Isolation of Enteric Organisms Day 1 Inoculation on differential and selective media Differential Media Medium EMB (Eosin Mehtylene

Blue)
Uninoculated: Burgundy (redpurple)

Bacteriostatic dye Eosin, Methylene blue

Fermentable carbohydrate

Indicator

Colony color LF-purple with green metallic sheen(E.coli) NLF-colorless

lactose

Eosin, methylene blue

MacConkey
Uninoculated: pink

Cyrstal violet, bile salt

lactose

Neutral red

LF-pink to red NLF-colorless LF-carrot orange(normal flora/coliforms) NLFgreen(stool pathogens) LF-red NLF-colorless

HEA (Hektoen Entric Agar)

Uninoculated: Muddy green

Bile salt

Lactose, sucrose, salicin

Bromthymol blue
With thiosulfate FeNH4 Citrate as H2S indicator

Deoxycholate

Bile salt

lactose

Neutral red

XLD (Xylose Lysine Deoxycholate)

Bile salt

Lactose, xylose, sucrose

Neutral red

LF-red NLF-colorless

Selective Media

Medium

Bacteriostatic dye

Fermentable carbohydrate

Indicator

Colony color LF-red NLF-white with black center (Salmonella) without black center(Shigella) SF-yellow
(V. cholerae)

SSA (Salmonella Shigella Agar)

Uninoculated: orange

Lactose w/ Na thiosulfate and Brilliant green Fe NH4 Citrate as H2S indicator Sucrose Oxgall, sodium w/ FeNH4 cholate Citrate as H2S indicator

Neutral red

TCBS (Thiosulfate Citrate Bile Salt Sucrose)

Uninoculated: green

Bromthymol blue

NSF-green
(V. parahemolyticus)

BSA (Bismuth Sulfite Agar)

Uninoculated: cream/off white

Brilliant green

Glucose

Bismuth sulfide

Black metallic sheen (S. typhi)

H2S indicators: HEA- sodium thiosulfate, ferric ammonium citrate SSA- sodium thiosulfate, ferric ammonium citrate TCBS- ferric ammonium citrate

Day Two From MacC Inoculate on TSI (Triple Sugar Iron) and LIA (Lysine Iron Agar)

TSI Reaction uninoculated: red orange A/AG Lactose and/or sucrose fermentation Glucose fermentation with acid and gas Lactose or sucrose fermented Glucose fermentation with acid and gas H2S production Lactose or sucrose not fermented Glucose fermentation with acid but no gas No H2S production Lactose or sucrose not fermented Glucose fermentation with acid and gas No H2S production K/A
+

Escherichia Klebsiella Enterobacter Citrobacter Shigella Providencia Serratia Anaerogenic E.coli Morganella

A/AG

K/A

K/AG

Lactose or sucrose not fermented Glucose fermentation with acid but no gas Lactose or sucrose not fermented Glucose fermentation with acid and gas H2S production

Salmonella typhi Salmonella Proteus Arizona Citrobacter Edwardsiella

K/AG+

TSI- screening medium for the identification of Enterobacteriaceae on the basis of glucose, lactose and sucrose fermentation and H2S production; detects LLF because this sugar is the one 1st attacked

Carbohydrate present-glucose, lactose and sucrose pH indicator-phenol red H2S indicator- ferrous sulphate or ferric ammonium citrate indicated by black ppt. in butt
Acid fermentation yellow color (A)

No acid fermentation red color (K) Gas production cracks or displacement of medium (G or encircle) H2S production blackening of butt (+)

LIA Reaction uninoculated: purple Lysine decarboxylation H2S production Note: lysine decarboxylation produces cadaverine. This cadaverine neutralizes the organic acids formed by glucose fermentation and butt reverts to alkaline state Lysine decarboxylation

K/K+

Salmonella Edwardsiella

K/K

Enterobacter Escherichia Klebsiella Serratia Citrobacter Enterobacter Shigella Proteus Providencia Morganella

K/A

No lysine decarboxylation Glucose fermentation

R/A

Lysine deamination No lysine decarboxylation Glucose fermentation

LIA-useful in the identification of Samonella, Proteus, Providencia and Morganella by virtue of its ability to decarboxylate or deaminate lysine and form H2S and ferment glucose Carbohydrate present glucose Amino acid present lysine H2S indicator ferric ammonium citrate pH indicator bromcresol purple
Lysine decaroxylation purple butt No lysine decaroxylation yellow butt Lysine deamination red slant H2S production blackening in the butt

Day Three From TSI Inoculate on TB (Tryptone Broth) indole production test (uninoculated: straw colored) MRVP (Methyl Red Voges Proskauer) methyl red test (straw colored) MRVP Voges Proskauer test (straw colored) SCA (Simmon Citrate Agar) citrate utilization test (green) UB (Urea Broth) urease test (straw colored) SIM (Sulfite Indole Agar) motility test, indole production (off white) 1. Indole Production Test: identification is based on the ability of organisms to produce indole for tryptophan Tryptophan
tryptophanase

indole + Kovacs/Erlichs rgt = red ring

Indicator Kovacs or Ehrlichs reagent (main ingredient: paminobenzaldehyde) Result positive (red ring) Negative (yellow ring) 2. Methyl Red Test: identification is based on the ability of organisms to produce and maintain acid end products Glucose Pyruvic acid lactic, acetic, formic, succinic pH less than 4.4 MR indicator Positive: Red solution Negative: yellow 3. Voges Proskauer Test: identification is based on the ability to produce neutral end product

Glucose Acetoin or acetyl methyl carbinol KOH in 0.3% creatine Alpha naphthol in ethyl alcohol Diacetyl or dimethyl carbinol Pink-red complex Negative yellow to brown

4. Citrate Utilization Test identification is based on the ability utilize citrate as its sole source of carbon Sodium citrate Indicator bromthymol blue Positive Prussian blue (Klebsiella) Negative green (E.coli) 5. Urease Test
urease

ammonia

NH4OH

Urea

CO2 + H2O + 2 NH3

(NH4)2CO3 Indicator phenol red Porsitive cherry red color (Proteus, Morganella, Providencia) Negative pale yellow 6. SIM: to test A) H2S production B) Indole production C) Motility