Identifying Bacteria based on
Enzymes and multiple test
media
Lab # 9
Medgar Evers College
Prof. Victor Santos
Aim
• To identify bacteria based on the enzymes they have that allow them
to carry out certain specific reactions!
• You will detect the presence of
• Amylase, proteases, lipases, tryptophanase, urease, and
phenylalanine deaminase.
Enzyme
Medium
used
Color change By product
detected
Amylase
(B. subtilis)
Starch agar
plate
Add iodine
We look for
look for clear clear zone
zone due to
starch
degradation
Proteases
(B.subtilis)
Skim milk
plate
Look for clear Clear zone of
zone due to
degradation
degradation of
casein protein
Lipases
(S.aureus)
Spirit blue
agar
We look for a
dark blue
precipitation
due to
lowering of
pH as a result
of fatty acids
being
released
Blue
precipitation
or sometimes
just depletion
of fat droplets
on the agar.
Tryptophanase
(E.coli)
Tryptone broth
We add kovac’s
reagent to detect
the indole ring
produced.
Tryptophan is
broken down
into indole,
pyruvate and
ammonia. We
detect the
indole.
Urease
(P.vulgaris)
Urea agar slant
When urea is
broken down it
releases ammonia
and carbon
dioxide. The
ammonia raises the
pH and the phenol
red indicator
causes a color
change from
yellow to bright
pink
We detect the raise
in pH due to the
ammonia being
released.
Phenylalanine Phenylalanine We look for a
deaminase
agar
greenish
precipitate
(P.vulgaris)
after the
addition of
10% ferric
chloride
Phenylalanine
is broken
down by the
enzyme into
ammonia and
phenylpyruvic
acid. The
ferric chloride
reacts with the
phenylpyruvic
acid to yield a
greenish color.
Multiple testing media
• These media are important because they save us time and money by
allowing us to test for several parameters at once.
• Examples; Kliger’s Iron Agar, SIM, and Litmus Milk
Kliger’s Iron Agar
• The Kliger’s medium contains 1.0 % lactose, 0.1 % glucose and the
amino acid cysteine, peptones, ferrous salts and the pH indicator
phenol red.
• This medium allows you to detect glucose fermentation, lactose
fermentation and hydrogen sulfide production.
• Inoculate by streaking and stabbing one tube with your unknown and
one with P. vulgaris, the control.
SIM
• This medium is 0.7% agar so its considered semi-solid to allow motile
organisms to be detected.
• This medium allows you to detect motility, hydrogen sulfide
production from the breakdown of cysteine, and detect the
breakdown of tryptophan.
• Inoculate one tube with unknown, and three controls; one with S.
aureus, and one with P. vulgaris and the last one with E. coli.
• YOU MUST STAB THESE!
Litmus milk
• Allows you to detect several properties!
A) Fermentation of sugar will lead to a pink color change due to acid formation.
B) The breakdown of proteins leads to ammonia formation which raises the pH and
leads to a blue color change.
C) Litmus reduction leads to a white color change. This happens due to a drop in
oxygen and the dye is then used as an electron acceptor becoming reduced and
changing the medium to a white color.
D) Coagulation of proteins can lead to curd formation.
E) Peptonization or the medium becomes translucent or sometimes brown due to
the breakdown of milk proteins.
F) Ropiness or the formation of thick slime due to the accumulation of waste
products and cells.
• Inoculate a tube of litmus milk with your unknown only!