Biochemical Tests for Identification
of Enterobacteriaceaecontinued
Clinical Microbiology Review 178
TEST PRINCIPLE INTERPRETATION OTHER
H 2 S production
Triple sugar iron
(TSI) agar
Indole
Sulfur-containing compounds = sodium
thiosulfate, cystine, methionine. Good test
to differentiate Salmonella(H 2 S pos) from
Shigella(H 2 S neg).
Contains 0.1% glucose, 1% lactose, 1%
sucrose, phenol red, sodium thiosulfate,
iron salt. Leave cap slightly loose. Record
as slant/butt, e.g., K/A. (Some record NC
[no change] for pink butt.)
Kligler’s iron agar (KIA) is same except no
sucrose.
Spot indole test commercially available.
Need source of tryptophan. Use colonies from
SBA or CHOC agar, not MacConkey. Pos =
blue. E. coli& Proteus vulgarisare pos.
Organisms that possess H 2 S-
producing enzymes produce color-
less H 2 S gas from sulfur-containing
compounds. H 2 S reacts with iron
salt in medium to form black
ferrous sulfide.
Sugar fermentation produces acid,
changes color of pH indicator.
Tryptophanase deaminates trypto-
phan. Indole produced. Kovacs’s
reagent (paradimethylamino-
benzaldehyde) forms pink-colored
complex with indole.
Black precipitate
Yellow = acid (A). Pink = alka-
line (K). Pink butt = glucose
not fermented. Yellow butt =
glucose fermented. Pink slant =
lactose/sucrose not fermented.
Yellow slant = lactose and/or
sucrose fermented. Black
precipitate = H 2 S produced.
Bubbles = gas production
Pos = pink
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