Biochemical Testing

Dr. Muhammad Asif Shaheen

Assistant Professor
Department of Microbiology,
GC University, Lahore
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
and 30% for Neisseria spp.
Principle of Catalase Test

 The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen
and water. The presence of the enzyme in a bacterial isolate is evident when a
small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of
oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble
production. The culture should not be more than 24 hours old.
 Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide
which is accumulated as an end product of aerobic carbohydrate metabolism.
 Purpose or Uses of Catalase Test
 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(which
cannot survive in the presence of oxygen) bacteria.
 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.
Procedure

 Tube Method
 Pour 1-2 ml of hydrogen peroxide solution into a test tube.
 Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24
hours test organism and immerse in the hydrogen peroxide solution.
 Observe for immediate bubbling.
 Slide Method
 Use a loop or sterile wooden stick to transfer a small amount of colony growth in
the surface of a clean, dry glass slide.
 Place a drop of 3% H2O2 in the glass slide.
 Observe for the evolution of oxygen bubbles.
Results interpretation
Positive: Copious bubbles produced, active bubbling
 Precautions of Catalase Test
 The test organisms should not be taken from blood agar culture. Red Blood cells
contain catalase and their presence will give a false positive test.
 Culture should be 18 to 24 hours old.
 Hydrogen peroxide must be fresh as it is very unstable.
 Iron wire loop should not be used.
 Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen
peroxide causing the reaction to be weakly positive; (a few bubbles elaborated
slowly). This should not be confused with a truly positive reaction.
 Do not add organism to reagent, particularly if iron-containing inoculating loops are
used. Iron containing loops will cause false positive test results if exposed to
hydrogen peroxide.
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).
Principle of Coagulase Test

 Coagulase is an enzyme-like protein and causes plasma to clot by converting

fibrinogen to fibrin. Staphylococcus aureus produces two forms of coagulase:
bound and free.
 Bound coagulase (clumping factor) is bound to the bacterial cell wall and reacts
directly with fibrinogen. This results in an alternation of fibrinogen so that it
precipitates on the staphylococcal cell, causing the cells to clump when a bacterial
suspension is mixed with plasma. This doesn’t require coagulase-reacting factor.
 Free coagulase involves the activation of plasma coagulase-reacting factor
(CRP), which is a modified or derived thrombin molecule, to from a coagulase-CRP
complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.
Slide Test (to detect bound
coagulase)
 Place a drop of physiological saline on each end of a slide, or on two separate
slides.
 With the loop, straight wire or wodden stick, emulsify a portion of the isolated
colony in each drops to make two thick suspensions.
 Add a drop of human or rabbit plasma to one of the suspensions, and mix gently.
 Look for clumping of the organisms within 10 seconds.
 No plasma is added to the second suspension to differentiate any granular
appearance of the organism from true coagulase clumping.
Tube Test (to detect free coagulase)

 Take 3 small test tubes and label as T (Test), P (Positive Control) and N
(Negative Control).
 Add .3-0.5ml Rabbit plasma in tube
 Mix single colony
 Incubate at 37 for 4 hours or at 25c for 24 hours.
 Check for clot formation
Organism Slide coagulase Tube coagulase

Staphylococcus aureus

+ (95%) +
Staphylococcus lugdunensis

+ -
Staphylococcus intermedius

- +
Staphylococcus epidermidis

- -
S. saprophyticus

- -
Precautions

 Don’t use human blood it may contain inhibitory substances

 Do incubate above 4 hours at 37
 Incubate for 24hours at 25C
 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.
 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.
 Principle
 The test is used to determine the ability of an organism to hydrolyze DNA. DNase agar
is a differential medium that tests the ability of an organism to produce an exo-
enzyme, called deoxyribonuclease. DNase are extracellular endonucleases that
cleave DNA and release free nucleotides and phosphate. DNase agar contains
nutrients for the bacteria, DNA, and mostly methyl green as an indicator. Methyl green
is a cation which binds to the negatively-charged DNA.
 Deoxyribonuclease allows the organisms that produce it to break down DNA into
smaller fragments.
 When the DNA is broken down, it no longer binds to the methyl green, and a clear
halo will appear around the areas where the DNase-producing organism has grown.
 the hydrolysis of DNA is observed by a clearing of the agar after addition of HCL
(oligonucleotides dissolves in acid but DNA salts are insoluble). The acid precipitates
unhydrolyzed DNA making the medium opaque. Therefore, DNase producing colonies
hydrolyze DNA and produce a clear zone around the growth.
 Media:
 DNase Medium
 Pancreatic digest of casein (10 g), yeast extract (10 g), deoxyribonucleic acid (2 g),
NaCl (5 g), agar (15 g), methyl green (0.5 g), pH 7.5.
 Method
 Using a sterile loop, inoculate the DNase agar with the organism to be tested on the
test area.
 Incubate the plate at 35-37°C for 24 hours.
 After incubation observe the color change in DNase with methyl green.
 In DNase agar without indicators:
 Flood the surface of agar with 1N HCL solution. Tip off the excess acid.
 Allow the reagent to absorb into the plate.
 Observe for clear zone around the colonies within 5 minutes.
Uses

 Used to determine the ability of an organism to hydrolyze

deoxyribonucleic acid.
 Used to differentiate Staphylococcus aureus which produces the enzyme
deoxyribonuclease from other Staphylococci which do not produce
DNase.
 It is also used to distinguish Serratia (positive) from Enterobacter sp.
 Moraxella catarrhalis (positive) from Neisseria
 Stenotrophomonas maltophila is Positive
 Vibrio cholera is positive
 Aeromonas is positive
Bile solubility test

 This helps to differentiate S. pneumoniae, which is soluble in bile and bile

salts, from other alphahaemolytic streptococci (viridans streptococci)
which are insoluble.
 Principle
 A heavy inoculum of the test organism is emulsified in physiological saline
and the bile salt sodium deoxycholate is added.
 This dissolves S. pneumoniaeas shown by a clearing of the turbidity within
10–15 minutes. Viridans and other streptococci
 are not dissolved and therefore there is no clearing of the turbidity.
 Method
 Required
 Sodium deoxycholate, 100 g/l (10% w/v)
 Physiological saline (sodium chloride, 8.5 g/l)
 Tube method
 Although the bile solubility test can be performed by testing colonies directly
on a culture plate or on a slide a tube technique is recommended because
the results are easier to read.
 1 Emulsify several colonies of the test organism in a tube containing 2 ml
sterile physiological saline, to give a turbid suspension.
 Divide the organism suspension between two tubes.
 To one tube, add 2 drops of the sodium deoxycholate reagent and mix.
 To the other tube (negative control), add 2 drops of sterile distilled water
and mix.
 Leave both tubes for 10–15 minutes at 35–37C.
 Look for a clearing of turbidity in the tube containing the sodium
deoxycholate
 Result
 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.
Limitations of Bile Solubility Test

 Bile salts will not induce clearing of a killed culture or one that is too acid.
Therefore saline suspensions of young cultures are used.
 The test should not be performed on old cultures, as the active enzyme
may be lost.
 Normal autolysis of S. pneumoniae may be inhibited by a high
concentration of bile salts being used. Evaporation may cause the reagent
to become more concentrated, therefore affecting the test.
 When performing the bile solubility tube test using saline or unbuffered
broth, it is essential to adjust the pH to neutral before adding the reagent
in order to avoid false negative reactions.
Bile Esculin Test

 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.
Principle

 Gram-positive bacteria other than some streptococci and enterococci are

inhibited by the bile salts in this medium. Organisms capable of growth in
the presence of 4% bile and able to hydrolyze esculin to esculetin.
Esculetin reacts with Fe3+ and forms a dark brown to black precipitate.
Thus the tolerance to the presence of bile and the hydrolysis of esculin
provide the means to presumptively identify organisms. If an organism can
hydrolyze esculin, the media will turn dark brown or black.
 Esculin + Acid → β-D-glucose + Esculetin
 Esculetin + Fe3+ → Dark Brown Color
Method

 Inoculate one to two colonies from an 18- to 24-hour culture onto the
surface of the slant.
 Incubate at 35°-37°C in ambient air for 24-48 hours.
 Observe for growth and blackening of the medium.
 Expected Results
 Positive: Growth and blackening of the agar slant
 Negative: Growth and no blackening of medium ; No growth
Limitations

 A heavy inoculum on BEA may cause interpretation of the bile esculin test
difficult to read. Excess inoculum decreases the ability of the bile to inhibit
growth of other gram-positive organisms that may hydrolyze esculin.
 There are a few streptococci that do not hydrolyze esculin but will grow in
the presence of bile. Growth without blackening of this medium does not
constitute a positive test.
Indole Test

 This test demonstrate the ability of certain bacteria to decompose the

amino acid tryptophane to indole, which accumulates in the medium.
 This is performed by a chain of a number of different intracellular
enzymes, a system generally referred to as “tryptophanase.”
Principle of Indole Test

 Tryptophan is an amino acid that can undergo deamination and

hydrolysis by bacteria that express tryptophanase enzyme. Indole is
generated by reductive deamination from tryptophan
 When indole is combined with Kovac’s Reagent (which contains
hydrochloric acid and p-dimethylaminobenzaldehyde in amyl alcohol)
the solution turns from yellow to cherry red. Because amyl alcohol is not
water soluble, the red coloration will form in an oily layer at the top of the
broth.
Procedure of Indole Test

 Take a sterilized test tubes containing 1-2 ml of tryptophan broth.

 Inoculate the tube aseptically by taking the growth from 18 to 24 hrs
culture.
 Incubate the tube at 37°C for 24-28 hours.
 Add 0.5 ml of Kovac’s reagent to the broth culture.
 Observe for the presence or absence of ring.
 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
 Escherichia coli
 Klebsiella oxytoca,
 Proteus sp. (not P. mirabilis)
 Provedencia
 Morganella
 Enterococcus faecalis, and Vibrio sp.
 Haemophilus influenzae,
 Edwardsiella sp., Flavobacterium sp., , Plesiomonas
shigelloides,Pasteurella multocida, Pasteurella pneumotropica,
Limitations of Indole Test

 Media containing glucose should not be used for indole testing due to the
formation of acid end products which have been shown to reduce indole
production.
 Media containing dye, such as MacConkey and EMB, are unsuitable
sources of inoculum due to possible carryover of dye and subsequent
interference of indole color interpretation.
Citrate Utilization test

 Simmons Citrate agar is an agar medium used for the differentiation of

Enterobacteriaceae based on the utilization of citrate as the sole source of
carbon.
 In the early 1920s, Koser developed a liquid medium formulation for the
differentiation of fecal coliforms from the coliform group. Simmons later
modified this formulation to produce a solid medium that eliminated
potential errors when interpreting growth.
 Composition
Sodium Chloride (NaCl) 5.0 gm

Sodium Citrate (dehydrate) 2.0 gm

Ammonium Dihydrogen Phosphate 1.0 gm

Dipotassium Phosphate 1.0 gm

Magnesium Sulfate (heptahydrate) 0.2 gm

Bromothymol Blue 0.08 gm

Agar 15.0 gm
 Principle of Simmons Citrate Agar
 Simmons Citrate agar is used to test an organism’s ability to utilize citrate as a source
of energy. Ammonium Dihydrogen Phosphate is the sole source
of nitrogen. Dipotassium Phosphate acts as a buffer. Sodium Chloride maintains
the osmotic balance of the medium. Sodium Citrate is the sole source
of carbon in this medium. Magnesium Sulfate is a cofactor for a variety of metabolic
reactions. Bacteriological agar is the solidifying agent. Organisms capable of utilizing
ammonium dihydrogen phosphate and citrate will grow unrestricted on this medium. If
citrate can be used, the microbe will accumulate alkaline/basic byproducts.
 Bacteria that can grow on this medium produce an enzyme, citrate-permease,
capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s
metabolic cycle for the production of energy. Growth is indicative of utilization
of citrate, an intermediate metabolite in the Krebs cycle.
 When the bacteria metabolize citrate, the ammonium salts are broken down
to ammonia, which increases alkalinity. The shift in pH turns the bromthymol
blue indicator in the medium from green to blue above pH 7.6.
Procedure

 After autoclaving add 4-5ml media in tubes

 Cool in slanted position (long slant, shallow butt).
 Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.
 The uninoculated medium will be a deep forest green due to the pH of the sample
and the bromothymol blue.
 Inoculate its slant and incubate for 24 hours
 Positive growth (i.e. citrate utilisation) produces an alkaline reaction and changes
the colour of the medium from green to bright blue.
Examples:
 Klebsiella,
 Enterobacter,
 Citrobacter,
 Serratia
 Proteus and Providencia species,
 Salmonella
 Negative test (i.e. no citrate utilisation) the colour of the medium remains unchanged.
Examples: Escherichia coli, Shigella,
Limitations of Simmons Citrate Agar

 Some organisms are capable of growth on citrate and do not

produce a color change.
 Growth is considered a positive citrate utilization test, even in
the absence of a color change.
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.
Principle of Urease Test

 Urea is the product of decarboxylation of amino acids. Hydrolysis

of urea produces ammonia and CO2. The formation of ammonia alkalinizes the
medium, and the pH shift is detected by the color change of phenol red from light
orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms
turn the entire medium pink within 24 hours.
Preparation

 Dissolve the ingredients in 100 ml of distilled water and filter sterilize

(0.45-mm pore size).
 Suspend the agar in 900 ml of distilled water, boil to dissolve completely.
 Autoclave at 121 degree C and 15 psi for 15 minutes.
 Cool the agar to 50 to 55 degree C.
 Aseptically add 100 ml of filter-sterilized urea base to the cooled agar
solution and mix thoroughly.
 Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes
during cooling until solidified.
Procedure of Urease Test

 Streak the surface of a urea agar slant with a portion of a well-isolated

colony or inoculate slant with 1 to 2 drops from an overnight brain-heart
infusion broth culture.
 Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air
for 24 hours
 Examine for the development of a pink color
 Interpretation

 Positive Reaction: Development of an intense magenta to bright pink color in

15 min to 24 h.
 Examples:
 Brucella spp, etc. 1 h
 Helicobacter pylori, --2 hours
 Proteus spp, 4 hours
 Provedencia 4-8 hours
 Morganella 4-8 hours
 Cryptococcus spp,
 Corynebacterium spp,
 Yersinia spp,
Limitations of Urease Test

 Some organisms rapidly split urea (Brucella and H. pylori with in minutes
to hours), while others react slowly.
 To detect Proteus species, the Urea Agar, Slants must be examined within
6 hours of inoculation for a reaction.
 Urea is light sensitive and can undergo autohydrolysis. Store at 2 to 8C in
the dark.
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.
 The cytochrome system is usually only present in aerobic organisms
which are capable of utilising oxygen as the final hydrogen receptor.
Principle of Oxidase Test

 Cytochrome containing organisms produce an intracellular oxidase enzyme. This

oxidase enzyme catalyzes the oxidation of cytochrome c. Organisms which contain
cytochrome c as part of their respiratory chain are oxidase-positive and turn the
reagent blue/purple.
 Organisms lacking cytochrome c as part of their respiratory chain do not oxidize
the reagent, leaving it colorless within the limits of the test, and are oxidase-
negative.
Procedure

 Oxidase Reagent:
 1% tetra-methyl-p-phenylenediamine dihydrochloride, in water
 Add 0.1 g reagent powder in 10ml of water
 Make fresh reagent daily( stable for 1 day only) or store at -20
 Method
 A strip of filter paper is soaked with a little freshly made 1% solution of the reagent.
 A speck of culture is rubbed on it with a platinum loop.
 A positive reaction is indicated by an intense deep-purple hue,
 Purpose/Uses of Oxidase Test
 It is used to differentiate Pseudomonads from related species.
 The test is used as an aid for the differentiation of (oxidase positive).
 Neisseria,
 Moraxella,
 Campylobacter,
 Vibrio,
 Brucella
 Pasteurella species
 Late Positive
 Stenotrophomonas maltophila
 Burkulderia cepacia
 Note
 To differentiate Micrococcus from Staph. 6% Oxidase reagent
should be used
 Micrococcus is weak positive
 Limitations of Oxidase Test
 The reagents used in the oxidase test have been shown to auto-oxidize, so
if reagent or filter paper become purple don’t use it
 Both bacteria and yeast grown on media containing high concentrations of
glucose show inhibited oxidase activity, so it is recommended to test
colonies grown on media without excess sugar, such as nutrient agar.
Tryptic soy agar is also an excellent media.
 Use of a nichrome or other iron containing loop may yield false-positive
reactions. Platinum loops are recommended.
 It is recommended to use colonies that are 18-24 hours old. Older colonies
will produce weaker reactions.
Result Interpretation of Oxidase Test

 Positive Result
 Development of a deep purple-blue/blue colour indicates oxidase
production within 10-30 seconds.
 Late positive or weak positive
 If turn blue within 30-60 seconds.
 Negative
 No color change until 60 sec.
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.
 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.
Hanging drop Method
 1. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners
using a toothpick. If too much Vaseline is used, it will be squeezed toward the
center and mix with the drop or squeeze out the edges and get on the objective
lens of the microscope.
 2. Place a loopful of the culture to be tested in the center of the prepared coverslip.
 3. Turn the clean concavity slide upside down (concavity down) over the drop on
the coverslip so that the Vaseline seals the coverslip to the slide around the
concavity.
 4. Turn the slide over so the coverslip is on top and the drop can be observed
banging from the coverslip over the concavity.
 5. Place the preparation in the microscope slide holder and align it using the naked
eye so an edge of the drop is under the low power objectives.
Principle of (Sulphide Indole Motility) Method

 Motility by bacterium is mostly demonstrated in a semi solid agar medium. In semi-

solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that
is easily recognized by the naked [Link] medium mainly used for this purpose
is SIM medium (Sulphide Indole Motility medium) which is a combination
differential medium that tests three different parameters, Sulfur Reduction, Indole
Production and Motility. This media has a very soft consistency that allows motile
bacteria to migrate readily through them causing cloudiness. The inoculum is
stabbed into the center of a semisolid agar deep.
Method of SIM

 Touch a straight needle to a colony of a young (18- to 24-hour) culture

growing on agar medium.
 Stab once to a depth of only 1/3 to ½ inch in the middle of the tube. Be sure
to keep the needle in the same line it entered as it is removed from the
medium.
 Incubate at 35°-37°C and examine daily for up to 7 days.
 Observe for a diffuse zone of growth flaring out from the line of
inoculation.
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.
Motility Indole Urea (MIU)

 The test organisms in MIU agar after incubation show either diffused growth or
turbidity extending away from stab inoculation line in case of motile organisms
while non-motile organisms appear as restricted growth along the stab-line.
Organisms possess urease, hydrolyses urea and releases ammonia and carbon
dioxide. Ammonia reacts in solution to form ammonium carbonate ,which is
alkaline leading to an increase in pH of the test medium. Phenol red present in
the medium changes its color from yellow to pink- red in alkaline pH. Indole,
skatole and indole acetic acid are produced from tryptophan present in casein
enzymic hydrolysate by the enzyme, tryptophanase .The indole formed reacts with
p-dimethyl amino benzaldehyde present in Kovac’s reagent to form quinoidal red-
violet compound.
Test Procedure For MIU Test

 Take well isolated single colony with an inoculating needle and stab the
medium leaving 1/3 part from the bottom of a tube.
 Use loose-fitting cotton plug in a test tube.
 Incubate at 37°C for 18-24 hours.
Observation

 Observe the tube for growth, motility and color change of the medium. Only check
indole formation taking reading of motility and urease reaction. Urease test
positive– A color change from yellow –orange to pink-red. No color change
indicates a negative reaction. Motility test – A positive reaction is shown by
clouding of the medium or by growth extension from the inoculating line. A negative
reaction is seen when the growth is restricted to the inoculating line. Indole test
positive– Record as Indole Positive Reaction if a pink-red color ring appears in a
test tube upon addition of Kovac’s reagent and as Indole Negative if there is no
color
Results

 Klebsiella penumonae and Acinetobacter baumannii are non-motile

 Most GNR are motile. like [Link], Enterobacter, Salmonella, Shigella,
Proteus, Pseudomonas etc.
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.
 All of these ingredients when mixed together and allowed solidification at an angle
result in a agar test tube at a slanted angle.
 The slanted shape of this medium provides an array of surfaces that are either
exposed to oxygen-containing air in varying degrees (an aerobic environment) or
not exposed to air (an anaerobic environment) under which fermentation patterns
of organisms are determined.
 Principle
 To facilitate the observation of carbohydrate utilization patterns, TSI Agar
contains three fermentative sugars, lactose and sucrose in 1%
concentrations and glucose in 0.1% concentration.
 Due to the building of acid during fermentation, the pH falls.
 The acid base indicator Phenol red is incorporated for detecting carbohydrate
fermentation that is indicated by the change in color of the carbohydrate
medium from orange red to yellow in the presence of acids. In case of
oxidative decarboxylation of peptone, alkaline products are built and the pH
rises.
 This is indicated by the change in colour of the medium from orange red to
deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the
medium detects the production of hydrogen sulfide and is indicated by the
black color in the butt of the tube.
 To facilitate the detection of organisms that only ferment glucose, the
glucose concentration is one-tenth the concentration of lactose or
sucrose. The meagre amount of acid production in the slant of the tube
during glucose fermentation oxidizes rapidly, causing the medium to
remain orange red or revert to an alkaline pH. In contrast, the acid
reaction (yellow) is maintained in the butt of the tube since it is under
lower oxygen tension.
 After depletion of the limited glucose, organisms able to do so will begin to
utilize the lactose or sucrose. To enhance the alkaline condition of the
slant, free exchange of air must be permitted by closing the tube cap
loosely.
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.
Method

 With a straight inoculation needle, touch the top of a well-isolated colony.

 Inoculate TSI by first stabbing through the center of the medium to the
bottom of the tube and then streaking the surface of the agar slant.
 Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air
for 18 to 24 hours.
 Examine the reaction of medium.
 Expected Results
 An alkaline/acid (red slant/yellow butt) reaction: It is
indicative of glucose fermentation only.
 An acid/acid (yellow slant/yellow butt) reaction: It indicates
the fermentation of glucose, 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 S
 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.
 Limitations
 It is important to stab the butt of the medium. Failure to stab the butt invalidates this
test. The integrity of the agar must be maintained when stabbing. Caps must be
loosened during this test or erroneous results will occur.
 TSI Agar must be read within the 18-24 hour stated incubation period. A false-
positive reaction may be observed if read too early. A false-negative reaction may be
observed if read later than 24 hours.
 An organism that produces hydrogen sulfide may mask acid production in the butt of
the medium. However, hydrogen sulfide production requires an acid environment,
thus the butt portion should be considered acid.
 TSI is not as sensitive in detecting hydrogen sulfide in comparison to other iron
containing mediums, such as Sulfide Indole Motility (SIM) Medium.
 TSI replaced KIA –Kligler's Iron Agar (KIA)
 The composition of Kligler’s Iron Agar (KIA) is identical to
Triple Sugar Iron Agar (TSI) except that 10 gm sucrose is added in
TSI.
 Carbohydrate Fermentation Test (Sugar
Fermentation Test)
 Principle
 The principle of carbohydrate fermentation states that the action of organism on a
carbohydrate substrate results in acidification of the medium, detected by a pH
indicator dye. Carbohydrate fermentation is the process microorganisms use to
produce energy. Most microorganisms convert glucose to pyruvate during glycolysis;
however, some organisms use alternate pathways. A fermentation medium consists
of a basal medium containing a single carbohydrate (glucose, lactose, sucrose,
mannitol etc.) for fermentation. However, the medium also contains various pH
indicators. In addition to a pH indicator to detect the production of acid from
fermentation, a Durham tube is placed in each tube to capture gas produced by
metabolism. The carbohydrate fermentation patterns shown by different organisms
are useful in differentiating among bacterial groups or species.
 Media
 The purple broth consists of peptone with the pH indicator bromcresol purple.
Specific carbohydrates are added in a concentration of 0.5-1%. This concentration
is recommended to ensure against depletion of the carbohydrate and reversal of
the fermentation reaction.
 Carbohydrates
 Glucose, lactose, sucrose, maltose, mannitol, galactose, starch, rhamnose,
aesculin, salicin, adonitol, dulcitol, sorbitol, cellobiose, xylose, mannose, trehalose,
inositol, raffinose, melibiose, and cellulose are carbohydrates commonly used as
substrate in the carbohydrate fermentation test.
 Method
 Allow medium to warm to room temperature prior to inoculation.
 Inoculate the Purple Broth (with carbohydrate of choice) with isolated colonies from
an 18-24 hour pure culture of the organism.
 Inoculate a control tube of Purple Broth Base in parallel with the carbohydrate
based media.
 Incubate inoculated media aerobically at 35-37ºC. for 3-5 [Link]: Increased
incubation up to 30 days may be necessary for some microorganisms.
 Observe daily for development of a yellow color in the medium.
 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.
 Fermentation Result of
Name of Gas Pro
Bacteria Gluc Arabino Cellobio Mannito duction
Lactose Sucrose Maltose Sorbitol Xylose
ose se se l
E. coli + ve + ve V + ve + ve -ve + ve + ve + ve + ve
K.
pneumonia + ve + ve + ve + ve + ve + ve + ve + ve + ve + ve
e
K. oxytoca + ve + ve + ve + ve + ve + ve + ve + ve + ve V
Proteus
+ ve -ve -ve -ve -ve -ve -ve -ve + ve + ve
mirabilis
Pseudomo
nas + ve -ve -ve -ve -ve -ve + ve -ve -ve -ve
aeruginosa
Salmonella
+ ve -ve -ve + ve -ve -ve + ve -ve + ve -ve
Typhi
Shigella
+ ve -ve -ve V V -ve + ve V -ve + ve
flexneri

Shigella
+ ve -ve -ve V V -ve -ve V -ve – ve
dysentery

+ ve = Positive fermentation -ve = Negative fermentation V = Variable

reaction
Decarboxylases test (lysine,
ornithine,
arginine)
 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.
 There are three decarboxylase enzymes that is routinely tested for –
 arginine decarboxylase,
 ornithine decarboxylase, and
 lysine decarboxylase.
 The production of lysine, arginine, ornithine decarboxylase by various members of
Enterobacteriaceae offers an important parameter to other biochemical tests for
differentiating bacteria within closely related groups.
 Principle of Decarboxylase Test
 Arginine, lysine, and ornithine decarboxylase media are used to detect an organism’s ability to
decarboxylate or hydrolyze an amino acid, forming an amine that produces an alkaline pH.
 The basal medium is usually Moeller’s formula and contains meat peptones and beef extract,
which supply nitrogenous nutrients to support bacterial growth.
 The media contains glucose as the fermentable carbohydrate and pyridoxal, which is an
enzyme cofactor that enhances decarboxylase activity. The pH indicators are bromcresol purple
and cresol red.
 Amino acids like arginine, lysine, and ornithine are singly added to the basal medium to detect
the production of enzymes that decarboxylate or hydrolyze these substrates.
 After the fermentation of glucose in the medium, acids are produced which lower the pH,
resulting in a change in color from purple to yellow.
 If the organism produces decarboxylase, decarboxylation, or hydrolysis of the amino acid
occurs in response to the acid pH.
 Decarboxylation results in alkaline end products (amines), causing the medium to revert to its
original color (purple).
 In the case of the organism that does not ferment glucose, the medium does not turn yellow
 Media:
 Peptic digest of animal tissue (5 g), beef extract (5 g), bromcresol purple (0.1 g), cresol
red (0.005 g), dextrose (0.5 g), pyridoxal (0.005 g), amino acid (10 g), pH 6.0.
 Method
 Prepare a suspension (≥McFarland No. 5 turbidity standard) in brain-heart infusion
broth from an overnight culture (18 to 24 hours old) growing on 5% sheep blood agar.
 Inoculate each of the three decarboxylase broths (arginine, lysine, and ornithine) and
the control broth (no amino acid) with 4 drops of broth.
 Add a 4-mm layer of sterile mineral oil to each tube.
 Incubate the cultures at 35°-37°C in ambient air.
 Examine the tubes at 24, 48, 72, and 96 hours.
 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.
 Control organisms

Results
Colour of
Control the control Ornithi
tube Arginine Lysine
ne

Klebsiella
Yellow – + –
pneumoniae

Enterobacter
Yellow + – +
cloacae
O-nitrophenyl-beta-D-
galactopyranoside (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.
 some bacteria lack permease enzymes and/or both enzymes. If the bacteria lack
the permease, they appear as late lactose fermenters or non-lactose fermenters.
Therefore, if the bacteria lack a permease enzyme, it is very difficult to
separate the lactose fermenter, late lactose fermenter, and non-lactose
fermenter using a traditional lactose fermentation test using lactose as a
substrate.
 In order to remove this difficulty, a new testing method using ‘o -Nitrophenyl -β- D-
galactopyranoside’ (ONPG) was devised called the ONPG test. The ONPG is a
colorless synthetic compound similar to lactose in structure which splits and
develops color in the presence of the β-galactosidase enzyme.
 Principle
 If ONPG is added in the culture medium as a substrate instead of the lactose, it is
acted upon by the β-galactosidase enzyme in a manner similar to the lactose. The
ONPG is structurally similar to lactose and can penetrate the bacterial cell without the
permease enzyme. Once inside the bacterial cell, the β-galactosidase enzyme breaks
the β-galactoside bond in the ONPG, releasing galactose and o-nitrophenol. The o-
nitrophenol is yellow in color and hence causes the development of visible yellow
color, indicating the bacteria is a lactose fermenter.
 As ONPG doesn’t require a permease enzyme, it gives similar results in a very short
time even though the test bacteria lack permease and contain only β-galactosidase.
 Culture Media
 The ONPG test can be performed by using ONPG broth or ONPG disk.
 For ONPG disk method
 Place an ONPG disk into a sterile tube and add 0.2 mL saline.
 Heavily inoculate the tube with a loopful of the test isolate.
 Incubate at 35-37°C for up to 4 hours.
 Examine for color change of the disk.
 B. For broth method
 Bring test medium to room temperature.
 Inoculate the test medium with heavy inoculum from a pure 18-24 hour culture.
 Incubate aerobically, with loose caps, at 35- 37ºC.
 Examine for a yellow color development at 1 hour.
 If no color change seen after an hour of incubation, continue incubation for up to 24
hours.
  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 Positive Bacteria

E. coli, Citrobacter freundii, Salmonella Choleraesuis, Shigella dysenteriae, Klebsiella
pneumoniae (and most Klebsiella spp.), Vibrio cholerae, Serratia marcescens, N.
lactamica, etc.
ONPG Negative Bacteria
Proteus vulgaris, P. mirabilis, Salmonella Typhimurium, Vibrio parahaemolyticus,
Providencia spp., Pseudomonas aeruginosa, N. gonorrhoeae, etc.
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.
 Principle
 This test is used to determine the ability of an organism to produce extracellular
proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate
connective tissue.
 The reaction occurs in two sequential steps: in first reaction gelatinases hydrolyze
gelatin into polypeptides and then polypeptides are further converted into amino
acids. The amino acid is taken up by the cell and used for metabolic purposes.
 The presence of gelatinases is detected using a nutrient gelatin medium. When an
organism produces gelatinase, the enzyme liquefies the growth medium by
hydrolyzing gelatin present in the medium.
 Method
 There are several methods for determining gelatinase production, all of which make use
of gelatin as the substrate. The standard and most commonly employed method is the
nutrient gelatin stab method.
 Inoculate the gelatin deep with 4 to 5 drops of a 24-hour broth culture.
 Incubate at 35°-37°C in ambient air for up to 14 days.
 Note: Incubate the medium at 25°C if the organism grows better at 25°C than at 35°C.
 Alternatively, inoculate the gelatin deep from a 24-hour-old colony by stabbing four or
five times, 0.5 inch into the medium.
 Remove the gelatin tube daily from the incubator and place at 4°C to check for
[Link]: Do not invert or tip the tube, because sometimes the only discernible
liquefaction occurs at the top of the deep where inoculation occurred.
 Refrigerate an un-inoculated control along with the inoculated tube. Liquefaction is
determined only after the control has hardened (gelled).
 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.
 Uses of Gelatin Hydrolysis Test
 Gelatin hydrolysis test is used to test the ability of an organism to produce
gelatinases.
 Gelatin hydrolysis test helps in the identification of
Serratia, Pseudomonas, Flavobacterium, and Clostridium.
 The test distinguishes the gelatinase-positive, pathogenic Staphylococcus
aureus from the gelatinase-negative, nonpathogenic Staphylococcus
epidermidis.
 The test can be used to differentiate genera of gelatinase-producing bacteria such
as Serratia and Proteus from other members of the family Enterobacteriaceae.
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.
 Principle of Methyl Red (MR) Test
 Some bacteria have the ability to utilize glucose and convert it to a stable acid like
lactic acid, acetic acid or formic acid as the end product.
 These bacteria initially metabolise glucose to pyruvic acid, which is further
metabolized through the ‘mixed acid pathway to produce the stable acid. The type
of acid produced differs from species to species and depends on the specific
enzymatic pathways present in the bacteria. The acid so produced decreases the pH
to 4.5 or below, which is indicated by a change in the color of methyl red from yellow
to red.
 In the methyl red test (MR test), the test bacteria is grown in a broth medium
containing glucose. If the bacteria has the ability to utilise glucose with production
of a stable acid, the color of the methyl red changes from yellow to red, when
added into the broth culture.
 Procedure of Methyl Red (MR) Test
 Inoculate MR broth with a pure culture of the organism.
 Incubate at 35°-37°C for a minimum of 48 hours in ambient air.
 Add 5 or 6 drops of methyl red reagent per 5 mL of broth.
 Observe for the color change in the broth medium.
 Expected Results
 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.
 Voges–Proskauer (VP) Test
 Voges and Proskauer, in 1898, first observed the production of a red color after the
addition of potassium hydroxide to cultures grown on specific media. Harden later revealed
that the development of the red color was a result of acetyl-methyl carbinol production. In
1936 Barrit made the test more sensitive by adding alpha-naphthol to the medium before
adding potassium hydroxide.
Principle of the 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.
 Culture Media
 The MR-VP broth, also known as Glucose Phosphate Broth, is used for performing
the VP test.
 Reagents
 5% Alpha-naphthol Solution (Barritt’s Reagent A) and 40% KOH or NaOH solution
(Barritt’s Reagent B) are required.
 Preparation of Barritt’s Reagent A
 Dissolve 5 grams of α-naphthol reagent in 100 mL of 95% ethanol. The reagent can
be stored for up to 3 weeks in a dark place at 4 to 8°C.
 Preparation of Barritt’s Reagent B
 Dissolve 40 grams of KOH pellet in 100 mL of sterile distilled water. The reagent can
be stored for up to 3 weeks at 4 to 8°C.
 Procedure of VP Test
 Using a sterile inoculating loop, pick up well-isolated colonies of sample bacteria from
18 to 24 hours old culture and inoculate the broth.
 Incubate the tubes aerobically for 18 to 24 hours at 35±2°C.
 Following incubation, transfer 2 mL of broth to a clean (sterile if possible) test tube.
 Add 6 drops of Reagent A (5% α-naphthol solution) and mix properly by shaking.
 Add 2 drops of Reagent B (40% KOH solution) and mix properly by shaking.
 Observe for the formation of red-pink color at the surface of the medium within 30
minutes. Continuously shake the tube vigorously during the 30-minute waiting period.
 If no color is developed (negative reaction) re-incubate the remaining broth for
additional 24 hours and test again.
 Result Interpretation of VP Test
 A positive result is indicated by the formation of a pink-red
color over the surface of the medium.
 A negative result is indicated by a lack of pink-red color over the
surface of the medium or the formation of the copper color.
 VP Test Results of Some Common
Bacteria

VP Positive Bacteria VP Negative Bacteria

Klebsiella spp.,
Enterobacter spp.,
Escherichia spp.,
Viridans Streptococci (except S.
Proteus vulgaris,
mitis, and S. vestibularis)
Citrobacter freundii,
Proteus mirabilis,
Morganella morganii,
Hafnia spp.,
Shigella spp.,
Serratia spp.,
Yersinia spp.,
Staphylococcus aureus,
V. parahaemolyticus
Listeria spp.,
V. cholerae
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).
 Principle
The nitrate reduction test is based on the detection of nitrite and its ability
to form a red compound when it reacts with sulfanilic acid to form a complex
(nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a
red precipitate (prontosil), which is a water-soluble azo dye.
 Method
 Determination of nitrate reduction to nitrite is a two step process. First, the
reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A
and B
 Inoculate the nitrate broths with bacterial suspension.
 Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours.
 After incubation look for N2 gas first before adding reagents.
 Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B.
 Observe for the reaction (color development) within a minute or less.
 If no color develops add zinc powder.
 Observe for at least 3 minutes for a red color to develop after addition of zinc.
 Expected Results
 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
 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.
 E coli is positive
 Proteus is negative
 Phenylalanine Deaminase Test
 Phenylalanine Deaminase Test (PDA) is the biochemical test used to determine
the ability of bacteria to synthesize the phenylalanine deaminase enzyme.
 Principle of Phenylalanine Deaminase Test
 Some bacteria have the capacity to synthesize phenylalanine deaminase enzyme,
which oxidatively deaminates (removes NH2) from the amino acid phenylalanine.
Upon deamination of phenylalanine by the deaminase enzyme, phenyl pyruvic acid
and ammonia are released. Thus released phenyl pyruvic acid reacts with the
chelating agent ferric chloride in the reagent and results in the formation of light to
deep-green colored complex, indicating a positive reaction.
 Positive Control: Proteus mirabilis ATCC 12453
 Negative Control: Escherichia coli ATCC 25922
 Novobiocin Susceptibility Test
 Novobiocin test is used to differentiate coagulase-negative staphylococci (CONS) and
presumptively identify the isolate as Staphylococcus saprophyticus (novobiocin
resistant).
 Principle of Novobiocin Test
 S. saprophyticus is second only to E. coli as the most frequent causative organism of
uncomplicated urinary tract infections (UTIs) in young sexually active women. So
when coagulase negative staphylococcus is isolated from a young sexually active
women, laboratory must further identify that isolate and find out is this a true
pathogen (Staphylococcus saprophyticus-novobiocin resistant) or a contaminant
(i.e. Staphylococcus epidermidis-novobiocin sensitive).
 Aseptically apply one 5ug novobiocin disk onto the inoculated agar surface and lightly
press down to ensure full contact with the medium.
Interpretation of Novobiocin test

 Resistant – zone size of < 12 mm

 Sensitive – zone size greater or equal to 16 mm
Optochin Susceptibility Test for the identification of
Streptococcus pneumoniae

 Using an inoculating loop, streak two or three suspect colonies of a pure

culture to be tested on 5% sheep blood agar plate
 Place an optochin disk (OP or P) disks (6 mm, 5 µg) within the streaked
area of the plate
 Incubate the blood agar plate at 35-37°C with ~5% CO2 (or in a candle-
jar) for 18 to 24 hours. (Culture do not grow well in ambient air, and larger
zones of inhibition occur)
 Reading the optochin test results
 Using a 6 mm, 5 µg disk, a zone of inhibition of 14 mm or greater indicates
sensitivity and allows for presumptive identification of pneumococci.
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.
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).
Method

 Using an inoculating loop, streak two or three suspected colonies of a

pure culture onto a blood agar plate.
 Using heated forceps, place a bacitracin disk in the first quadrant (area of
heaviest growth). Gently tap the disk to ensure adequate contact with the
agar surface.
 Incubate the plate for 18 to 24 hours at 35°-37°C in ambient air for
staphylococci and in 5% to 10% carbon dioxide (CO2) for streptococci
differentiation.
 Look for a zone of inhibition around the disk.
Expected Results

 Positive: Any zone of inhibition greater than 10 mm; susceptible.

 Negative: No zone of inhibition; resistant