PRACTICAL

Identification of
Gram-Negative
bacterium
Bambang Isbandrio

Sasaran Belajar
Mahasiswa dapat menjelaskan tentang:
strategi identifikasi bakteri Gram-negatif
jenis-jenis tes untuk memastikan spesies
bakteri Gram-negatif penyebab infeksi
menginterpretasikan hasil pemeriksaan
sensitivitas AB Gram-negatif

Diagnosis
infeksi

First day preliminary

findings
Growth
characteristics
Gram stain
Catalase
Oxidase
Motility

Catalase

Presence of catalase enzyme which breaks

down peroxide radicals
Place a few drops of hydrogen peroxide solution
on a loopful of your isolate on a glass slide and
watch for the appearance of bubbles- they may
be very small
Bubbles = positive/ no bubbles=negative

Oxidase test

Motility test

Presumptive follow up
Gram Negative Rods
Oxidase negative
Catalase positive
Motile/ Nonmotile
One of the members of
enterobacteriaceae

Oxidase Positive
Catalase Positive
Motile
Vibrio or
Pseudomonas etc

Members of
enterobacteriaceae:

oxidase Negative

Escherichia
Klebsiella
Citrobacter
Salmonella
Shigella
Proteus
etc

Nonterobacteriaceae
Oxidase positive :

Vibrios
Pseudomona
s

SELECTIVE MEDIA
ENRICHMENT
BROTHS
Rappaport

SS

Hektoen

Mac
Conkey

Selenite
mannitol
salt

STOOLS

Sabouraud
Gentachloram

Campylosel

Yersinia
Cin
Candida
ID

SM ID

CHROMOGENIC MEDIA

Clostridium
difficile

Isolation & Culture: Colony

Size
Shape
Color
Surface features

Smooth - Rough

Transparency
Hemolysis
Smell

A gram negative rod

on BA/Macconkey
agars

Non hemolytic, moist, irregular,

flat, centrally raised, 3-5 mm
colonies,

Lactose fermenting,
Flat,irregular centrally raised,
moist 2-4 mm colonies

Enterocc + Candida
Haemophilus + Gardnerella
Sta. aureus + Strep. G
E. coli + Ps. aeruginosa

MacConkey (MAC) Agar

Peptone
1.7%
Polypeptone 0.3%
Lactose1
1.0%
Bile salts2
0.15%
Crystal violet2
Neutral red3
Sodium chloride
Agar
1.35%
pH=7.1

0.5%

Differential medium for lactose fermentation

Inhibit gram positives and fastidious gram-negatives; MAC agar

selective for
gram-negatives

Red color at pH < 6.8

Members of
enterobacteriaceae:

oxidase
LF
NLF
Negative(example)
Escherichia
Salmonella
Enterobacter
Klebsiella

Shigella
Proteus

Alpha hemolysis

Incomplete hemolysis
Greenish darkening of
the agar under the
colonies
Displayed by
Streptococcus
pneumoniae
Caused by peroxide
produced by the
bacteria
http://gold.aecom.yu.edu/id/micro/hemolysis.htm

Beta hemolysis

Complete
hemolysis, giving
a clear zone with
a clean edge
around the colony.
Streptococcus
pyogenes
Caused by the
presence of
hemolysine
http://gold.aecom.yu.edu/id/micro/hemolysis.htm

Gamma hemolysis

No hemolysis
No change in the
blood agar around
the colony
Ex: enteroccocus
faecalis

http://gold.aecom.yu.edu/id/micro/hemolysis.htm

Swarming appearance of
Proteus

24

Tests to read on
second day

Check whether the

culture is pure
Repeat catalase,
oxidase,gram and
motility to confirm
previous days findings

Biochemical Reactions

Sugar Fermentation

H2S Test

Citrate utilization

Identification

Triple sugar iron agar

0.1% glucose
1% lactose
1% sucrose
Ferrous sulfate
pH indicator: phenol red

Triple Sugar
Iron Agar

TSI Reactions of the

Enterobacteriaceae

A/A + g = acid/acid plus gas (CO 2)

A/A = acid/acid
A/A + g, H2S = acid/acid plus gas, H2S

Alk/A = alkaline/acid
Alk/A + g = alkaline/acid plus gas
Alk/A + g, H2S = alkaline/acid plus gas,
H 2S
Alk/A + g, H2S (w) = alkaline/acid plus
gas, H2S (weak)

A/A + g

Escherichia coli
Klebsiella pneumoniae
Klebsiella oxytoca
Enterobacter aerogenes
Enterobacter cloacae

K/A
Serratia marcescens1, 2
Yersinia enterocolitica2
145% of strains
2Non-lactose, sucrose fermenter

K/A + g, H2S
Citrobacter freundii
Proteus vulgaris1
1Non-lactose, sucrose fermenter

Alk/A

Shigella
Providencia

Alk/A + g

Salmonella serotype Paratyphi

A

Alk/A + g, H2S

Salmonella (most serotypes)

Proteus mirabilis
Edwardsiella tarda

Alk/A + g, H2S (w)

Salmonella serotype Typhi

Biochemical
tests

TSI: A/A, Gas +, H2S Negative

SIM: H2S,Neg, Indole: Positive.,
Motile
ItMotility;
could be(PW) Motile
ESCHERICHIA, CITROBACTER,OR
ENTEROBACTER
Glucose
and Gas)

Macconkey: LF

Lactose

: Positive

Sucrose

: Negative / Positive

Mannitol

Indole
Gram: Gram Negative
rods
MR
LAO : +-+
VP
Catalase: Positive
Melonate: Citrate
Oxidase ; Negative
KCN ; _

Urease

One of the members

of

: Positive (Acid

: Positive
: Positive
: Positive
: Positive
: Negative
: Negative

So it is Escherichia coli

API System

Escherichia coli

Pseudomonas aeruginosa

Haemophilus influenzae

API System
Actinomyces pyogenes

Staphylococcus xylosus

Listeria monocytogenes

Pseudomonas and related organisms

Aerobic gram-negative nonfermentative rods
Pseudomonas aeruginosa: opportunistic infections of
multiple sites
Burkholderia cepacia: RT infection in cystic fibrosis
patients, UTI, opportunistic infections
Burkholderia pseudomallei: opportunistic pulmonary
infections or sepsis
Stenotrophomonas maltophilia: opportunistic infections
Acinetobacter baumannii: opportunistic infections of RT
Moraxella catarrhalis: opportunistic RT infections

Pseudomonas

Structure and
Physiology
Gram-negative rods.
Motile with polar flagella.
Obligate aerobe.
Oxidase-positive.
Do not ferment carbohydrates.
Resistant to multiple drugs.

P. aeruginosa
Forms round colonies with a
fluorescent greenish color, sweet
odor, and -hemolysis.
Pyocyanin- nonfluorescent
bluish pigment;
pyoverdin- fluorescent
greenish pigment;
pyorubin, and pyomelanin
Some strains have a prominent
capsule (alginate).
Identification of P. aeruginosa is usually based on oxidase test
and its colonial morphology: -hemolysis, the presence of
characteristic pigments and sweet odor, and growth at 42 oC.