase system that will
catalyse the transport of electrons between electron donors in the bacteria and a
redox dye- tetramethyl-p-phenylene-diamine. The dye is reduced to deep purple
color. This test is used to assist in the identification of Pseudomonas, Neisseria,
Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella, all of
which produce the enzyme cytochrome oxidase.

Uses
The oxidase test is used to determine if an organism possesses the cytochrome
oxidase enzyme.
The test is used as an aid for the differentiation of Neisseria, Moraxella,
Campylobacter and Pasteurella species (oxidase positive).
It is also used to differentiate pseudomonads from related species.

Expected Results
Positive Result
Development of a deep purple-blue/blue color indicates oxidase production
within 5-10 seconds.
Negative Result
No purple-blue color/No color change.

Oxidase Positive Organisms: Pseudomonas, Neisseria, Alcaligens, Aeromonas,

Campylobacter, Vibrio, Brucella, Pasteurella, Moraxella, Helicobacter pylori,
Legionella pneumophila, etc.
Oxidase Negative Organisms: Enterobacteriaceae (e.g. E. coli)
 PHENOL RED FERMENTATION TEST:
Fermentation media are used to differentiate organisms based on their ability to ferment
carbohydrates incorporated into the basal medium.
Phenol Red Broth Medium with various added carbohydrates serves as a differential
medium by aiding in differentiation of various species and genera by their ability to ferment
the specific carbohydrate, with the production of acid or acid and gas.
The carbohydrate source can vary based on test requirements. The common broth media
used are:
Phenol Red Glucose Broth
Phenol Red Lactose Broth
Phenol Red Maltose Broth
Phenol Red Mannitol Broth
Phenol Red Sucrose Broth

Objective
To determine the fermentation reactions of pure cultures of microorganisms.
Uses
It is recommended to determine the fermentation reaction of carbohydrates for
the differentiation of microorganisms.
It is useful in identifying Gram negative bacilli, especially Enterobacteriaceae.
Result Interpretation
I. Acid production:
Positive: After incubation the liquid in the tube turns yellow (indicated by the change in
the color of the phenol red indicator). It indicates that there is drop in the pH because of
the production of the acid by the fermentation of the carbohydrate (sugar) present in the
media.
Negative:The tube containing medium will remain red, indicating the bacteria cannot
ferment that particular carbohydrate source present in the media.
II. Gas Production
Positive:A bubble (small or big depending up the amount of gas produced) seen in the
inverted Durham tube.
Negative: No bubble in the inverted Durham tube i.e. bacteria does not produce gas from
the fermentation of that particular carbohydrate present in the media i.e. anaerogenic
organism.
 PHENYLALANINE AGAR TEST:
Biochemical tests are the tests used for the identification of bacteria species based on the
differences in the biochemical activities of different bacteria. Bacterial physiology differs
from one type of organism to another. The differences in carbohydrate metabolism, protein
metabolism, fat metabolism, production of certain enzymes, ability to utilize a particular
compound etc. help them to be identified.
On such test is the the phenylalanine agar test which tests for the ability of some specific
species to convert amino acid phenylalanine to phenylpyruvic acid; an important reaction
in the differentiation of Enterobacteriaceae. Based on this criterion, the test is carried out
for the differentiation of Proteus and Providencia group from other members of
Enterobacteriaceae which lack such an activity.

Objective
To determine the ability of an organism to oxidatively deaminate phenylalanine
to phenylpyruvic acid.
Uses
It is recommended for use in the differentiation of gram-negative enteric bacilli based on
the ability of the microorganisms to produce phenylpyruvic acid by oxidative
deamination. The genera Morganella, Proteus, and Providencia can be differentiated
from other members of the Enterobacteriaceae family.
Result Interpretation
Positive: Green color develops on slant after ferric chloride is added within 1-5 minutes
after applying ferric chloride reagent.
Negative: Absence of a green color reaction. Negative results will take on a yellow color
due to the color of the ferric chloride.
 POTASSIUM HYDROXIDE TEST:
The KOH String Test relies on the differential resistance to 3% potassium hydroxide
between gram positive and negative cells, where a portion of a colony is mixed with
a small volume of 3% KOH. If the cells lyses, the liberated cellular DNA makes the
mixture viscous or “stringy.” The positive string test indicates a gram negative
organism. Hence the alternative name for the test is “String Test”.
The potassium hydroxide test may thus aid in differentiation between Gram positive
and Gram negative organisms and is a useful complement to the Gram stain and the
antibiotic disc test.

Objective
To differentiate between Gram negative and Gram positive organisms

Uses
In laboratories where large numbers of cultures have to be processed, the
above test may be used in addition to Gram stain for preliminary
differentiation.
It is a useful complement to the Gram stain and the antibiotic disc test.

Result Interpretation
Positive: Organisms become thick, stringy and form long strands within
the first 30sec. This is seen in Gram negative bacteria.
Negative: Organisms leave the suspension unaltered or absence of
stringing. This is seen in Gram positive bacteria.
 PYRUVATE BROTH TEST:
Biochemical tests are the tests used for the identification of bacterial species based
on the differences in the biochemical activities of different bacteria. Bacterial
physiology differs from one type of organism to another.
The ability of bacteria to form organic compounds by metabolizing certain
carbohydrates and related compounds is a widely used method for the
identification of microorganisms.
One such test is the pyruvate broth test which tests for the ability of some specific
species to utilize the substrate pyruvate.

Objective
To determine the ability of an organism to utilize pyruvate to produce
acidic end products.

Uses
The test aids in the differentiation between Enterococcus faecalis
(positive) and Enterococcus faecium (negative).
It can be used as a part of an identication tests for other organisms
capable of utilizing pyruvate.

Result Interpretation
Positive: A positive pyruvate utilization result is indicated by a color
change of the broth from greenish-blue to yellow.
Negative: A negative pyruvate utilization result is indicated by no color
change and the resultant broth remaining a greenish-blue color.
 PYR TEST:
PYR (Pyrrolidonyl Aminopeptidase) Test is used for the detection of pyrolidonyl
arylamidase (also called pyrrolidonyl aminopeptidase) activity in Streptococcus
pyogenes(group A strep), Enterococcus spp., some coagulase-negative
staphylococci, and some Enterobacteriaceae. It is also known as PYR (L-
pyrrolidonyl-β-naphthylamide) which serve as a substrate for the detection of
pyrrolidonyl peptidase.

Uses
It is used for the presumptive identification of group A streptococci
(Streptococcus pyogenes).
It is used for the rapid differentiation of enterococci from group D ß-
hemolytic streptococci.
It also differentiate some Staphylococci (positive haemolyticus from
negative S. auricularis).
It is used in the identification of E. coli, separating it from other indole
positive, lactose positive, gram-negative rods.
Result Interpretation
Positive: Bright pink or cherry-red color within 1-2 minutes.
Examples: Group A Streptococci (Streptococcus pyogenes), Group D Enterococci
(Enterococcus faecalis and Enterococcus faecium), Coagulase negative
Staphylococcus species such as S. hemolyticus, S. lugdunensis, S. schleiferi.,
Enterobacter, Citrobacter, Klebsiella, Yersinia and Serratia, Aerococcus, Gamella,
Lactococcus, most Corynebacterium (Arcanobacterium) hemolyticum.
Negative: No color change or a blue color due to a positive indole reaction.
Examples: Group B Streptococci (Streptococcus agalactiae), Streptococcus mitis,
S. bovis, S. equinus, S. milleri.
 SALT TOLERANCE TEST:
The ability of the bacteria to grow in the presence of variable amount of
Sodium Chloride (NaCl) has been used to characterize several bacteria. It
takes into account the organism’s ability to tolerate various osmotic
concentrations. E. faecalis, E. zymogenes, E. liquifaciens, and E. durans are
among the Enterococcus species that are salt tolerant.
Objective
To determine the ability of an organism to grow in high concentrations of
salt.
Uses
It is used to differentiate enterococci (positive) from non-enterococci
(negative).
It is used to differentiate non-beta-hemolytic strains of catalase- negative,
gram-positive cocci (i.e. Enterococcus and Aerococcus) based on their
ability to grow in a 6.5% sodium chloride broth.
Aerococcus species such as A. viridans and A. urinae can also grow in
6.5% NaCl, therefore salt tolerance broth can also be used to differentiate
Aerococcus species from other similar organisms such as Stomatococcus
and Helcococcus.
Result Interpretation
Positive: Visible turbidity in the broth, with or without a color change from
purple to yellow. Note: Turbidity alone is indicative of a positive test
Negative: No turbidity and no color change after 72 hours of incubation.
 STARCH HYDROLYSIS TEST:
Starch is a complex polysaccharide found abundantly in plants and usually
deposited in the form of large granules in the cytoplasm of the cell.
Starch consists of 2 components—amylose and amylopectin, which are present in
various amounts.
The amylose consists of D-glucose units linked in a linear fashion by α-1,4 linkages.
It has 2 non-reducing ends and a reducing end.
Amylopectin is a branched polysaccharide. In these molecules, shorter chains of
glucose units linked by α-1,4 are also joined to each other by α-1,6 linkages.
The major component of starch can be hydrolyzed by a-amylase, which is present in
some bacteria while well known in case of fungi.
The ability to degrade starch is used as a criterion for the determination of amylase
production by a microbe.
Objective
To determine the ability of an organism to hydrolyze starch
To differentiate organism based on their α- amylase enzyme activity

Uses
It aids in the differentiation of species of genera Corynebacterium,
Clostridium, Bacillus, Bacteroides, Fusobacterium, and members
ofEnterococcus spp
Result Interpretation
Positive test:A clear zone around the line of growth after addition of iodine
solution indicates that the organism has hydrolyzed starch.
Negative test:A blue, purple, or black coloration of the medium (depending
on the concentration of iodine).
 SULPHUR REDUCTION TEST:
SIM medium (Sulphide Indole Motility medium) which is a combination
differential medium that tests three different parameters, Sulfur Reduction,
Indole Production and Motility.
As the name suggests, it is commonly used to test a microbe for the ability to
produce the gas hydrogen sulfide (H2S). The “S” in SIM stands for sulfur

Objective
To test for the ability of an organism to reduce sulphur
To differentiate gram-negative enteric bacilli on the basis of sulfide production

Uses
It is used to differentiate sulfur reducing members of the genera
Salmonella, Shigellaand Proteus from the negative Moranella morganii and
Providencia rettgeri.
The production of hydrogen sulphide is a useful diagnostic test in the
identification of enteric bacteria.

Result Interpretation
Positive: Darkening of the medium (a black precipitate) or blackening of the
line of inoculation indicates the presence of bacteria producing hydrogen
sulfide.
Negative: A negative H2S test is denoted by the absence of blackening.
 SXT TEST:
Although bacitracin susceptibility has routinely been used for the presumptive
identification of group A streptococci, it has been observed that streptococcal
groups vary in their susceptibilities to Taxo A (bacitracin, 0.04 units) and SXT
(trimethoprim [1.25 mg] plus sulfamethoxazole [23.75 mg],) disks.
Group A beta hemolytic strep is susceptible to the A disc, but resistant to SXT
Group B beta hemolytic strep is resistant to both A and SXT discs
Other types of beta-hemolytic streps are resistant to the A disc, but sensitive to SXT
(example Group C)
Objective
To differentiate among Streptococcal groups based on their susceptibility to
sulfamethoxazole/ trimethoprim (SXT) antibiotics.
Uses
SXT disc susceptibility test in conjunction with bacitracin is used for the
presumptive identification of beta hemolytic streptococci on blood agar
(presumptively identifying beta-hemolytic streptococci as either group A, B or
not group A and B).
The resistance to SXT is used for the primary recovery of groups A and B
streptococci from specimens with mixed culture. Their resistance allows them to
selectively grow out from contaminating bacteria that are inhibited by this
antibiotic.
Bacitracin/SXT susceptibility tests are still in use where facilities for serologic
group determination are unavailable.
Result Interpretation
Susceptible: A zone of inhibition around the disc indicates susceptibility to
sulfamethoxazole/ trimethoprim.
Resistant: The absence of a zone of inhibition around the SXT disk
indicates resistance to sulfamethoxazole/ trimethoprim.
 THE TRIPLE SUGAR IRON (TSI) TEST:
The Triple Sugar Iron (TSI) test is a microbiological test named for its ability to test
a microorganism’s ability to ferment sugars and to produce hydrogen sulfide.
An agar slant of a special medium with multiple sugars constituting a pH-sensitive
dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium
thiosulfate and ferrous sulfate or ferrous ammonium sulfate is used for carrying out
the test.

Objective
To determine the ability of an organism to ferment glucose, lactose, and
sucrose, and their ability to produce hydrogen sulfide.

Uses
The test is used primarily to differentiate members of the Enterobacteriaceae
family from other gram-negative rods.
It is also used in the differentiation among Enterobacteriaceae on the basis of
their sugar fermentation patterns.

Result Interpretation
An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose
fermentation only.
An acid/acid (yellow slant/yellow butt) reaction: It indicates the
fermentation of dextrose, lactose and/or sucrose.
An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate
fermentation results.
Blackening of the medium: Occurs in the presence of H2
Gas production: Bubbles or cracks in the agar indicate the production of
gas ( formation of CO2and H2)

Triple sugar iron agar. A, Acid slant/acid butt with gas, no H2S
(A/A). B, Alkaline slant/acid butt, no gas, H2S-positive (K/A
H2S+). C, Alkaline slant/alkaline butt, no gas, no H2S (K/K). D,
Uninoculated tube.
 UREASE TEST:
Urea Agar was developed by Christensen in 1946 for the differentiation of enteric
bacilli. The urease test is used to determine the ability of an organism to split urea,
through the production of the enzyme urease.

Uses
This test is used to differentiate organisms based on their ability to hydrolyze
urea with the enzyme urease.
This test can be used as part of the identification of several genera and species
of Enterobacteriaceae, including Proteus, Klebsiella, and some Yersinia and
Citrobacter species, as well as some Corynebacterium species.
It is also useful to identify Cryptococcus spp., Brucella, Helicobacter pylori, and
many other bacteria that produce the urease enzyme.
Directly, this test is performed on gastric biopsy samples to detect the presence
of H. pylori.

Result Interpretation
Positive Reaction: Development of an intense magenta to bright pink color
in 15 min to 24 h.
Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp,
Helicobacter pylori, Yersinia spp, Brucella spp.

Negative Reaction: No color change.

Examples: Escherichia, Shigella, Salmonella.
 VOGES–PROSKAUER (VP) TEST:
The Voges-Proskauer (VP) test is used to determine if an organism produces
acetylmethyl carbinol from glucose fermentation. If present, acetylmethyl
carbinol is converted to diacetyl in the presence of ∝- naphthol, strong
alkali (40% KOH), and atmospheric oxygen. The ∝-naphthol was not part of
the original procedure but was found to act as a color intensifier by Barritt
and must be added first. The diacetyl and quanidine-containing compounds
found in the peptones of the broth then condense to form a pinkish red
polymer.

Result Interpretation
Positive Reaction: A pink-red color at the surface
Examples: Viridans group streptococci (except Streptococcus vestibularis),
Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio
eltor, Vibrio alginolyticus.

Negative Reaction: A lack of a pink-red color

Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia,
Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and
Vibrio parahaemolyticus .

A copper color should be considered negative. A rust color is a weak

positive reaction
 X AND V FACTOR TEST:
Haemophilus spp are small, pleomorphic, gram-negative bacilli or coccobacilli with
random arrangements.
A clinically important species of the genus, influenzaeis a fastidious organism
which grows best at 35-37°C with ~5% CO2 and in the presence of special
accessory growth factors called X and V factors.
X factor comprises protoporphyrin IX, also called haemin or other iron-containing
porphyrins. These are required for growth because X-dependent strains are unable
to convert d-aminolaevulinic acid to protoporphyrin. They are heat stable.
V factor comprises nicotinamide adenine dinucleotide (NAD) or nicotinamide
adenine dinucleotide phosphate (NADP). They are heat labile.
However, different species of Haemophilushave varying requirements for X and V
growth factors.
For example. Haemophilus parainfluenzaerequires V factor only for grow while
Haemophilus ducreyi requires only X factor without need of V factor.
Objective
To differentiate among Haemophilus species based on their characteristic
utilization of X and V growth factors.
Uses
They are used for the differentiation of Haemophilus species, including
Aggregatibacter aphrophilus based upon their requirements for the growth
factors.

Result Interpretation
Positive:
Growth around the XV disk only shows a requirement for both factors.
Growth around the V disk, no growth around the X disk, and light growth around
the XV disk shows a V factor requirement.
Negative:
Growth over the entire surface of the agar indicates no requirement for either X
or V factor.