Clinical Microbiology Laboratory

. 305

2555

TSI (A/A, Gas-,H2S+ or A/A, Gas+,H2S+)

LDA
+

INDOLE

LDC

Proteus vulgaris

ODC

Salmonella arizonae

Citrobacter freundii

Proteus mirabilis

Proteus penneri

..
TSI (K/A, Gas-,H2S+ or K/A, Gas+,H2S+)
INDOLE
+

LDC

LDC

Mannitol

LDA

P.vulgaris

C.freundii

Edwardsiella tarda
E.coli

Proteus mirabilis
Citrobacter freundii

Malonate
E.hoshinae

LDA

+
+

Citrate
+

Salmonella spp.
( group by antiserum)

GAS

S.pulloruma

S.typhia

a Salmonella antiserum group D

Motility

S.gallinaruma

TSI (K/A,Gas-,H2S- or K/A,Gas+,H2S-

LDA
+

Citrate
+

Urease

+
Indole

Morganella morganii

Mannitol

Gas

P.rettgeri P.stuartii

Motility

Indole
+

+
-

+
ODC

S.odorifera
biogroup 1

S.odorifera
biogroup 2

ODC
+

C.diversus

Yellow pigment Citrate

E.coli
+
+

E.fergusonii +

E.agglomerans

Gelatin

E.coli
+

Y.pseudotuberculosis
Salicin

Shigella or E.coli
by antiserum

E.hermannii Sorbitol
-

K.rhinoscleromatis Urease

Malonate
+

Citrate
LDC

LDC

P.stuartii +

P.alcalifaciens

LDC

Y.pestis (pinpoint colony)

K.ozanae Shigella or E.coli
by antiserum

Enterobacter spp VP Salmonella paratyphi A

Serratia spp.

Hafnia alvei

Salmonella spp

TSI
A/A,G-,H2Sor
A/A,G+,H2S-

Indole

TSI
A/A,G-,H2S+
or
A/A,G+,H2S+

Family Enterobacteriaceae
Motile
LDA
LDC
Citrate
Ureae
Organisms

+
-

+/+

+/-/+

-/+

+
+
+
+/-

+
+
+
+
-

+
-

+
+
+
+/+
-

+
+
+/+
+
-

+
+
-/+
+/+
+/-

Indole

Motile

LDA

LDC

Citrate

Ureae

+
-

+
+
+
+
+

+
+
+
-

-/+
+/+
+

+
+
+
-

E.coli
Enterobactre (gelatin -)
Serratia (gelatin -)
Hafnia alvei (rare)
Klebsiella pneumoniae
Klebsiella oxytoca
Klebsiella sp.
Citrobacter diversus
Kluvera ascorbata (rere)
Proteus penneri
Yersinia enterocolitica (Motile 25 C)

Organisms
Proteus vugalis
Proteus mirabilis (rare)
Proteus penneri
Citrobacter freundii
Salmonella arizonae (malonate +)

TSI
K/A,G-,H2Sor
K/A,G+,H2S-

Indole
+
+
-/+

Motile
+
+/-

LDA
-

LDC
+/-

Citrate
+
-

Ureae
+/-

+/-/+

+/+

-/+
-/+

+
+
+
-

+
+
+
+
+
+

+
+
+
-

+
+
-

+
+
-

+
+
-

Organisms
Citrobacter diversus
E.coli
Shigella gr.A,B,C (acetate -)
E.coli (acetate +)
Shigella sonnei
Klebsiella sp.
Enterobacter (gelatin -)
Serratia (gelatin +)
Providencia rettgeri
Providencia sp.
Morganella morganii
Hafnia alvei
Salmonella sp.
Salmonella paratyphi A

TSI
A/A,G-,H2S+
Or
A/A,G+,H2S+

Indole
+
+
-

Motile
+
+
+
+
+

LDA
+
+
-

LDC
+
+

Citrate
+
-/+
+/+

Ureae
+/+
+
-

Organisms
Citrobacter freundii
Edwardsiella tarda
Proteus vugalis (rare)
Proteus mirabilis
Salmonella sp. (malonate -)
Salmonella arizonae (malonate +)
Salmonella typhi

 Family Enterobacteriaceae

Organism
Escherichia coli
E.vulneris
E.fergusonii
E.hermanii
Shigella
Edwardsiella tarda
Salmonella sp
Salmonella typhi
Salmonella paratyphi A
Salmonella cholerasuis
Salmonella arizonae
Citrobacter freundii
C.diversus
C.amalonaticus
Klebsiella pneumoniae
K.ozaenae
K.rhinoschleromatis
K.oxytoca
Enterobacter cloacae
E.aerogenes
E.sakazakii
E.agglomerans
E.gergoviae
Hafnia alvei

Slant
A(K)
K(A)
K
K(A)
K
K
K
K
K
K
K(A)
K(A)
K(A)
K(A)
A
A(K)
A(K)
A
A
A
A
A(K)
A
K(A)

TSI
Butt
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A

SIM
Gas H2S Indole Motile
+
+
V
+
+
+
+
+
+
+
+
V
+
+
+
+
+
+
+
- +W
+
+
V
+
+
V
+
+
+
+
+
+
+
+
+
+
+
+
+
+
V
+
+
+
+
+
+
+
+
V
V
+
+
+
+
+
+

LIA
CT URE
LDA LDC
+
+
+
V
+
+
+
+
+
V
+
+
+
V
+
V
V
V
+
+
+
V
V
+
+
+
+
V
+
+
+
V
V
+
+
+
+
-

Organism
Serratia marcescens
S.liquefaciens
S.rubidaea
S.odorifera
Proteus vugalis
P.penneri
P.mirabilis
Morganella morganii
Providencia
alcalifaciens
P.rustigianii
P.stuartii
P.rettgeri
Cedecea davisae
C.neteri
C.lapagei
Ewingella americana
Kluyvera ascorbata
Tatumella ptyseos
Yersinia enterocolitica
Y.frederiksenii
Y.intermedia
Y.kristensenii
Y.pestis
Y.pseudotuberculosis

TSI
Slant Butt Ga
s
A
A
V
A
A
V
A
A
V
A
A
A
A
V
A
A
V
(K)A A
+
K
A
+
(K)A A
V
(K)A A
V
(K)A A
(K)A A
A
A
A(K)
A(K)
A
A
A
A
A
K
K
K

A
A
A
A
A
A
A
A
A
A
A
A

V
+
+
+
V
V
V
-

SIM
H2S Indole Motile

LIA
CT URE
LDA LDC

+
V
+
-

+
+
+
+
+
+

+
+
+
+
+
V
+
+
+
V
V
+

+
+
+
V
+
+
+
+

+
V
V
V
-

+
+
+
+
V
V
+
V
+
+

V
+
+
+
+
V
+

+
V
+
+
V
-

+
+
V
V
+
-

+
-

+
+
+
+
+
V
-

V
V
V
V
+

 Family Vibrionaceae
Organism
Aeromonas sp.
Plesiomonas shigelloides

Vibrio cholerae
V.mimicus
V.metschnikovii
V.hollisae
V.damsela
V.fluvualis
V.furnissii
V.alginolyticus
V.parahaemolyticus
V.vulnificus

Slant
A(K)
K
K(A)
A
A
K
K
A
A
A
K
A(K)

TSI
Butt
A
A
A
A
A
A
A
A
A
A
A
A

SIM
Gas H2S Indole Motile
V
V
+
+
V
+
+
+
+
V
V
+
V
V
V
+
V
V
V
+
+
+
+
+

LIA
CT URE
LDA LDC
V
V
+
+
+
+
+
V
V
V
+
+
+
+
V
+
-

 Aeromonas Plesiomonas
Oxidase
+
+
-

TSI
LIM
Slt But H2S Gas Lys Ind

+
- +
d +

Species
A.hydrophila
A.sobria
A.veronii
A.caviae
A.schubertii
A.trota
A.shigelloides

d
d

d
+
d

d
+
d

Mot

organism

+
+
d

Aeromonas
Plesiomonas
Enterobacteriaceae

Lys

Orn

Arg

VP

Ara

Man

Ino

Esc

Glu/Gas

0/129*

+
+
+
+
+
+

+
+

+
+
+
+
+
+

+
+
+
d
-

+
+
-

+
+
+
+
+
-

+
+
+
-

+
+
+
+
-

+= 90% positive; -=90% negative; d=10-89% positive.

*Vibriostatic agent : 2, 4-diamino-6, 7-diisopropyl pteridine.

 Aeromonas sp.
Oxidase-positive, Glucose-fermenting,
Gram negative rods, resistant to 0/129
Vibriostatic Agent (150g)
Esculin hydrolysisa
Indole
production

+ Gas from Glucose

+

A.schubertii

VogesProskauer

A.trota

Acid from
arabinose

A.caviae

Resistance to
Cephalothinc
(30 g)

Acid from
sucrose

(TSI)

+
A.hydrophila
+

A.jandaei
A.sobria

A.veronii

a : agar formulation only

b : Aerokey II can be modified to end here with an identification of A.sobria
c : Kirby-Bauer disk diffusion method only

A.hydrophila

 Vibrio sp.
Colonies Oxidase
TSI
LIM
or TCBS
Slant Butt H2S Gas LDC IND
+
A
A
- +
+
+
A
A
- +
+
+
A
A
- +
+
Yellow +
A
A
- d
+
A
A
d
+
A
A
+ d
+
K
A
+ +
Green +
K
A
+ +
+
K
A
+ +

Species
V.cholerae
V.alginolyticus
V.mimicus
V.parahaemolyticus
V.vulnificus
V.fluvialis
V.furnissii

NaCl
0% 1%
+
+
-

+
+
+
+
+
+
+

Arabinose

+
-

O rganism
MT
+
+
+
+
+
+
+
+

Lactose

Salicin

(+)

V.cholerae
V.alginolyticus
V.carchariae
V.cincinatiensis
V.fluvialis
V.furnissii
V.mimicus
V.parahaemolyticus
V.vulnificus

Gas from
Glucose

V.hollisae

+
d
+

1% Nacl
8% Nacl
10% Nacl
LDC+1% NaCl
ODC+1% NaCl
ADH+1% NaCl
VP+1% NaCl

+
+
+
d

+
+
+
+
+
+

+
+
+
-

+
+
+
-

+
+
d

+
d
d
-

+
d
d
d
+

+
+
+
-

+
d
+
+
-

+
+
d
-

+
d
+
+

+
-

+
d
d
d

+
+
+
+
-

Arabinose
Lactose
ONPG
Salicin
Cellobiose
Oxidase
Nitrate to nitrite
D-Mannitol
m-Inositol

+
+
+
+
S/R
S/R

+
+
+
R
S

+
d
d
+
+
+
R
S

+
d
+
+
+
R
S

d
+
+
d
S
.

+
+
+
+
+
+
+
R
.

d
d
+
d
S
S

+
+
+
S
S

+
+
+
+
R
S

(d)
d
+
+
+
+
d
S
S

+
+
S
.

+
+
+
d
.

d
d
+
d
d
+
+
+
R
R

+
d
+
+
+
S
S

0/129 : 10 g
150g

+ = 90% positive, - = 90% negative, d = 10-89% positive, ( ) = delayed reaction

Plesiomonas

V.damsela

Aeromonas

V.vulnificus

+
-

V.mimicus

+
-

V.metschnikovii

+
-

V.cincinatiensis

+
+
-

V.carchariae

+
-

V.furnissii

+
-

V.fluvialis

+
+

V.alginolyticus

Sucrose
Gas from glucose
0% NaCl

TEST

V.cholerae

V.parahaemolyticus

Vibrio sp.


1. Oxidase Test

Cytochrome C oxidase oxidize

1. Kovacs oxidase reagent (1% tetramethyl-p-phenylenediamine dihydrochloride)

2.

Nutrient agar Kovacs oxidase reagent

platinum loop

Cytochrome C oxidase 10
Vibrionaceae (vibrio, Aeromonas, Plesiomonas) Pseudomonas ()
genus Neisseria
Control
Positive
Pseudomonas aeruginosa
Negative
Escherichia coli

oxidase Aeromonas sp., Vibrio sp.
Stainless steel Nichrome inoculating loops wires false positive

2. Triple sugar iron agar (TSI agar)

TSI
TSI 3 0.1% glucose Lactose
Sucrose 1% slant glucose slant
lactose / sucrose
slant ( phenol red ) butt
24 glucose ( phenol red
) ( TSI K/A)
2
( TSI A/A)
ferment TSI N/N, K/N
A/N

TSI Sodium thiosulfate ferrous sulfate
H2S ferrous ion ferrous sulfide
Sodium thiosulfate

thiosulfate
H2S ferrous
reductase
ions
from bacteria

ferrous sulfide
(black ppt.)

0.1% glucose, 1% iactose, 1% sucrose

phenol red ( indicator)
peptone
sodium thiosulfate
ferrous ammonium sulfate
13x100 .
4 . 1210C 15 slant
butt 1 anaerobic
alkaline slant ferment glucose
()

 needle slant 370C

18-24 .
TSI
K/A
glucose lactose sucrose
A/A
glucose lactose / sucrose
A/AG,H2S
glucose lactose / sucrose gas
hydrogen sulfide
K/K, K/N, N/N glucose lactose sucrose
Control
Citrobacter freundii
=
A/AG,H2S
Providencia rettgeri
=
K/A
Pseudomonas aeruginosa =
K/K
3. Lysine Indole Motile Medium (LIM)
family Enterobacteriaceae
Vibrionaceae stab
35-370C 18-24

1. Motility test
0.4%

stab
stab
Control
Positive
Proteus vulgaris
Negative
Klebsiella pneumoniae

2. Indole test:
tryptophanase tryptophan indole

p-dimethylaminobenzaldehyde Kovac
tryptophan

tryptophanase
from bacteria

Indole

p-dimethyl
aminobenzaldehyde
in Kovacs reagent

Rosindol dye (red colour)

Kovacs reagent
Kovacs reagent
control
Positive
Proteus vulgaris
Negative
Klebsiella pneumoniae
3. Lysine decarboxylase test (LDC)
glucose glucose
indicator decarboxylase

indicator
Fermentation

Glucose

Bromcresol purple

acid

yellow

by bacteria
Lysine

Lysine

Bromcresol-purple

cadaverine
decarboxylase

Purple

Control
Positive
Negative

Enterobacter aerogenes
Enterobacter cloacae

4. Lysine deaminase test (LDA)

(1/4 )
(1/4 )
3 genera Proteus Morganella
LIM medium
Decarboxylase medium base (Difco)

peptone
yeast extract
dextrose
bromcresol purple

Providencia
9 g.
5 g.
3 g.
1 g.
0.2 g.

Tryptone
10
g.
Ferric ammonium citrate
0.5 g.
L-lysine monohydrochloride
10
g.
Agar
2.5 g.

1000 ml.
pH 6.8
13x100 . 3 .
1210C 15 deeped media ()
4. Citrate

citrate
metabolism indicator
enzyme

sodium citrate

from bacteria

CO2
Na+

Na++H2O
OH

Na2CO3
NaOH

alkaline
Bromthymol blue
blue colour

 (Simmon citrate medium)

Sodium citrate
Bromthymol blue ( indicator)
slant
Control
Positive
Negative

Enterobacter aerogenes
Escherichia coli

5. Voges-Proskauer Test (VP)

acetyl-methyl carbinol (acetoin)

glucose oxidise KOH Diacetyl
Diacetyl -naphthol (catalyst) arginine ( Peptone)
Creatine complex

1. -naphthol; 5% in absolute ethyl alcohol

2. 40% KOH
3. MR/VP broth
MR/VP broth (formulated by Clark and Lubs)
Polypeptone
7 g.
Glucose
5 g.
Dipotassium phosphate
5 g.
Distilled water to
1 ml.
Final pH = 6.9

24 inoculate MR/VP medium 0.3-0.5 .

incubate water bath 35oC 4 -naphthol 3 40% KOH
1 10-15


10-15
Enterobacter, Serratia, Klebsiella pneumoniae, Hafnia
E.coli

1 . Copper-like colour
false positive
6. urease

urease urea ammonium carbonate

indicator
Urea

Urease

(pH 6.8) from bacteria

2NH3

Phenol red

Dark pink

(pH 8.1)

Urea
Phenol red (indicator)
urea base 100 .

agar 15 900 . (Autoclave)

5oC sterile urea base
slant
rapid method urea
( agar) 10 . 0.5 .
2

1. Malonate, Acetate

2. lactose, sucrose, arabinose, salicin

pH simple carbohydrates

 10-12 (110oC)
25
7. Carbohydrate Fermentation Test

1. 20% carbohydrate broth (peptone 10 g; meat extract 3 g; NaCI 5 g,

1 )
2. Buffer-salt solution (pH 7.0) 1% phenol red indicator

carbohydrate 1 (0.04 .) ; buffer-salt solution 0.1 . heavy

bacterial cell suspension buffer-salt solution 35oC 4 .

phenol red indicator

carbohydrate buffer salt solution

phosphate buffer
8. Motile-Nitrate-Pyocyanin medium (MNP)
Nonfermentative gram-negative
bacteria motility medium MNP
3 stab
incubate 35-37oC 18-24 (22oC)
Pseudomonas fluorescens incubate

2.1 Motility test : LIM

2.2 Nitrate test : nitrate solution A B 5

Nitrate

Nitrate
reductase

Nitrite

NO,NO2

N2 (gas)

Sulfanilic
(sol.A)
naphthylamine (sol.B)
Red color
(Positive result)

Zinc dust
Red color
(negative result)

1-2 nitrate reduce nitrite

( nitrite ) unreduced nitrate
Zinc dust reduce nitrate nitrite
nitrate
2.3 Pyocyanin production test
: pyocyanin pigment
Pseudon, onas aeruginosa
: pyocyanin pigment
1. MNP: MNP medium
Nitrate medium9 g.
Agar..2 g.
Sodium chloride..5 g.
Mangnesium chloride1.4 g.
Potassium sulfate10 g.
Glycerol10 g.
..1000 g.
pH 7.2
13x100 . 3 .
121oC 15 15
2. nitrate reduction
Solution A
Sulfanilic acid.8 g.
Acetic acid (5N), 30%..1 litre
Solution B

-Naphthylamine.5 g.
Acetic acid (5N), 30%..1 litre

 Acetic acid 5N
: Glacial acetic acid 1 + 25

Nitrate reductiion test nitrate

medium motility Pyocyanin pigment
P.aeruginosa
9. Bile Solubility test

bile salt
Streptococcus pneumoniae -hemolytic streptococcus

1. 2% sodium deoxycholate, pH 7.0 ()

2. Blood agar plate
3.

1. Plate technique:
5% blood agar plate 18-24 . 2%
deoxycholate 1 incubate
35oC 30 S.pneumoniae
2. Direct Method:
(blood specimens)
2 1 (chain)
2% sodium deoxycholate 1
S.pneumoniae bile salt
Streptococci
* zone of optochin < 18 mm

10. 6.5% NaCl slant (J. Clin. Micro. 7;238, 1978)

Brain heart infusion agar..26 g.
Glucose.5 g.
Sodium chloride.30 g.
Bromcresol purple (1% ethanol).1 ml.
Proteose peptone..5 g.
.500 g.
pH = 7.0

13x100 . 3 .
slant

NaCl 6.5%
Bromcresol purple
6.5% NaCl (broth)
1. Phenol red broth base (Gibco)1.5 g.
Peptone10 g.
Sodium chloride5 g.
Phenol red.0.018 g.
2. Dextroxe.0.5 g.
3. Sodium chloride6.5 g.
4. Beef extract0.1 g.
5. Distilled water...100 g.
pH = 7.4

13x100
. 2 . 121oC 15 15

11. Esculin Hydrolysis

glycoside esculin esculetin

glucose group D Streptococci, Listera monocytogenes nonfermentative gram-negative rods

1. Esculin solution
Esculin (Sigma).5.0 g.
Ferric ammonium citrate.0.5 g.
NaCl8.1 g.
K2HPO4.0.4 g.
KH2PO4.0.1 g.
.1
2. 12x75 .

esculin solution 0.2 . 35oC

30

hydrolyse glycoside esculin esculetin ferric

ammonium citrate Group D Streptococci, Listeria monocytogenes
non-fermentative gram-negaive rod
Bile esculin agar ()

13x100 .
3 . 121oC 15 15

12. Catalase test

catalase enzyme H2O2 O2 H2O

3% Hydrogen peroxide
slide 3% H2O2 1

( Catalase
)
13. Plasma (For coagulase test)
slide coagulase test undiluted plasma
tube coagulase test Trypticase soy broth
1:4 ( clot 4-20 )
0.5 . 4oC

(Tube coagulase test)

1.
Enterococci gram negative rod P.aeruginosa citrate

2. 20 S.aureus
Staphylokinase clot
slide tube coagulase test

14. PR Glucose / PR Mannitol

1. Phenol red broth base (Difco)..1.6 g.
Proteose peptone No. 3.10 g.
Bacto-Beef extract1 g.
Sodium chioride.5 g.
Phenol red..0.025 g.
2. Agar..0.5 g.
3. Glucose Mannitol..1 g.
4. Distilled Wster..100 ml.

13x100
. 3 . 100oC 10 10
Deep medium

()
15. PPR Medium ( pigment streptococci group B)
Proteose Peptone No.3..3 g.
.5 g.
NaCl0.5 g.
Agar1 g.
..100 ml.

tube
13x100 . 2 . 1

loop needle
incubate 35oC 24





16. Gelatin liguefaction

Gelatinase enzuyme Gelatin

Nutrient gelatin

Stab Nutrient gelatin 37oC 24 .

17. Lecithinase test

Lecithinase hydrolyse lecithin end product diglyceride

phosphorylcholine

Egg yolk agar

Egg yolk agar 18-24 . 37oC

zone colonies
zone
18. Phenylalanine deaminase

35oC 18-24 . 10% ferric

chloride 4-5

19. Dnase test agar

1. Dnase test agar w/methyl green 42 gm. Dnase test agar 42 gm.
5% methyl green 1 ml.
2. 1,000 ml.
121oC 15 15 50oC

loop 1-2 .
5 . 35oC 18-24 .

clear zone

20. Carbohydrate broth (manitol, sorbitol, arabinose, raffinose)
1. Brain heart infusion 37
2. () 10
3. 1.6 Bromcresol purple in 95% ethanol 1 .
4. 1,000 .
13x100 . 3 .
10 10
:
:
21. Arginine dihydrolase
1. Moellers decarboxylase 10.5
2. L-arginine 10 ( DL-arginine 20 )
3. 1,000 .
pH 6.0-6.5 13x100 .
3 . 10 10 mineral oil
( 10 .)

 :
: (
arginine dihydrolase)
22. Rapid carbohydrate utilization test for identification of Neisseria sp.
1. Buffered balanced salt indicator solution (BSS)
KH2PO4
0.01
K2HPO4
0.04
KCl
0.8
Phenol red (1% aqueous solution)
0.4
Distilled water
100
pH=7 sterile
4oC

g.
g.
g.
ml.
ml.

2. 20% carbohydrate solution

glucose, maltose, sucrose, lactose fructose
sterile freezer

1. BSS carbohydrate solution

suspend BSS 0.3 ml. ( 2 loop)

2. 0.1 ml. BSS sterile pasteur

pipette 20% carbohydrate solution 1
3. sterile pasteur pipette cell suspension 1 1
4. incubate water bath (37oC)
5. 15-30 4
6. :


1. Collins C, Patricia ML, and Grange JM. Coollins and Lynes Microbiology Method, 7 th .
Great Britain: Butterworth-Hrinemann Ltd., 1995.
2. Howard BJ, Keiser JF, Smith TF, et al. Clinical and pathrogenic microbiology, 2nd . St.
Louis: The C.V mosby Company, 1994.
3. Koneman EW, Alen SD, Janda WM, et al. Color atlas and text book of diagnostic
microbiology, 4th . Philadelphia: J.B. Lippincott, 1992.
4. Hawkey PM and Lewis DA. Medical bacteriology: a practical approach. Oxford
University Press, 1989.
5. Robert BW and Elvyn GS. Diagnostic Microbiology, 2nd . The C.V. Mosby Company,
1966.
6. Gobat BP. Diagnostic Microbiology: Laboratory Manual. USA. Mosby, 1998.
7. , , .
Routine culture
MSRV medium, , , 19-23
..2535.
8. , ,
,
, , 2529.
9. . .
, : .2540.
10. , , .
.
, 2538.