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Exercise

Isolation of Enteric Organisms

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Culture Media for Isolation of

Enterics

EosinMethylene Blue

MacConkey

HektoenEnteric Agar

Salmonella-Shigella Agar

ThiosulfateCitrate Bile SaltSucrose

Bismuth SulfiteAgar

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Culture Media for Isolation of

Enterics

TripleSugar Iron

LysineIron Agar

Tryptonbroth

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Culture Media for Isolation of

Enterics

Methyl Red

VogesPros-kauer

Simmon

Citrate

Agar

UreaBroth

orUreaAgar

SIM

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Day 1

Inoculate

onEMB Mac-

Conke

y

HEA

SSA

TCBS

BSA

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Day 2

Interpretresults of

plates Inoculate on TSI andLIA from either EMB,

Mac, or HEA

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Uninoculated EMB

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EMB

Selective forGram-negative

bacteriaDifferentiates

LF from NLF

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EMB

Carbohydrateincorporated

Lactose

IndicatorsEosin and methylene blue

(inhibit Gram positive bacteria)

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InterpretationIf lactose is fermented,precipitated eosin andmethylene blue are absorbed,resulting in purple color in themedium

A classical green metallicsheen is produced by E. coli

Nonlactose fermentersproduce colorless colonies.

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Lactose Fermenters on EMB (pink to purple

colonies)

Escher ich ia co l i: Enterobacter cloacae:

K lebs iel la pneumon iae

(E. co liGreen metallic sheen)

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Nonlactose Fermenter on EMB

(colorless to yellowish colonies)

Salmonella typhi: Shigel la sonnei: Proteus

vulgar is

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Pseudomonas aerug inosaon

EMB

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MacConkey

Uninoculated

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MacConkey Agar

Selective for Gramnegative bacteria

Differentiates LF

from NLF

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Principle

Bile salt

Neutral red

Indicator

Crystal violet

Inhibit Grampositivebacteria

Lactose

Carbohydrate

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Interpretation

If lactoseisfermente

d

Medium is

acidified and

bile saltis

precipita-ted

Precipita-ted dye

is

absorbed

producin

g a pinkto redcomplex

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Lactose Fermenter 0n

MacConkey

Escher ich ia co l i: Enterobacter cloacae:

K lebs iel la pneumon iae

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Nonlactose Fermenter on

MacConkey

Salmonella typhi: Shigel la sonnei: Proteus

vulgar is

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-green colonies = pyocyanin) on

MacConkey

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Uninoculated HEA

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Hektoen Enteric Agar

Purpose

Selective forstool

pathogens

Inhibitnormal flora

of the lowerGI tract

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Principle

Inhibit Gram positive bacteria Inhibit Gram negative coliformsBile salts

Carbohydrate sourcesLactose, sucrose, and

salicin

Indicator

Blue (>7.6), green (6-7.6), yellow (

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Interpretation

Green colonies (with orwithout black center

Nonfermenters

Pathogens

Yellow colonies

Normal flora

Except Yersinia enterocolitica(sucrose fermentation)

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Lactose Fermenter on HEA

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Xylose Lysine Deoxycholate Agar

Purpose

Isolation anddifferentiation of

stool pathogens

Inhibition of

normal floracoliforms

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Principle

Sodiumdeoxycholate

Inhibits Gram positivebacteria

Partially inhibits E. coli

Inhibits swarming of

Proteus

Phenol red

Indicator

Yellow in acidicenvironment

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Principle

Xylose

Carbohydrate

Yellow colonies(fermenter)

Red colonies(nonfermenter)

Lysine

Red colonies

(decarboxylation)

Ferricammonium

citrate H2S

production

blackening

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This is a XLD agar plate culture of Enterobacter

sakazakii after 24 hours of growth at 36 C.

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Salmonella-Shigella Agar

PurposeInhibits

normal floracoliforms

Differentiation of stool

pathogens

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Principle

Bile salts

Inhibits Grampositive bacteria

Brilliant green agar

and bile salts Inhibit Gram

negativecoliforms

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Principle

Carbohydrate

Lactose

Indicator Red in acidic condition (LF)

Colorless (NLF)Neutral red

H2S indicator Black-centered colonies

Sodium thiosulfateand ferric

ammonium citrate

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Interpretation

Pink to red

Normalflora

coliforms

Colorlesswithout black

center

Shigella

Colorless withblack center

Salmonella

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Nonlactose Fermenter on SSA

Shigella

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SS agar plate culture ofEnterobacter sakazaki i

after 24 hours of growth at 36 C.

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Thiosulfate Citrate Bile Salt Sucrose

Agar

PurposeIsolate

most Vibriospecies

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Principle

CarbohydrateSucrose

Inhibit Gram positive bacteria

Inhibit LFOxgall and

cholate

IndicatorBromthymol

blue

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Interpretation

Yellow

Sucrosefermenter

V. cholerae

Green

Nonsucrosefermenter

V.

parahemolyticus

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TCBS

Sucrose fermenter yellow

Nonsucrose fermenter - green

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Bismuth Sulfite Agar

Most entericNo growth

S. typhiMetallic colonies with black ring

PurposeIsolation of S. typhi

S t phi E coli and Prote s sp bacteria are

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S. typhi, E. coli, and Proteus sp. bacteria are

cultured on bismuth sulfite agar; 48hrs

incubation.

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Day 3

Interpret TSI andLIA

Inoculate on TB,

MRVP, SCA, UB,and SIM

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Triple Sugar Iron

PurposeInitial step inidentifyingenterics

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Principle

Proteinsources

Beef and

yeastextracts,peptone

Permit

growth ofmost

bacteria

Carbohydrate

Lactose,glucose,sucrose

Indicator

Phenolred

H2Sindicator

Ferroussulfate

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TSI Reaction

Slant

Aerobic

Alkaline (red)

Oxidativedecarboxylation ofpeptides and amino

acids

Butt or deep

Anaerobic

Acid (yellow)

Minimal amino aciddegradation (fewamines)

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Carbohydrate Fermentation

Small amount of acid

Alkaline slant overacid deep (K/A)

Glucose

Large amount of acid

Acid slant over acidbutt (A/A)

Glucose,

lactoseand/orsucrose

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Noncarbohydrate Fermenter

Alkaline slant overalkaline deep (K/K)

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H2S Production

Sodium thiosulfate

Ferric sulfate and ferricammonium citrate

Black precipitate ofFerrous sulfide

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Gas Production

Fermentation

Presence of cracks in the

medium or pulling away ofthe medium

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Uninoculated TSI

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Acid Slant : Acid Butt

Alk li Sl t

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Alkaline Slants

Acid Butt: Salmonel la& Shigel la

Alkaline Butt: Pseudomonas

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Possible TSI ReactionsReaction Carbohydrate fermented Typical Organisms

A/AG Glucose with acid and gas

Lactose and/or sucrose with

acid and gas

Escherichia

Klebsiella

Enterobacter

K/AG+ Glucose with acid and gas

Lactose or sucrose notfermented

H2S production

Salmonella

ProteusCitrobacter

K/A Glucose with acid, no gas

Lactose and glucose not

fermented

Shigella, Providencia

Serratia

Anaerogenic E. coli

K/K Glucose, lactose, or sucrose

not fermented

Pseudomonas

Alcaligenes

A/AG+ Glucose fermented with gas

Lactose or sucrose fermented

H2S production

Citrobacter freundii

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LIA Reaction

Purpose

Ability to

Deaminaselysine

Decarboxylatelysine

Produce H2S

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Lysine Iron Agar

Protein, glucose, lysine,sulfur

Ferric ammonium citrate

Bromcresol purple

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Principle

Glucose fermentationYellow deep

Lysine decarboxylation

Alkaline cadaverinePurple deep

Lysine deanimationRed slant

H2S productionBlack ppt

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LIA

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Interpretation

K/K Lysine decarboxylase positive Purple/purple

K/A Lysine decarxylase negative Purple/yellow

R/A

Deaminase positive

Red/yellow

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IMViC Reaction

I

Indole

productiontest

M

Methyl red

test

Vi

Voges

Proskauertest

C

Citrate

utilizationtest

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Indole Production Test

Purpose

Distinguish enterics based on the ability toproduce indole from tryptophan

Speciation of Proteus

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Principle

Tryptophan in peptone isoxidized to indole, skatole,

and indole-acetic acid

Intracellular enzyme thatmetabolize tryptophan is

tryptophanase

Detected by the indicator

alcoholic p-dimethylaminobenzaldehyde

Kovacs or Ehrlich reagent

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Principle

Tryptophan

Red ringIndole

Tryptophanas

eIndole + pyruvic

acid + ammonia

P-

dimethylami

nobenzalde

hyde

INDOLE

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INDOLE

POSITIVE: E. co li& P. vu lgaris

NEGATIVE INDOLE

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NEGATIVE INDOLE

E. clo acae, K . pneumon iae, Ps. aerugin osa, S. sonnei,

S. typhi

Methyl Red Voges Proskauer

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Methyl Red Voges Proskauer

Test

Purpose

Dextrose broth buffered withpeptone

Glucose

Pyruvic acidand acetoin

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Methyl Red Test

Pyruvic acidMixed acid

fermentationpH > than

4.4

MR

indicatorMR positiveRed

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Methyl Red Test

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Voges-Proskauer Test

Acetoin(acetylmethyl

carbinol)Diacetyl

VP positive Pink redcomplex

KOH +

air

Naphthol +

creatine

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Voges-Proskauer Test

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Citrate Utilization Test

Purpose

Utilization of citrate as sole source ofcarbon

Identification of lactose fermenters

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Principle

Sodiumcitrate

Ammonia

Ammonium

hydroxide

Bromthymol

blue

Blue due to increase in

pH

SIMMON'S CITRATE AGAR

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SIMMON'S CITRATE AGAR

Citrate sole carbon source. Methylene blue as a pH

indicator

NEGATIVEP. vulgaris , S. so nnei, E. coli

POSITIVE

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S. typh i. E. clo aceae, K . pneumoniae, Ps.

aeruginosa

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Sulfite Indole Motility Agar

Motility Test

H2S Production

Indole Production

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SIM (SULPHUR:INDOLE:MOTILITY) MEDIUM

SULPHUR

POSITIVE: P. vu lgaris& Salmonel la

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MOTILITY

Determined by turbidity in semi solid media

POSITIVE: E. co li, E. cloaceae, Ps. aerug ino sa

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NON MOTILE

Demonstrated by growth on the inoculum line

S. sonnei, K. pneumon iae

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Urease Test

Purpose

Splits urea into lakalineend products

Identification of rapid andweak urease producer

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Principle

Urease

Urea

+ H2O

NH3 (NH4)2 CO3

CO2

H2O

POSITIVE RESULT

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POSITIVE RESULT

Klebsiel la& Proteus

UREA TUBES

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UREA TUBES

Determination of Urease production

NEGATIVE RESULT

E. co li; Enterobacter, Salmonella, Shigel la&

Pseudomonas

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Urease test

This is a sheep blood agar plate culture of

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Enterobacter sakazakii after 24 hours of growth at

36 C.

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SCA

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E.coli on Hektoen Enteric Agar