Biochemical Tests

Enterobacteriaceae
Dr.T.V.Rao MD

Dr.T.V.Rao MD 1
 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
Dr.T.V.Rao MD 2
 Initial Grouping of the Enterobacteriaceae
(VP=Voges Proskauer,
PDA=Phenylalanine Deaminase)
GENERA VP PDA
Klebsiella POSITIVE NEGATIVE
Enterobacter POSITIVE NEGATIVE
Serratia POSITIVE NEGATIVE
Hafnia POSITIVE NEGATIVE
Pantoea POSITIVE NEGATIVE
Dr.T.V.Rao MD 3
 Initial Grouping of the
Enterobacteriaceae
GENERA VP PDA

Proteus1 NEGATIVE POSITIVE

Morganella NEGATIVE POSITIVE

Providencia NEGATIVE POSITIVE

1
Proteus mirabilis: 50% of strains VP positive
Dr.T.V.Rao MD 4
 Initial Grouping of the
Enterobacteriaceae
GENERA VP PDA
Escherichia NEGATIVE NEGATIVE
Shigella NEGATIVE NEGATIVE
Edwardsiella NEGATIVE NEGATIVE
Salmonella NEGATIVE NEGATIVE
Citrobacter NEGATIVE NEGATIVE
Yersinia NEGATIVE NEGATIVE
Dr.T.V.Rao MD 5
 Initial Grouping of the
Enterobacteriaceae1
GENERA INDOLE CITRATE
Escherichia POSITIVE NEGATIVE
2
Shigella POSITIVE NEGATIVE
3
Yersinia POSITIVE NEGATIVE
Edwardsiella POSTIVE NEGATIVE
1
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 6
3
Yersinia enterocolitica 50% positive
 Initial Grouping of the
Enterobacteriaceae 1
GENERA INDOLE CITRATE
Salmonella NEGATIVE POSITIVE2
Citrobacter NEGATIVE POSITIVE

1
VP negative, PDA negative
2
Salmonella serotype Paratyphi A and Typhi
negative

Dr.T.V.Rao MD 7
 Key Characteristics of the
Enterobacteriaceae
TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

/
E
coli
A/A
+ +   +    + + +/
 +
/
    +       
Shi Ak/
A- A
C

         + 
Shi Ak/
D A +
     + + + 
Ed Ak/
A + + +
    
Sal Ak/ +/
A + + + + + + 
/
    +  + 
Cit A/A +/ +/
Ak/ + + + +
A
Yer A/A
+    +/
  
Dr.T.V.Rao MD
+/

RT
(1)  +  8
(1) RT=room temperature
 Key Characteristics of the
Enterobacteriaceae
TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

     
Kle A/A
pne + + + + + +
    
Kle A/A
oxy + + + + + + +
    
En A/A
aer + + + + + + +
   
En A/A +/
cloa + + + + + + +
    
Serr A/A
(1) + + + + + + +
     
Haf Ak/
A + + + + + +
Pan A/A
+ /+  +/ /+ +/ /+ /+    
Alk/
A
(1) Produces DNase, lipase, and gelatinase

Dr.T.V.Rao MD 9
 Key Characteristics of the
Enterobacteriaceae
TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR

 + +  + + +s  + 
Prot Ak/ +/ +/
mir A
a

 +/ +  + /+ + + +s   
Prot A/A
vulg

 +   +  + + +  +
Mor Ak/
A

    + + + + +   
Pro Ak/
v A
s = swarming motility

Dr.T.V.Rao MD 10
Biochemical Characteristics of
Escherichia coli and Shigella
E. coli E. coli O157:H7 Shigella
TSI A/Ag A/Ag Alk/A
Lactose + + –
ONPG + + –/+1
Sorbitol + – +/–
Indole + + +/–
Methyl re + + +
VP – – –
Citrate – – –
Lysine + + –
Motility + +
Dr.T.V.Rao MD
– 11
Biochemical Characteristics of
Salmonella
Most Serotypes Typhi Paratyphi A
TSI Alk/A Alk/A Alk/A
H2S (TSI) + + (weak) –
Citrate + – –
Lysine + + –
Ornithine + – +
Dulcitol + – +
Rhamnose + – +
Indole – – –
Methyl red + + +
VP – – –
Dr.T.V.Rao MD 12
 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
Dr.T.V.Rao MD 13
 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-dimethylamino-
benzaldehyde, 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
oxytoca, and Proteus vulgaris.
Dr.T.V.Rao MD 14
 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. Kovac’s reagent—contains hydrochloric
acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in
color.
Reading Results: Kovac’sDr.T.V.Rao
reagent MD
reacts with indole and 15
creates a red color at the top part of the test tube.
Reading the Result
Indole

Dr.T.V.Rao MD 16
 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.
MR—tests for acid end products from glucose fermentation.
VP—tests for acetoin production from glucose fermentation.
Media and Reagents Used:
Glucose Broth
Methyl Red indicator for acid
Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B:
Potassium Hydroxide, & Deionized
Dr.T.V.RaoWater.
MD 17
 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.
Dr.T.V.Rao MD 18
 MR/VP continued
Reading Results:
MR— a + result is red (indicating pH below 6) and a – result is yellow
(indicating no acid production)
VP—A + result is red after VP reagents are added (indicating the
presence of acetoin) and a – result is no color change.

Methyl Red: left – and right + MD

Dr.T.V.Rao VP: left + and right – 19
 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.
Dr.T.V.Rao MD 20
 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: Simmon’s Citrate Agar
contains sodium citrate (carbon source), ammonium
ion (nitrogen source), & pH indicator—bromthymol
blue.
Dr.T.V.Rao MD 21
 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.
Dr.T.V.Rao MD 22
 IMViC Reactions
I M Vi C
Escherichia coli + + – –
Edwardsiella tarda + + – –
Proteus vulgaris + + – –
Klebsiella pneumoniae – – + +
Klebsiella oxytoca + – + +
Enterobacter spp. – – + +
Serratia marcescens – – + +
Citrobacter freundii – + – +
Citrobacter koseri + +
Dr.T.V.Rao MD
– + 23
 Urease-Producing
Enterobacteriaceae
Proteus
Morganella
Providencia rettgeri
Klebsiella pneumoniae
Klebsiella oxytoca
Enterobacter cloacae
Yersinia enterocolitica
Dr.T.V.Rao MD 24
 Urea Hydrolysis
How to Perform Test: Inoculate Urea broth
with inoculating loop.
Property it tests for: This test is done to
determine a bacteria’s ability to hydrolyze urea
to make ammonia using the enzyme urease.
Media and Reagents Used: Urea broth
contains a yeast extract, monopotassium
phosphate, disodium phosphate, urea, and
phenol red indicator.Dr.T.V.Rao MD 25
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.
Dr.T.V.Rao MD 26
 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.
Dr.T.V.Rao MD 27
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.
Dr.T.V.Rao MD 28
 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.
Dr.T.V.Rao MD 29
 Amino Acid Decarboxylation 1

Lysine → Cadaverine

Ornithine → Putrescine

Arginine → Citrulline → Ornithine →

Putrescine
1Conversionof arginine to citrulline is a
dihydrolase reaction

Dr.T.V.Rao MD 30
 Amino Acid Decarboxylation
Tube Amino Acid Color Interpretation
Base None Yellow Broth acidified1
1 Lysine Purple Positive
2 Ornithine Yellow Negative
3 Arginine Yellow Negative
1Indicates organism is a viable glucose
fermenter, and pH of broth medium
sufficiently acidified to activate decarboxylase
enzymes.
Dr.T.V.Rao MD 31
 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.
Dr.T.V.Rao MD 32
 Amino Acid Decarboxylation
Lys Orn Arg
Klebsiella + – –
Enterobacter +/– + +/–

Escherichia + +/– –/+

Salmonella + + +

Dr.T.V.Rao MD 33
 Amino Acid
Decarboxylation
Lys Orn Arg
E. aerogenes + + –
E. cloacae – + +
P. Mirabilis – + –
P. vulgaris – – –
Shigella D – + –
Shigella A-C – – _

Dr.T.V.Rao MD 34
 H2S-Producing
Enterobacteriaceae
Salmonella
Edwardsiella
Citrobacter
Proteus

Dr.T.V.Rao MD 35
 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.
Dr.T.V.Rao MD 36
 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
Dr.T.V.Rao MD 37
 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.
Dr.T.V.Rao MD 38
 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.
Dr.T.V.Rao MD 39
Additional Biochemical Reactions
for the Enterobacteriaceae1

Fermentation of mannitol, dulcitol, salicin, adonitol,

inositol, sorbitol, arabinose, raffinose, rhamnose,
maltose, xylose, trehalose, cellobiose, alpha-
methyl –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).
Dr.T.V.Rao MD 40
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