Biochemical tests

1. Biochemical Tests
Enterobacteriaceae
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1

2. 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

3. 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

4. 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

5. 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

6. Initial Grouping of the
Enterobacteriaceae1
GENERA INDOLE CITRATE
Escherichia POSITIVE NEGATIVE
Shigella
Yersinia
POSITIVE2
POSITIVE3
NEGATIVE
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.
3
Yersinia enterocolitica 50% positive
Dr.T.V.Rao MD 6

7. Initial Grouping of the
Enterobacteriaceae1
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

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

9. Key Characteristics of the
Enterobacteriaceae
TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR
Kle
pne
A/A
+ +  +  +  +  +  
Kle
oxy
A/A
+ +  + + +  +  +  
En
aer
A/A
+ +  +  +   + + + 
En
cloa
A/A
+ +  +  +  +/ +  + +
Serr
(1)
A/A
+ +  +  +   + + + 
Haf Ak/
A + +  +     + + + 
Pan A/A
Alk/
A
+ /+  +/ /+ +/ /+ /+    
(1) Produces DNase, lipase, and gelatinase
Dr.T.V.Rao MD 9

10. Key Characteristics of the
Enterobacteriaceae
TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR
Prot
mir
a
Ak/
A  + + +/  +/ + + +s  + 
Prot
vulg
A/A
 +/ +  + /+ + + +s   
Mor Ak/
A  +   +  + + +  +
Pro
v
Ak/
A     + + + + +   
s = swarming motility
Dr.T.V.Rao MD 10

11. 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

12. 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

13. 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

14. 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

15. 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’s reagent reacts with indole and
creates a red color at the top part of the test tube.
Dr.T.V.Rao MD 15

16. Reading the Result
Indole
Dr.T.V.Rao MD 16

17. 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 Water.
Dr.T.V.Rao MD 17

18. 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

19. 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 + VP: left + and right –
Dr.T.V.Rao MD 19

20. 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

21. 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

22. 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

23. 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

24. Urease-Producing
Enterobacteriaceae
Proteus
Morganella
Providencia rettgeri
Klebsiella pneumoniae
Klebsiella oxytoca
Enterobacter cloacae
Yersinia enterocolitica
Dr.T.V.Rao MD 24

25. 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

26. 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

27. 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

28. 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

29. 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

30. Amino Acid Decarboxylation1
Lysine → Cadaverine
Ornithine → Putrescine
Arginine → Citrulline → Ornithine →
Putrescine
1Conversion of arginine to citrulline is a
dihydrolase reaction
Dr.T.V.Rao MD 30

31. 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

32. 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

33. Amino Acid Decarboxylation
Lys Orn Arg
Klebsiella + – –
Enterobacter +/– + +/–
Escherichia + +/– –/+
Salmonella + + +
Dr.T.V.Rao MD 33

34. 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

35. H2S-Producing
Enterobacteriaceae
Salmonella
Edwardsiella
Citrobacter
Proteus
Dr.T.V.Rao MD 35

36. 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

37. 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

38. 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

39. 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

40. 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

41. Programme Created for Medical
and Paramedical students in
Microbiology
Email
doctortvrao@gmail.com
Dr.T.V.Rao MD 41