C.

TSI (Triple Sugar Iron)

Reaction
Principle: Composition of TSI agar

0.1% Glucose: fermentation = butt: yellow; slant: red

1.0 % lactose/1.0% sucrose: fermentation = butt, slant:
yellow
Iron: Ferrous sulfate: H2S formation
Phenol red: indicator of acidification = acidic: yellow;
alkaline: red
Peptone: source of nitrogen

C. TSI (Triple Sugar Iron)

Reaction
Procedure
Inoculate
organisms
into TSI slant
and butt

Examine color of
slant and tube:
reaction that
took place

Incubate at
35C for 24
hours
Examine
tubes:
bubbles and
cracks = CO2/
H2

Examine
tubes:
blackening =
production of
H2S

Interpretation of Results
Reaction

(+) Positive Result

(-) Negative Result

Production of H2S

black color of ferrous

sulfide

no black color in medium

Production of CO2/ H2

bubbles or cracks

no bubbles or cracks

Slant/butt reactions: Acid (A) = yellow; Alkaline (K) = red; None

(NC)= no color change
Identification

Interpretation

K/NC

glucose, lactose and sucrose non-fermenter

K/K

glucose, lactose and sucrose non-fermenter

K/A

glucose fermentation only, gas (+ or -), H2S (+ or -)

A/A

glucose, lactose /sucrose fermenter gas (+ or -),

H2S (+ or -)

Theoretical and Experimental Results

Organism

Experimental

E. coli

no black spots in
medium

(-) H2S

(-) H2S

no
breakage/bubbles

(-) gas: CO2/H2

(+) gas: CO2/H2

A/A

fermentation of
lactose/sucrose and
glucose

A/A: glucose,
lactose /sucrose
fermenter gas

no black spots in
medium

(-) H2S

(-) H2S

presence of bubbles

(+) gas: CO2/H2

(+) gas: CO2/H2

A/A

fermentation of
lactose/sucrose and
glucose

K/NC: glucose,
lactose and
sucrose nonfermenter

P.
auruginosa

Theoretical

D. Production of acetyl-methyl-carbinol
(Voges-Proskauer Test) and mixed acids
(Methyl Red Test) from Glucose
(MR-VP Test)

Principle

MR Test

Principle
VP test

butylene glycol pathway:

forms acetyl methyl
carbinol (acetoin)
At atmospheric oxygen
and 40% KOH, acetoin is
converted to diacetyl
with alpha-naphthol as a
catalyst to bring out a
red complex

Procedure
Inoculate
organisms into
MR-VP broth
(replicates)

Incubate at
35C for 48
hours

Set 2: VP test
+12drps of 5% alpha
naphthol in absolute ethyl
alcohol
+4drps of 40% KOH

Set 1: MR test
+5drps
methyl red

Interpretation of Results
Test

(+)
Positive
Result

(-) Negative
Result

Positive and
negative results
for MR test

MR: mixedacid
remains
turns red
fermentatio
yellow
n
VP:
remains
presence of turns red
yellow
diacetyl

Positive and
negative results
for VP test

Theoretical and Experimental Results

Organism

E. coli
P.
aeruginos
a

MR test

VP test

EXP

EXP

THE
O

THEO

+ + -