Isolation of Enteric Organisms

Exercise

Culture Media for Isolation of Enterics

Eosin Methylene Blue SalmonellaShigella Agar
Thiosulfate Citrate Bile Salt Sucrose Bismuth Sulfite Agar

MacConkey

Hektoen Enteric Agar

Culture Media for Isolation of Enterics

Triple Sugar Iron

Lysine Iron Agar

Trypton broth

Culture Media for Isolation of Enterics

Methy l Red Voges Proskauer

Simm on Citrat e Agar

Urea Broth or Urea Agar

SIM

Day 1

EM B

Inoculate on
MacConke y

HE A

SS A

TCB S

BS A

Day 2

Interpret results of plates

Inoculate on TSI and LIA from either EMB, Mac, or HEA

Uninoculated EMB

EMB

Selective for Gram-negative bacteria

Differentiates LF from NLF

EMB

Indicators
Eosin and methylene blue (inhibit Gram positive bacteria)

Carbohydrate incorporated Lactose

Interpretation If lactose is fermented,

precipitated eosin and methylene blue are absorbed, resulting in purple color in the medium

A classical green metallic sheen is produced by E. coli

Nonlactose fermenters produce colorless colonies.

Lactose Fermenters on EMB (pink to purple colonies)

Escherichia coli: Enterobacter cloacae:

Klebsiella pneumoniae (E. coli Green metallic sheen)

Nonlactose Fermenter on EMB (colorless to yellowish colonies)

Salmonella typhi: Shigella sonnei: Proteus

vulgaris

Pseudomonas aeruginosa on EMB

MacConkey
Uninoculated

MacConkey Agar

Selective for Gram negative bacteria Differentiates LF from NLF

Principle
Inhibit Gram positive bacteria
Bile salt Neutral red Indicator Crystal violet Lactose

Carbohydrate

Interpretation Mediu m is acidifie d and bile salt is precipit a-ted

Precipita -ted dye is absorbe d producin g a pink to red complex

If lactose is fermente d

Lactose Fermenter 0n MacConkey

Escherichia coli: Enterobacter cloacae:

Klebsiella pneumoniae

Nonlactose Fermenter on MacConkey

Salmonella typhi: Shigella sonnei: Proteus vulgaris

Pseudomonas aeruginosa (Bluegreen colonies = pyocyanin) on MacConkey

Uninoculated HEA

Hektoen Enteric Agar

Purpose
Selective for stool pathogens

Inhibit normal flora of the lower GI tract

Principle
Bile salts
Inhibit Gram positive bacteria Inhibit Gram negative coliforms

Lactose, sucrose, and salicin

Carbohydrate sources

Bromthymol blue

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

Sodium thiosulfate H2S detection and ferric ammonium Black-centered colonies citrate

Interpretation
Green colonies (with or without black center Yellow colonies

Nonfermenters

Normal flora

Pathogens

Except Yersinia enterocolitica (sucrose fermentation)

Lactose Fermenter on HEA

Xylose Lysine Deoxycholate Agar

Isolation and differentiation of stool pathogens

Purpose
Inhibition of normal flora coliforms

Principle
Sodium deoxycholate
Inhibits Gram positive bacteria Partially inhibits E. coli Inhibits swarming of Proteus

Phenol red
Indicator Yellow in acidic environment

Principle
Xylose
Carbohydrate Yellow colonies (fermenter) Red colonies (nonfermenter)

Lysine
Red colonies (decarboxylation)

Ferric ammonium citrate

H 2S production blackening

This is a XLD agar plate culture of Enterobacter sakazakii after 24 hours of growth at 36 C.

Salmonella-Shigella Agar

Purpose

Inhibits normal flora coliforms

Differentiatio n of stool pathogens

Principle
Brilliant green agar and bile salts

Bile salts

Inhibits Gram positive bacteria

Inhibit Gram negative coliforms

Principle
Carbohydrate

Lactose

Neutral red

Indicator Red in acidic condition (LF) Colorless (NLF)

Sodium thiosulfate H2S indicator Black-centered colonies and ferric ammonium citrate

Interpretation
Pink to red Colorless without black center

Colorless with black center

Salmonella Normal flora coliforms Shigella

Nonlactose Fermenter on SSA

Shigella

SS agar plate culture of Enterobacter sakazakii after 24 hours of growth at 36 C.

Thiosulfate Citrate Bile Salt Sucrose Agar

Purpose

Isolate most Vibrio species

Principle
Sucrose Oxgall and cholate
Carbohydrate

Inhibit Gram positive bacteria Inhibit LF

Bromthymol Indicator blue

Interpretation

Yellow
Sucrose fermenter V. cholerae

Green
Nonsucrose fermenter V. parahemolyticu s

TCBS
Sucrose fermenter yellow

Nonsucrose fermenter - green

Bismuth Sulfite Agar Purpose

Isolation of S. typhi

S. typhi
Metallic colonies with black ring

Most enteric
No growth

S. typhi, E. coli, and Proteus sp. bacteria are cultured on bismuth sulfite agar; 48hrs incubation.

Day 3
Interpret TSI and LIA Inoculate on TB, MRVP, SCA, UB, and SIM

Triple Sugar Iron

Purpose

Initial step in identifying enterics

Principle
Protein sources Carbohydrate Indicator

H2S indicator
Ferrous sulfate

Beef and yeast extracts, peptone

Permit growth of most bacteria

Lactose, glucose, sucrose

Phenol red

TSI Reaction
Slant
Aerobic Alkaline (red) Oxidative decarboxylation of peptides and amino acids

Butt or deep
Anaerobic Acid (yellow) Minimal amino acid degradation (few amines)

Carbohydrate Fermentation
Glucose
Small amount of acid Alkaline slant over acid deep (K/A)

Glucose, lactose and/or sucrose

Large amount of acid Acid slant over acid butt (A/A)

Noncarbohydrate Fermenter

Alkaline slant over alkaline deep (K/K)

H2S Production

Sodium thiosulfate
Ferric sulfate and ferric ammonium citrate Black precipitate of Ferrous sulfide

Gas Production

Fermentation
Presence of cracks in the medium or pulling away of the medium

Uninoculated TSI

Acid Slant : Acid Butt

Alkaline Slants Acid Butt: Salmonella & Shigella Alkaline Butt: Pseudomonas

Possible TSI Reactions

Reaction A/AG Carbohydrate fermented Glucose with acid and gas Lactose and/or sucrose with acid and gas Glucose with acid and gas Lactose or sucrose not fermented H2S production Glucose with acid, no gas Lactose and glucose not fermented Glucose, lactose, or sucrose not fermented Glucose fermented with gas Lactose or sucrose fermented H2S production Typical Organisms Escherichia Klebsiella Enterobacter Salmonella Proteus Citrobacter

K/AG+

K/A

Shigella, Providencia Serratia Anaerogenic E. coli Pseudomonas Alcaligenes Citrobacter freundii

K/K A/AG+

LIA Reaction

Purpose Ability to
Deaminase lysine Decarboxylate lysine Produce H2S

Lysine Iron Agar

Protein, glucose, lysine, sulfur Ferric ammonium citrate

Bromcresol purple

Principle
Yellow deep Purple deep Red slant Black ppt
Glucose fermentation Lysine decarboxylation Alkaline cadaverine Lysine deanimation

H2S production

LIA

Interpretation
K/K
Lysine decarboxylase positive Purple/purple Lysine decarxylase negative Purple/yellow Deaminase positive Red/yellow

K/A R/A

IMViC Reaction
I
Indole production test

M
Methyl red test

Vi
Voges Proskauer test

C
Citrate utilization test

Indole Production Test

Purpose

Distinguish enterics based on the ability to produce indole from tryptophan

Speciation of Proteus

Principle
Tryptophan in peptone is oxidized to indole, skatole, and indole-acetic acid Intracellular enzyme that metabolize tryptophan is tryptophanase

Detected by the indicator alcoholic pdimethylaminobenzaldehyde

Kovacs or Ehrlich reagent

Principle
Tryptophan

Tryptophanas e
Indole + pyruvic acid + ammonia Indole
Pdimethylami nobenzalde hyde

Red ring

INDOLE POSITIVE: E. coli & P. vulgaris

NEGATIVE INDOLE E. cloacae, K. pneumoniae, Ps. aeruginosa, S. sonnei, S. typhi

Methyl Red Voges Proskauer Test

Purpose

Dextrose broth buffered with peptone

Glucose

Pyruvic acid and acetoin

Methyl Red Test

Pyruvic acid
Mixed acid fermentation pH > than 4.4

Red

MR positive

MR indicator

Methyl Red Test

Voges-Proskauer Test
Acetoin (acetylmethyl carbinol)
KOH + air

Diacetyl

Naphthol + creatine

VP positive

Pink red complex

Voges-Proskauer Test

Citrate Utilization Test

Purpose

Utilization of citrate as sole source of carbon

Identification of lactose fermenters

Principle

Ammonium hydroxide

Ammonia
Bromthymol blue

Sodium citrate

Blue due to increase in pH

SIMMON'S CITRATE AGAR Citrate sole carbon source. Methylene blue as a pH indicator NEGATIVE P. vulgaris, S. sonnei, E. coli

POSITIVE S. typhi. E. cloaceae, K. pneumoniae, Ps. aeruginosa

Sulfite Indole Motility Agar Motility Test H2S Production Indole Production

SIM (SULPHUR:INDOLE:MOTILITY) MEDIUM SULPHUR POSITIVE: P. vulgaris & Salmonella

MOTILITY Determined by turbidity in semi solid media POSITIVE: E. coli, E. cloaceae, Ps. aeruginosa

NON MOTILE Demonstrated by growth on the inoculum line S. sonnei, K. pneumoniae

Urease Test
Purpose Splits urea into lakaline end products

Identification of rapid and weak urease producer

Principle
Urease

NH3

(NH4)2 CO3

Urea + H2O

CO2 H2O

POSITIVE RESULT Klebsiella & Proteus

UREA TUBES Determination of Urease production NEGATIVE RESULT E. coli; Enterobacter, Salmonella, Shigella & Pseudomonas

Urease test

This is a sheep blood agar plate culture of Enterobacter sakazakii after 24 hours of growth at 36 C.

SCA

E.coli on Hektoen Enteric Agar