Biochemical Tests

Enterobacteriaceae
Dr.T.V.Rao MD

Dr.T.V.Rao MD

Tests To Know
Common Study Tests
Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production
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Initial Grouping of the Enterobacteriaceae (VP=Voges Proskauer, PDA=Phenylalanine Deaminase)

GENERA Klebsiella Enterobacter Serratia Hafnia Pantoea

VP POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE

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PDA NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE

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Initial Grouping of the Enterobacteriaceae

GENERA Proteus1 Morganella Providencia
1

VP NEGATIVE NEGATIVE NEGATIVE

PDA POSITIVE POSITIVE POSITIVE

4

Proteus mirabilis: 50% ofMD strains VP positive Dr.T.V.Rao

Initial Grouping of the Enterobacteriaceae

GENERA Escherichia Shigella Edwardsiella Salmonella Citrobacter Yersinia VP NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE
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PDA NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE

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Initial Grouping of the Enterobacteriaceae1

GENERA Escherichia Shigella Yersinia Edwardsiella
1

INDOLE POSITIVE POSITIVE 3 POSITIVE POSTIVE

2

CITRATE NEGATIVE NEGATIVE NEGATIVE NEGATIVE

VP negative, PDA negative 2 Shigella groups A, B, and C variably positive for indole production (25-50%), group D Shigella negative. Dr.T.V.Rao MD 3 Yersinia enterocolitica 50% positive

Initial Grouping of the 1 Enterobacteriaceae

GENERA Salmonella Citrobacter
1

INDOLE NEGATIVE NEGATIVE

CITRATE POSITIVE2 POSITIVE

VP negative, PDA negative 2 Salmonella serotype Paratyphi A and Typhi negative

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Key Characteristics of the Enterobacteriaceae

TSI E coli Shi AC Shi D Ed Sal Cit A/A Ak/ A Ak/ A Ak/ A Ak/ A ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

+ + + + + +/ / + / + + + + + + + + + + + + + + + + + + + +/ +/ +/ / + +
+/ RT (1)

A/A Ak/ A Yer A/A

+/ Dr.T.V.Rao MD

+
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(1) RT=room temperature

Key Characteristics of the Enterobacteriaceae

TSI Kle pne Kle oxy En aer En cloa Serr (1) Haf Pan A/A A/A A/A A/A A/A Ak/ A A/A Alk/ A ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

+ + + + + + +

+ + + + + +
/+

+ + + + + +

+/ /+ +/ /+ /+

+ + + + +

+ +
+/

+ + + +

+ + + + +

+ + + +

+
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(1) Produces DNase, lipase, and gelatinase

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Key Characteristics of the Enterobacteriaceae

TSI Prot mir a Prot vulg Mor Pro v Ak/ A A/A Ak/ A Ak/ A ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

+ +

+/

+/

+ + +s +

+/ + + /+ + + +s + + + + + + + + + + +

s = swarming motility
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Biochemical Characteristics of Escherichia coli and Shigella

E. coli
TSI Lactose ONPG Sorbitol Indole Methyl re A/Ag + + + + +

E. coli O157:H7
A/Ag + + + +

Shigella
Alk/A /+1 +/ +/ +

VP
Citrate Lysine Motility

+ +

+
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Biochemical Characteristics of Salmonella

Most Serotypes TSI H2S (TSI) Citrate Lysine Ornithine Alk/A + + + + Typhi Alk/A + (weak) + Paratyphi A Alk/A +

Dulcitol
Rhamnose Indole

+
+

+
+

Methyl red
VP

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IMViC Reactions
I = Indole production from tryptophan M = methyl red test in which acidification of glucose broth (pH<4.4) due to formation of mixed carboxylic acids (lactic, acetic, formic) from pyruvate results in pH indicator methyl red turning red Vi = positive Voges-Proskauer test due to formation of acetoin from pyruvate in glucose broth C = ability to utilize citrate as single carbon source
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Indole Reaction
Enterobacteriaceae that possess tryptophanase can utilize tryptophan by deamination and hydrolytic removal of the indole side chain. Free indole is detected by p-dimethylaminobenzaldehyde, whose aldehyde group reacts with indole forming a red-colored complex. Production of indole from tryptophan is an important biochemical property of Escherichia coli, many strains of group A, B, and C Shigella, Edwardsiella tarda, Klebsiella Dr.T.V.Rao MD 14 oxytoca, and Proteus vulgaris.

Indole Test
How to Perform Test: Inoculate Tryptone broth with
inoculating loop.

Property it tests for: This test is performed to help

differentiate species of the family Enterobacteriaceae. It tests for the bacteria species ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one.

Media and Reagents Used: Tryptone broth contains

tryptophan. Kovacs reagentcontains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcoholyellow in color.

Reading Results: Kovacs reagent reacts with indole and

creates a red color at the top part of the test tube.
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Reading the Result

Indole

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Methyl Red/Voges Proskauer (MR/VP)

How to Perform Tests: Inoculate 2 glucose broths with
inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube.

Properties they test for: Both tests are used to help

differentiate species of the family Enterobacteriaceae.
MRtests for acid end products from glucose fermentation.

VPtests for acetoin production from glucose fermentation.

Media and Reagents Used:

Glucose Broth Methyl Red indicator for acid Voges Proskauer reagentsA: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water. Dr.T.V.Rao MD
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Voges-Proskauer Reaction
Acetoin and butylene glycol are detected by oxidation to diacteyl at an alkaline pH, and the addition of naphthol which forms a red-colored complex with diacetyl.

The production of acetoin and butylene glycol by glucose fermentation is an important biochemical property used for the identification of Klebsiella, Enterobacter, and Serratia.
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MR/VP continued
Reading Results:
MR a + result is red (indicating pH below 6) and a result is yellow (indicating no acid production)

VPA + result is red after VP reagents are added (indicating the presence of acetoin) and a result is no color change.

Dr.T.V.Rao Methyl Red: left and right + MD

VP: left + and right

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Citrate Utilization
Citrate is utilized by several of the Enterobacteriaceae as a single carbon source. To test this ability bacteria are incubated in medium that contains only citrate as a source of carbon. Ammonium phosphate is available as a nitrogen source.
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Citrate Test
How to Perform Test: Inoculate slant with inoculating
loop.

Property it tests for: This test is used to help

differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source.

Media and Reagents Used: Simmons Citrate Agar

contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicatorbromthymol blue.
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Citrate Test Reading

Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a result remains green
Left positive and right negative.
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IMViC Reactions
I Escherichia coli Edwardsiella tarda Proteus vulgaris + + + M + + + Vi + C +

Klebsiella pneumoniae

Klebsiella oxytoca
Enterobacter spp. Serratia marcescens

+
+ +

+
+ +

Citrobacter freundii
Citrobacter koseri

+
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+
+
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Urease-Producing Enterobacteriaceae
Proteus Morganella

Providencia rettgeri
Klebsiella pneumoniae

Klebsiella oxytoca
Enterobacter cloacae

Yersinia enterocolitica
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Urea Hydrolysis
How to Perform Test: Inoculate Urea broth
with inoculating loop.

Property it tests for: This test is done to

determine a bacterias ability to hydrolyze urea to make ammonia using the enzyme urease. contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator.
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Media and Reagents Used: Urea broth

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Urease Test
Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.
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Reactions for Identification of Genera and Species1

Decarboxylation of amino acids Motility

Urease activity
Hydrogen sulfide (H2S) production
1Voges-Proskauer,

phenylalanine

deaminase, indole, and citrate reactions are useful to both cluster Enterobacteriaceae

and identify to genus and species.

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Amino Acid Decarboxylation

Enterobacteriaceae contain decarboxylases with substrate specificity for amino acids, and are detected using Moeller decarboxylase broth overlayed with mineral oil for anaerobiosis. Moeller broth contains glucose for fermentation, peptone and beef extract, an amino acid, pyridoxal, and the pH indicator bromcresol purple.
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Amino Acid Decarboxylation

If an Enterobacteriaceae contains amino acid decarboxylase, amines produced by decarboxylase action cause an alkaline pH, and bromcresol purple turns purple.

Lysine, ornithine, and arginine are utilized. A base broth without amino acid is included in which glucose fermentation acidifies the broth, turning the bromcresol purple yellow.
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Amino Acid

1 Decarboxylation

Lysine Cadaverine

Ornithine Putrescine
Arginine Citrulline Ornithine Putrescine
1Conversion

of arginine to citrulline is a dihydrolase reaction

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Amino Acid Decarboxylation

Tube Amino Acid Color Interpretation Base None Yellow Broth acidified1

1
2 3

Lysine
Ornithine Arginine

Purple Positive
Yellow Negative Yellow Negative

1Indicates

organism is a viable glucose fermenter, and pH of broth medium sufficiently acidified to activate decarboxylase enzymes.
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Amino Acid Decarboxylation

Decarboxylation patterns are essential for the genus identification of Klebsiella, Enterobacter, Escherichia, and Salmonella. Decarboxylation patterns are also essential for the species identification of Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, and Shigella sonnei.
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Amino Acid Decarboxylation

Lys Klebsiella + Orn Arg

Enterobacter +/
Escherichia Salmonella + +

+
+/ +

+/
/+ +

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Amino Acid Decarboxylation

Lys E. aerogenes + Orn + Arg

E. cloacae
P. Mirabilis P. vulgaris Shigella D

+
+ +
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+
_
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Shigella A-C

H2S-Producing Enterobacteriaceae Salmonella Edwardsiella Citrobacter Proteus

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Hydrogen Sulfide (H2S)

In presence of H+ and a sulfur source (sodium thiosulfate, sulfur-containing amino acids and proteins) many Enterobacteriaceae produce the colorless gas H2S.

For detection of H2S a heavy-metal (iron or lead) compound is present that reacts with H2S to form black-colored ferrous sulfide.
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Systems for H2S Detection1

Lead acetate paper
SIM tube (peptonized iron) Hektoen and SS2 agar (ferric ammonium citrate) XLD3 agar (ferric ammonium citrate)

Triple-sugar-iron agar (ferrous sulfate)

1In

order of decreasing sensitivity

2Salmonella-Shigella

3Xylose-lysine-deoxycholate
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Bacterial Motility
Many but not all Enterobacteriaceae demonstrate flagellar motility.

Motility can be measured by use of <0.4% semisolid (soft) agar or microscopic examination of drops of broth containing bacteria and hanging from cover slips.
Shigella and Klebsiella are non-motile, and Yersinia is non-motile at 35oC but motile at 22o-25oC.
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Motility Agars
Sulfide-indole-motility (SIM) is a semisolid motility agar that contains peptonized iron for detection of H2S and tryptophan for indole production. Pure motility agar lacks an H2S indicator and tryptophan for indole production, and contains tetrazolium salts that are reduced to red formazan complexes to enhance visual assessment of motility.
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Additional Biochemical Reactions 1 for the Enterobacteriaceae

Fermentation of mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, arabinose, raffinose, rhamnose, maltose, xylose, trehalose, cellobiose, alphamethyl D-glucoside, erythritol, melibiose, arabitol, glycerol, mucate, and mannose Utilization of malonate, acetate, and tartrate Gelatin hydrolysis, esculin hydrolysis, lipase, and DNase Growth in KCN Yellow pigment
1JJ

Farmer, Enterobacteriaceae: Introduction and Identification, ASM Manual, 8th Edition (2003).
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Programme Created for Medical and Paramedical students in Microbiology Email

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