Tests for Identification of Bacteria

TRIPLE SUGAR IRON (TSI) AGAR TEST

Used to identify and distinguish Enterobacteriaceae
by their ability to catabolize lactose, sucrose, or
glucose and to liberate sulfides
Contains 1%lactose and sucrose, 0.1% glucose,
ferrous ammonium sulfate, sodium thiosulfate and
phenol red
Phenol red
o pH indicator
o detects acid production from carbohydrate
fermentation
fermentation acid formation turns yellow
K = ALKALINE; A = ACID

K
A

red slant_
yellow butt

Interpretation: Glucose fermentation

A
A

yellow slant
yellow butt

Some Bacteria and their appearance on TSI slant:

IMViC TEST
Mnemonic used for a group of individual tests used
in Microbiology. It is especially helpful in
distinguishing members of Enterobacteriacieae.
IMViC = Indole, Methyl red, Voges-Proskauser, and
Citrate. The small i is just inserted for ease of
pronunciation
INDOLE TEST
Broth contains the amino acid tryptophan
Bacteria containing the enzyme, tryptophanase
facilitates reaction

Interpretation: Glucose and

Lactose/Sucrose fermentation
-

(+) H2S
H2S formation = blackening of agar

Formation of red/fuchsia
ring at the surface of the
media = (+) indole test
Memory aid: indOle, the
O looks like a ring
Bacteria that are indole
(+): Escherichia coli,
Proteus vulgaris

METHYL RED TEST

-

(+) Gas
Gas formation = Splitting of agar
-

All
enteric
bacteria
catabolize glucose but end
products vary depending
on enzyme pathways
Methyl red detects pH
change to acid range as a
result of acidic end
products (lactic a., acetic
a., formic a.)
Mixed acid fermenters acidify this medium
Distinguish Escherichia coli (a mixed acid fermenter)
vs. Enterobacter aerogenes
pH 6 yellow (-); pH 4 red (+)

1 | A.S.S.

PHENYLALANINE DEAMINASE TEST

VOGES-PROSKAUER TEST
-

identifies bacteria that

ferment glucose to 2,3butanediol
Barritts reagent (40%
KOH, 5% -naphthol in
absolute ethanol) detects
the presence of acetoin
(acetyl-carbinol), a
precursor in the synthesis
of 2,3-butanediol
Red color (+) VP test
Bacteria that are VogesProskauer (+): Klebsiella spp., Enterobacter aerogenes
CITRATE TEST
Determine the ability of the
organism to utilize sodium
CITRATE as its ONLY carbon
source & inorganic ammonium
as its nitrogen source
Bacteria turn the Bromothymol
blue indicator from green to
BLUE.
(+) = BLUE
Memory aid: Citrate Cyan =
BLUE

LYSINE DECARBOXYLASE TEST

-

UREASE TEST
Used to distinguish members of Proteus from other
NON-LACTOSE
fermenting
enteric
bacteria
(Salmonella, Shigella)

Urea hydrolyzed ammonia accumulate

Alkalinize medium turn deep pink/purplish red
(+) urease test = deep
pink
Proteus is (+) urease

LACTOSE FERMENTATION TEST

Medium
contains
lactose. (+) Lactose
fermenter = acidic byproducts

turn
YELLOW
Gas
produced
is
detected by inverted
tube (Durham tube)

Used to differentiate among certain enteric bacteria,

such as Escherichia coli and Proteus vulgaris. P.
vulgaris produces the enzyme phenylalanine
deaminase

To see if the microbe can use the amino acid lysine as

a source of carbon and energy for growth. Use of
lysine is accomplished by the enzyme lysine
decarboxylase.
When glucose is fermented, the butt of the medium
becomes acidic (yellow). If the organism produces
lysine decarboxylase,
cadaverine is formed.
Cadaverine
neutralizes the
organic acids formed
by glucose
fermentation, and
the butt of the
medium reverts to
the alkaline state
(purple). If the
decarboxylase is not
produced, the butt
remains acidic
(yellow).
MOTILITY TEST
Usually administered as
part of the SIM (Sulfide,
Indole, Motility) test
Growth not confined to the
stab line or presence of
turbidity indicates a
positive result (motile
bacteria).

2 | A.S.S.

OXIDASE TEST
Test used to determine if a bacterium produces
certain cytochrome c oxidases

Dark purple = oxidase (+)

Colorless/light pink =
oxidase (-)
N. gonorrhea, P.
aeruginosa, Vibrio spp. =
oxidase (+)
Enterobacteriaciae
=
oxidase (-)

EOSIN METHYLENE BLUE (EMB) AGAR

Slightly selective for gram-negative bacteria
DIFFERENTIAL
medium,
distinguish
lactose
fermenters
Escherichia coli has a distinct green metallic sheen
appearance
Lactose fermenter acid prodn dark colored
colonies
Non-lactose fermenter deaminate CHON
alkaline colorless colonies

MAC CONKEY AGAR

is a culture medium selective for Gram-negative
bacteria and DIFFERENTIAL for LACTOSE fermentation
lactose fermenter = Pink colonies

HEKTOEN ENTERIC AGAR (HEA)

is a selective and differential agar primarily used to
recover Salmonela spp. and Shigella spp.
Has indicators for lactose fermentation and H2S
production
Contains various sugars (lactose, sucrose, salicin)
which are not utilized by Salmonella and Shigella
(only utilized by lactose enterics). Also contains
PEPTONE, which the non-lactose fermenters prefer
Also has thiosulfate/ferric ammonium citrate which
produce a black precipitate in the presence of H2S,
distinguishing Salmonella vs Shigella
2 color changes:
o 1. Lactose/Peptone utilization
Lactose acidify red/yellow (e.g. E. coli)
Peptone alkalinize BLUE agar (Salmonella
and Shigella)
o 2. H2S production
(-) H2S green colonies Shigella
(+) H2S BLACK colonies Salmonella

SELENITE BROTH
Is an ENRICHMENT medium for
maximum Salmonella spp.
isolation/recovery from specimens
Red precipitate indicates Salmonella
growth

WIDALS TEST
-

Serological test
(agglutination) for enteric
fever whereby bacteria
causing typhoid fever are
mixed with serum
containing specific
antibodies obtained from
an infected individual.
Identifies Salmonella
Detects antibodies against
O and H antigen and notes
for rise in antibody titer
(+) = titer against O > 1:320;
H > 1:640
3 | A.S.S.

VIBRIO CHOLERAE AGGLUTINATING SERA TEST

slide and/or tube agglutination tests to determine
whether the culture is positive for the O antigen of
the lipopolysaccharides of Vibrio cholerae O1 that
exist in two forms termed Inaba and Ogawa

Content and photo References:

MOODLE (Microbiology & Parasitology) http://cm.dlshsi.edu.ph/course/index.php?categ
oryid=6
Classroom Lectures
Wikipedia
Google Images

Transcribed by:

ALKALINE PEPTONE WATER BROTH

ENRICHMENT medium used for the
cultivation of Vibrio species from feces
and other infected materials

"Don't have my name inscribed into the stone. Just

say: 'Here lies love.'"

SKIRROWS SELECTIVE MEDIUM (SSM)

A selective solid medium used for the cultivation and
isolation of Campylobacter jejuni from fecal
specimens
Addition of antibiotics such as vancomycin, polymyxin
B, trimethoprim (Prestons agar) refrains normal stool
flora overgrowth and enhances C. jejuni recovery.

HIPPURATE TEST
Tests for bacterias
ability to hydrolyze
HIPPURATE
(+) = deep blue/purple
(-) = faint blue/no color
change
Hippurate positive
bacteria: Campylobacter
jejuni, Listeria
monocytogenes,
Gardnerella vaginalis,
Group B streptococci

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