MICROBIOLOGY LABORATORY
EXERCISE 7.a --IMViC TEST
Biochemical test for identification of enteric
organisms:
Basis: metabolic action of microorganisms
on the culture media
Used for the identification of enteric organisms/
gram-negative bacilli
One of the earliest sets of test used for the
identification of enteric bacilli
includes such organisms as Klebsiella,
Enterobacter, Citrobacter and Escherichia coli
 acid fermentation pathway
This acronym stands for
•I - Indole
•M- Methyl red
•V - Voges – Proskauer
•( i ) is inserted for euphony
•C – Citrate
INDOLE
Indole, a benzyl pyrrole, is one of the metabolic
degradation product of amino acid tryptophan
Indole positive bacteria produce tryptophanase,
an enzyme that is capable of hydrolyzing and
diaminating tryptophan, thus producing:
- indole
- pyruvic acid
- ammonia
Materials:
2% Peptone broth tube
Test organisms
Ether
indicator: Erlich/Kovac's reagent
(para-dimethyl-aminobenzaldehyde)
Procedure:
-Inoculate 1 loopful of the test organism into the
tube of peptone broth.
Incubate at 370C for 24-48 hours.
-Next laboratory period, add 1 ml. of ether.
-Shake well and allow to stand for a few minutes
until the ether rises to the surface.
-Gently add about, 1cc. of Kovac’s or Erlich’s
reagent down the side of the tube so that it forms a
ring between the medium and the ether layer.
Observation:
Positive result
•Bright red or purple ring
•If indole has been produced
by the organism it will, being
soluble in ether, it will be concentrated in the
ether layer and upon the addition of Erlich’s
reagent, a positive result is the
production of a purple ring at the
junction of the medium and the ether layer.
Negative result – Yellow color
- no red or purple ring
METHYL RED TEST:
All enterics oxidize glucose for energy; however the
end products vary depending on bacterial enzymes
Both the MR and VP tests are used to determine
what end products result when the test organism
degrades glucose
MR test is a quantitative test for acid production,
requiring positive organism to produce strong acids
(lactic, acetic, formic) from glucose via the mixed
End result is based on the final pH reached
only those organism that can maintain low ph of
about
ph 4-4.5 can be called methyl red – positive
organisms that are MR (+) are always VP (-)
Materials:
MR-VP broth medium (contains 10% glucose)
Test organisms
Methyl red ph indicator
Procedure:
Inoculate 1 loopful of the test organism into a tube
MR-VP medium.
Incubate for 24-48 hours at 370C.
Next laboratory period, add 5 to 10 drops of methyl
red reagent.
Mix thoroughly and observe the results.
Observation:
žPositive result – cherry red/bright red color
•ph 4-4.5
•Ex. Salmonella, Escherichia,
Citrobacter, Proteus, Morganella
and Providencia
žNegative result – Yellow color
•At neutral pH the growth of the bacteria is not
inhibited
•The bacteria thus begin to attack the peptone in the
broth, causing the pH to rise above 4.5
•At this pH, methyl red indicator produce a yellow
color
•Ex. Enterobacter and Klebsiella
 Negative result
•Remains Yellow to Amber; no change in color
Ex. E. coli

CITRATE TEST:

VOGES-PROSKAUER TEST: ža test depends upon the ability of the organism, to

utilize citrate as the sole source of carbon and
-is a test for the detection of acetyl-methyl carbinol energy growth
(acetoin) which is also a degradation product of ž
glucose Materials:
•Solid media : Simmon’s Citrate Agar
Materials: •Liquid media: Kosher’s Media
MR-VP medium (contains 10% glucose) •Test Organism
Test organism žSimmon's media contains bromthymol blue, a pH
Potassium Hydroxide indicator with a range of 6.0 to 7.6
Alpha-napthanol reagent •uninoculated Simmon's citrate agar has a pH of 6.9,
so it is an intermediate green color (neutral pH)
•Growth of bacteria in the media leads to
When these reagents are added to a broth in which development of a Prussian blue color at more alkaline
acetyl methyl carbinol is present, they turn a pH's (around 7.6) (positive citrate)
burgundy color/crimson red color (a positive VP ž
test) Procedure:
organisms that are VP (+) are always MR (-) Inoculate the test organism on the medium by stab
streaking.
acetyl-methyl carbinol + Potassium Hydroxide Incubate at 370C for 24 - 48 hours.
↓ Observe.
dimethyl-carbinol
↓ Observation:
guanidine compounds žPositive result
↓ •Deep blue/ Prussian blue color
Crimson red •indicating that the test organism
has been able to utilize citrate for energy source
• Ex. Enterobacter, Klebsiella, Salmonella,
Procedure: Citrobacter
Microorgan Indole MR
and Providencia VP Citrate
ism
-Inoculate MR-VP medium with 1 loopful of the test ž
E. coli + + - -
organism žNegative result
- •Retains its original
Enterobacter - color (Green)
- + +
Incubate for 48 hours at 370C. •Ex. Escherichia, Shigella and Morganella
Citrobacter - + - +
-
Next laboratory period add 0.6 ml. 5% alpha- ž
napthol reagent.
-Mix and shake the mixture lightly.
-
Add 0.2 ml (5drops). of 40% potassium hydroxide
reagent (KOH).
-Mix and shake the mixture lightly.
-
Shake the tube gently to expose the medium to
atmospheric oxygen and allow the tube to remain
undisturbed for 10 to 15 minutes.
Exercise 7.b
Observation: Lysine-Iron agar test:
ž
Positive result Intended Use
•Crimson Red color •Lysine Iron Agar is used for the differentiation of
•Presence of Acety methyl carbinol enteric organisms based on their ability to
Ex. Enterobacter and Klebsiella decarboxylate or deaminate lysine and to form
ž hydrogen sulfide
Components:
Hydrogen Sulfide formation:
ž-Organisms which produce hydrogen sulfide gas will
exhibit a black precipitate in the butt of the tube
ž
ž-LIA is not as reliable an indicator of H2S as are KIA
and TSI
ž
ž-Splitting of the agar in the butt indicates gas
production.

Procedures:
-
Inoculate the organism into the Lysine Iron Agar
medium

-
Lysine deamination: Stab the butt twice all the way down, then streak the
•Reaction in the slant slant using a straight wire needle
•
Lysine decarboxylation -
•Reaction in the butt
Incubate at 37OC for 18-24 hours.
ž-is an aerobic process which occurs on the slant
of the media
ž-If the bacterium can deaminate Lysine in the
Reading LIA reactions:
slant, it will produce an unknown orange product
ž-This orange product will react with the Brom-
ž
cresol Purple
SLANT BUTT
ž-If the organism has the ability to deaminate
ž
lysine, the ammonia produced will react with the
ferric ammonium citrate to produce a dark red purple slant purple butt
color on the slant of the tube P/P - /+ lysine deaminase lysine decarboxylase
negative positive
-Positive deamination: red slant
-Negative deamination: purple slant purple slant yellow butt
žThe genus that will give a positive red slant are P/Y -/- lysine deaminase lysine decarboxylase
negative negative
Proteus, Providencia, Citrobacter and Morganella
red slant yellow butt
+/
Lysine decaboxylation: R/Y
-
lysine deaminase lysine decarboxylase
ž -is an anaerobic process which occurs in the positive negative
butt of the media
ž -Due to overneutralizing the acid formed
-you cannot have a (+) slant with a (+) butt!
from glucose fermentation
ž -The bacteria that is inoculated into the butt
Exercise 7.c
of this tube can ferment the glucose, producing a
Sulfide Indole motility test (SIM):
low or acidic pH
-This induces the bacteria to form the
ž-A semi-solid culture medium used to detect:
carboxylase enzyme which removes the -COOH •sulfide formation
from the lysine molecule, creating an amine end •indole production
product, that is alkaline in pH •motility
ž ž-Used for the for the identification of
ž -If the organism has the ability to Enterobacteriaceae
decarboxylate lysine, it produces an amine end-
product which reacts with the pH indicator to give a Procedures:
purple color in the butt of the tube - Stab the Sulfide-Indole-Motility (SIM) medium with
(Positive decarboxylation: purple the organism once with a straight wire needle to the
butt) bottom of the tube
(Negative decarboxylation: yellow -Incubate at 37 C for 24 hours.
butt) -Read the motility before adding Ehlichs / Kovac’s
žOrganisms that can decarboxylate lysine : reagent.
Salmonella, Klebsiella, Enterobacter,
Serratia Observations:
 Test organism
A. Hydrogen sulfide production
Procedures:
-The sulfide reaction is indicated by -
blackening of the medium along the line of Inoculate 5 loopful of the test organism into the urea
inoculation or in the bottom of the tube christensen broth.
-the reaction is derived from organisms bearing -
sulfur-containing amino acids Incubate at 370C for 24 hours.
-Hydrogen sulfide reacts with ferrous sulfate to -
form ferrous sulfide which is seen as a black Observe for color change.
colored substance
ž(+) H2S organisms are always motile Observation:
-POSITIVE
B. Motility Determination •dark pink color
•The pH indicator phenol red changes
- to a deep pink color in the
POSITIVE TEST (MOTILE) presence of ammonia turning the medium alkaline
žmotile organisms migrate from the stab line and •particularly helpful in identifying Proteus from other
diffuse into the medium, causing turbidity species due to the rapid action of the enzyme by
žThey may exhibit fuzzy streaks of growth at the Proteus compared to other non-lactose fermenting
stab line enterics
- •Also (+) to Klebsiella, Haemophilus and Citrobacter
NEGATIVE TEST (NON-MOTILE)
žbacterial growth accentuated along stab line, -NEGATIVE
surrounding mediums remains clear •If the medium retains its original color
-
CONTROL MEDIUM (Uninoculated) ž
žno growth, medium remains clear
ž
ž
ž

C. Indole Production Exercise 7.e

ž CYTOCHROME OXIDASE TEST:
-Add about 13drops of Ehrlichs / Kovac’s reagent
to the tube The cytochromes are iron-containing
hemoproteins that act as the last link in the chain of
- aerobic respiration by transferring electrons
POSITIVE TEST (hydrogen) to oxygen, with the formation of water.
a red or purple ring indicates that indole is
produced at the junction of the medium and the Cytochrome oxidase or Warburge’s respiratory
reagent enzyme – the enzyme which reduces molecular
oxygen to water to compete the last link in the chain
-NEGATIVE TEST of aerobic respiration
a yellow color is observed.
The test is most helpful in screening colonies
Exercise 7.d suspected of being one of the Enterobacteriaceae (all
Urease test: negative) and in identifying colonies suspected of
being a pseudomonas specie or a neisseria specie
ž-Urease, an enzyme produced by some which are all positive. (Pseudomonas, Vibrio,
microorganisms, attacks the nitrogen and carbon Aeromonas, Neisseria and Comamonas)
bond in amide compounds and forms the alkaline
end product ammonia žThe cytochrome oxidase test utilize certain reagent
ž-Microorganism that possess the enzyme urease dyes, such as the “redux dye: tetramethyl-
have the capability of hydrolyzing urea with the paraphenylenediamine dihydrochloride”, that
release of ammonia and carbon dioxide substitute for oxygen as artificial electron acceptors.
Urea + Water ž
Ammonia & Carbon dioxide ž
ž In the reduced state the dye is colorless,
Materials: however, in the presence of cytochrome oxidase and
atmospheric oxygen, tetramethyl-
Urea Christensen broth
paraphenylenediamine dihydrochloride is oxidized,
forming indophenol blue color.
Materials:
Culture plate of organism
Cytochrome oxidase paper strip (Pathotec oxidase
strip) (tetra-methyl-para-phenylene diamine
dihydrochloride)
Procedures:
-The test is commonly performed by one of the two
methods:
-The direct plate technique in which 2 to 3 drops
reagent are directly poured into the isolated
bacteria colonies growing on the plate medium.
ž
-The indirect paper strip procedure. In which a few
drops of the reagent are either added to a filter
paper trip or commercial disk or strips impregnated
that dried reagent are used.
ž control.
-In either method, a loopful of suspected colony is
smeared into the reagent zone of the filter paper.
Observations:
Bacterial colonies having cytochrome oxidase
actively will develop a deep blue color to purple to
black
at the inoculation site within seconds.
Exercise 7.f
COAGULASE TEST:
ž Some bacteria produce coagulase, an enzyme
and as a precursor of thrombin like substance
which coagulate plasma.
žThere are two kinds of coagulase which could be
present in the microorganism
– the free coagulase and
– the bound coagulase.
žThis enzyme is demonstrated in vitro by two
methods.
•The Test Tube Method
•The Slide Method
ž-Differentiate pathogenic from non-pathogenic
Staphylococcus.
Tube Method:
The method measures free coagulase.
ž ž
Materials:
•Citrated or heparinized human or rabbit plasma is
diluted with isotonic saline or other suitable diluent
•Overnight broth culture of microorganism.
ž EXERCISE NO. 7.h
Procedures:
-Place about 0.5ml of diluted plasma in each of 2 test
tubes.
-To one tube add 5 drops of an overnight broth culture of
the organism.
-Do not place anything in the other tube which serves as
control
Incubate broth tube at 370C. Examine at 24 hours.
Result:
Positive Coagulase – a clot is formed
Negative Coagulase – no clot is formed
This usually detects bound coagulase, which is the
clumping factor attached to the cells and acts directly on
the fibrinogen.
ž
ž
Procedures:
-
Divide the slide into two sections with grease pencil.
-Place a small drop of normal saline on each area.
-Emulsify one or two colonies of microorganism on blood
agar plate on each drop to make a smooth suspension.
-Add a drop of undiluted plasma to one of the drop of saline
and stir gently with a wire loop.
-Do not put anything in the other drop that serves as
-Observe for clumping of the microorganism
microscopically.
Positive - clumping / Negative - no clumping
Exercise 7.g
CATALASE TEST:
ž-Some bacteria produce the enzyme catalase, which is an
iron porphyrin protein
ž-This enzyme is responsible for the decomposition of
hydrogen peroxide to water and oxygen
H2O2 CATALASE H 2 O + O2
ž
Procedures:
-Emulsify about 1 or 2 colonies of Staphylococcus organism
from a plate culture with a drop of saline on a slide.
-Add a drop of hydrogen peroxide.
-Observe for air bubbles
ž
Observation:
ž
žPositive test
•presence of air bubbles
•Bubbling upon the addition of hydrogen peroxide is
indicative of the presence of catalase for this organism.
•Note, most aerobic organism make catalase.
•Differentiates Staphylococcus from Streptococcus
All Staph (+) All Strep (-)
Negative test – no air bubbles
Carbohydrate Fermentation
-Bacteria ferment carbohydrates in different ways
depending upon the enzymes they process.
-In bacteriologic test systems, acidity therefore is
an indication of fermentation and is detected by observing
color change of pH indicators as acid products are
formed.
ž
Materials:

1. Lactose broth 1% (with Durham’s tube)

2. Sucrose broth 1% ž
3. Mannitol broth 1%
4. Dextrose semi-solid agar 5% ž
5. Slant agar culture with Coliform, Klebsiella
and E. coli

Indicator dye is “Bromcresol purple”

ž
Procedures:
1) Arrange the tubes properly and label each with
the corresponding sugar.
2) Inoculate the test organism into this different
carbohydrate test tube.
3) Incubate at 370C for 24 hours.
4) Next laboratory period observe the color changes
and gas production.

Acid production
-phenol red turns into yellow color
-No acid production-color remains purple

EXERCISE NO. 7.i.

Triple Sugar Iron Reaction

The TSI reaction provides a means for the

presumptive identification of gram negative enteric
bacteria. The rationale of this test is the fact that many
of the gram negative bacteria can be differentiated on
the basis of:
1) Acid production from fermentation of carbohydrates
2) Gas formation
3) Production of hydrogen sulfide (H2S)
Indicator - Phenol red

Materials:

Triple Sugar Iron Agar tubes

Culture of enteric bacteria
ž
Procedures:

1) Fish out a colony from plate culture of bacteria and

inoculate it by making a stab to the butt portion of
the TSI medium.
Then pull the needle up to the surface of the agar and
make a zigzag streaking on the slant portion. Cover the
tube loosely with a cotton plug.

2) Incubate the tubes for 24 hours and observed the

reaction produced in each test tube. Record
your observation.

Yellow-acid Red-alkaline

-red slant/red butt – at least 2 sugars fermented

-red slant/yellow butt – only one sugar is fermented,
usually Lactose
-yellow slant/yellow butt – no sugar fermented
-black discoloration - presence of ferrous sulfide (H2S)