•Indole.
•Methyl Red.
•Voges Proskauer.
•Citrate.
IMViC.
Urease
•Triple Sugar Iron.
•Kligler Iron Agar.
TSI & KIA.
Motility.
Principle.
• Indole is a component of the amino acid “Tryptophan”.
• Some bacteria have an enzyme (Tryptophanase).
• Function: Breaking Tryptophan, releasing indole.
• The presence of indole reflects the presence of enzyme.
Media.
• Tryptophan broth.
• Peptone broth.
Reagent.
• Kovac’s reagent.
Inoculate the test
tubes of
Tryptophan broth
with a loopful of
bacterial growth.
Incubate tubes at
35º-37ºC for 24
hours.
After incubation
period, add 5
drops of kovac’s
reagent.
Read the results
immediately.
Purpose.
• To determine the ability of bacteria to produce
ACIDS as final products from glucose fermentation.
Media.
• MR/VP broth.
Reagent.
• Methyl Red indicator.
Inoculate the
test tubes of
MR/VP broth
with a loopful of
bacterial growth.
Incubate tubes
at 35-37ºC for
48 hours.
After incubation
period, add 4
drops of Methyl
Red reagent.
Read the results
immediately.
Purpose.
• To determine the ability of bacteria to produce ACETOIN
as final products from glucose fermentation.
Media.
• MR/VP broth.
Reagent.
• Alpha Naphthol.
• 40% KOH
Inoculate the test
tubes of MR/VP
broth with a
loopful of
bacterial growth.
Incubate tubes at
35-37ºC for 48
hours.
After incubation
period, add 6
drops of Alpha
Naphthol & 2
drops of 40%
KOH
(IN THIS ORDER)
Loosen the cap of
the tube and put
it in a slant
position for 15 to
45 minutes
Read the results
after 15 minutes
•If positive – report as
positive.
•If negative – keep it
for another 30
minutes
What happens when alpha naphthol and 40%
KOH are added to the test tube?
•Acetoin, if present, is oxidized to diacetyl, in
the presence of oxygen and alkaline
conditions.
•Alpha naphthol catalyzes the reaction between
diacetyl and peptone to produce a complex of
pink colored compound.
Purpose.
•To determine if the organism is capable of utilizing Citrate as the ONLY source of
carbon.
Principle.
•Bacteria can break the conjugate salt of citrate into organic acids and CO2.
•CO2 Combines with sodium from the conjugate salt to form sodium carbonate (a
basic compound).
Media.
•Simmon’s citrate.
Reagent.
•Bromothymol blue pH indicator (incorporated in the medium).
Inoculate the
slant of test tube
with an organism
(Fish tail).
Incubate the
tube for 1-2
days at 35-37ºC.
After incubation,
read the results
immediately.
Purpose.
• To determine the presence of urease enzyme
Principle.
• Urea may be broken down by Urease into CO2 and ammonia.
• Ammonia turns the medium alkaline (pH>7).
Media.
• Urea media broth or slant.
Reagent.
• Phenol red pH indicator (incorporated in the medium).
Inoculate the tube
medium with
bacteria.
•If slant agar, do the fish
tail.
Incubate the tubes
at 35-37ºC for 24
hours.
After incubation,
observe color
change and read
results.
Why do
they
move?
•Toward beneficial things
like O2 and nutrients.
•Away from deleterious
chemicals and toxins like
O2
What is the
structure
they use?
How do
they move?
•Most motile bacteria use
Flagella.
•Spirochetes are helical
bacteria that have an
internal structure that is
responsible for spiral
rotation.
•When flagella rotate
counterclockwise they act
like Propeller.
•As a result bacteria move
through the liquid.
Purpose.
• To determine the ability of bacteria to move (Motile Vs Non-motile bacteria).
Media.
• Semi-solid (less than 1% of Agar).
• Motility media.
Procedure.
• With a sterile needle, inoculate bacteria within the media (single stab).
• Incubate for 24 hours at 35-37ºC.
• After incubation, read the results.
• Motile bacteria can move away from the original site of stabbing.
Purpose.
• Used to differentiate between bacteria in terms of:
• 1-Fermentation of Glucose, Lactose and/or Sucrose.
• Acids are the final products of fermentation.
• 2-Production of gas.
• 3-Production of hydrogen sulfide (H2S)
Note.
• TSI & KIA differ in that:
• TSI agar contains glucose, lactose and sucrose.
• KIA contains only glucose and lactose.
Components of TSI include:
• 1% Lactose.
• 1% Sucrose.
• 0.1% Glucose.
• pH sensitive dye (Phenol red – pH indicator)
• Sodium thiosulfate.
• Ferrous sulfate / Ferric ammonium sulfate (H2S
indicator)
If bacteria can ferment glucose but not
lactose.
•Acid is enough to turn the butt yellow, but not the
slant.
If bacteria can ferment both glucose and
lactose.
•Fermentation of glucose occurs first, resulting in a
yellow butt.
•Lactose fermentation occurs next, resulting in a
yellow slant.

Where does H2S come from?
◦ H2S is a toxic gas that is produced by some bacteria
form:
 Breakdown of sulfur-containing amino acids, or
 The use of sodium thiosulfate during anaerobic
respiration (as in our experiment).

What is the reaction?
◦ When H2S is produced, it reacts with IRON in the
medium.
 Source of iron is Ferrous sulfate / Ferric ammonium
sulfate.
◦ The result is a black precipitate, which is ferric
sulfide (FeS).
What are the produced gases?
•H2 and CO2.
Results.
•Detection by the presence of cracks or
bubbles in the medium.
•Figure is shown later in slides.
1
2
3
4
Tube (1):
•A/A, G
Tube (2):
•K/A
Tube (3):
•K/A, H2S
Tube (4):
•K/K
Example
KIA
IMViC
Urease
Motility
Escherichia coli
A/A/G
++--
-
+
Klebsiella pneumonia
A/A/G
--++
+
-
Enterobacter aerogenes
A/A/G
--++
-
+
Salmonella typhi
K/A, G, H2S
-+-+
-
+
Proteus vulgaris
K/A, G, H2S
-+-+
+
+
Shigella sonnei
K/A
++--
-
-
Pseudomonas aeruginosa
K/K
---+
-
+