PENJELASAN PRAKTIKUM

MIKROBIOLOGI BLOK KARDIOVASKULER

Kupang, 18 Februari 2016

Optochin
resistant

Bile Soluble -

Blood agar

Tes Katalase

Mannitol Salt
Agar

DNAse Test

Bacitrasin
Resistant

Blood Agar

Tes Katalase

ovobiocin Resistant

Mannitol Salt Agar

Katalase Test
Koagulase Test

Bile
Optochin
Soluble + Sensitive

Tes Katalase

Blood agar

Blood Agar

Bacitrasin
Sensitive

Tes Katalase

CAMP Test

V
Blood agar

Mannitol Salt
Agar

Novobiocin
Oxacilin
Sensitive DNAse Test
Resistant

STAPHYLOCOCCUS
Frequent Human Infection
S. aureus
S. epidermidis
S. hemolyticus
S. lugdunensis
S. saprophyticus

Rare Human Infection

S. capitis
S. caprae
S. saccharolyticus
S. warneri
S. pateuri
S. hominis
S. auricularis

STAPHYLOCOCCUS SP

Bulat dengan diameter

kira 1 m

Bersusun berkelompok
secara tidak beraturan

Gram positif

Non-motil

Non spora

Katalase +

STAPHYLOCOCCUS
AUREUS

Bakteri gram +

Menghasilkan pigmen lipochrom

S. aureustermasuk bakteriosmotoleran

Faktor Virulensi
Koagulase +
Lekosidin
Enterotoksin
Toksin Sindroma Syok Toksik-1
Hemolysin

Sudah banyak kasus MRSA

S. aureus colonies on sheep blood agar. Zone of beta-hemolysis

around the colonies

CATALASE TEST

Differentiate Staphylococci
from Streptococci and
Enterococci (catalase
negative)

Detect presence of cytochrome

oxidase : H2O2 O2 + H2O

Test procedure:

Slide catalase place growth

from colonies on glass slide;
add 3% hydrogen peroxide;
vigorous bubbling seen (+ve)

Tube catalase pick growth

from media (not blood agar),
immerse in 3% hydrogen
peroxide

COAGULASE TEST

To differentiate S. aureus from other

Staphylococcus sp

S. aureus have bound coagulase and free

coagulase

Bound coagulase (clumping factor) present

on surface of cell wall

free coagulase - excreted extracellularly

SLIDE COAGULASE TEST

Slide coagulase test

Detect bound
coagulase

React directly with

fibrinogen in plasma

Test procedure: Mix

growth suspension with
EDTA-rabbit plasma;
observe agglutination
(+ve)

Slide coagulase test. Rapid method for

identifying S. aureus. Saline control on
left

COAGULASE TEST

Some S. aureus do not produce bound

coagulase; some coagulase-negative staph
produce weak slide coagulase test (S.
delphini, S. schleiferi)

Must confirm with tube coagulase

Do not take growth from mannitol salt agar

high salt content cause autoagglutination

TUBE COAGULASE

Tube coagulase

Indirect detection of
extracellular coagulase

Excreted coagulase
detect coagulasereacting factor in
plasma form a
complex

Complex will react with

fibrinogen to form fibrin
(clot formation)

MEDIUM MSA

Beef
extract1.0 g

Peptone (Difco) orPolypeptone(BBL)10.0

g

NaCl
75.0 g

Mannitol
10.0 g

Phenol red
0.025 g

Agar

Mannitol salt agar - Mannitol fermentation indicated by

change in phenol red indicator to yellow

DNASE TEST

The purpose is to see if the microbe can

useDNAas a source of carbon and energy
for growth. Use ofDNAis accomplished by
an enzyme calledDNase.

NOVOBIOCIN SENSITIVITY

Useful for presumptively distinguishing

Staphylococcus saprophyticusfrom
other CoNS

CHARACTERISTIC

S. aureus

Pigment

Yellow to
white

White

White to pale
gray

Coagulase production

Yes

No

No

Mannitol
fermentation

Yes

No

No

Sensitive

Sensitive

Resistance

Hemolysis

Novobiocin sensitivity

S. epidermidis S. saprophyticus

STREPTOCOCCUS
PHYLOGENETIC
GROUP

SPECIES

LANCEFIELD GROUP

TYPE OF
HAEMOLYSISA

Pyogenic group

Str pyogenes
Str. agalactiae
Str. equisimilis

A
B
C

Mitis group

Str. pneumoniae
Str mitis
Str. oralis
Str. sanguis
Str. gordonii

O
O
Not identified
H
H

Anginosus group

Str. anginosus
Str. intermedius

G, F (and A)

Salivarius group

Str. salivarius

None

Bovis group

Str. bovis

or none

Mutans group

Str. mutans
Str. sabrinus

Not designated
Not designated

None
None

GRAM-STAIN

Gram-positive
spherical or ovoid
cocci

Diplococci to long
chains (in broth
cultures)

old cultures or dead

bacteria may
appear gramnegative

STREPTOCOCCUS
PYOGENES

Gram +

Berbentuk rantai
yang panjangnya
bervariasi

Ada yang berkapsul

hialuronik

Hemolisin

Protein M,F,dan G

Kapsul hialuronik

Substansi C dan
antigen membran
sitoplasma

Eksotoksin

Spreading factor
(proteinase,
streptokinase,
nuklease, dll)

Bersifat fakultatif
anaerob
Tumbuh subur pada
keadaan
mikroaerofilik,
dengan temperatur
optimum 37C

FAKTOR VIRULENSI

HEMOLYSIS

-hemolysis complete hemolyis of rbc

Hemolysin induced

Marker for pathogenicity

-hemolysis greenish discolouration;

partial hemolysis

Hydrogen peroxide induced oxidizes

haemoglobin to green methaemoglobin

HEMOLYSIN

Hemolyse red blood cells

Beta-hemolytic group A S. pyogenes

produce 2 types of hemolysin

Streptolysin S observed as complete lysis

around colonies on blood agar, Oksigen
stabil, non-imunogenik

Streptolysin O

inactivated by oxygen ( Oksigen-labil)

Imunogenik

Antibodies against streptolysin O (ASTO)

formed following infection

CULTURE
CHARACTERISTICS

Alpha-hemolysis greenish discoloration of blood agar

Beta-hemolysis clearing around colonies

BACITRACIN
SUSCEPTIBILITY

Presumptive
identification of Grp
A -hemolytic
streptococci
(susceptible to
bacitracin)

0.04U bacitracin
disc zone of
inhibition 10 mm or
larger (susceptible)

CAMP TEST
CHRISTIE ATKINS MUNCH-PETERSEN

CAMP TEST

CAMP test

CAMP factor
produced by
majority of S.
agalactiae

Act
synergistically
with beta
hemolysin
produced by S.

IDENTIFICATION OF NONBETA-HEMOLYTIC
STREPTOCOCCI
Optochin test

For identification of
S. pneumoniae.

Optochin disks
applied on
streaked surface of
blood agar

Zones >14 mm
with 6 mm disc
sensitive
(S. pneumoniae)

DIFFERENTIATION OF NON-BETAHEMOLYTIC STREPTOCOCCI

Streptococcus

Optochin
suscept

Bile
soluble

Bile esculin

S. pneumoniae

S. bovis

Other viridans group

-a

Occasional strains may produce weak reaction

BILE ESCULIN TEST

BILE SOLUBILITY TEST

Principle:

S. pneumoniae produce a self-lysing enzyme to inhibit the

growth

The presence of bile salt accelerate this process

Procedure:

Add ten parts (10 ml) of the broth culture of the organism to be
tested to one part (1 ml) of 2% Na deoxycholate (bile) into the test
tube

Negative control is made by adding saline instead of bile to the

culture

Incubate at 37oC for 15 min

Record the result after 15 min

BILE SOLUBILITY TEST

Results:

Positive test appears

as clearing in the
presence of bile while
negative test appears
as turbid

S. pneumoniae soluble
in bile whereas S.
viridans insoluble

e.g. S. epidermidis

bakteri aerob : adalah bakteri yang

menggunakan oksigen molekular sebagai
terminal aseptor elektron selama respirasi.

bakteri anaerob: adalah bakteri yang

tidak menggunakan oksigen molekular
sebagai terminal aseptor elektron. Bakteri
ini mendapat energi dengan melakukan
fermentasi
atau
dengan
melakukan
respirasi anaerobik.

bakteri fakultatif anaerob: adalah

golongan bakteri yang bisa mendapat
energi dengan respirasi atau fermentasi,
tergantung pada keadaan lingkungannya.

Berdasarkan Produksi
enzim
1. Oksidase: berdasar dihasilkannya atau tidak
oksidase, bakteri dibagi atas bakteri positif oksidase
dan negatif oksidase.

2. Hemolisin adalah enzim yang dapat memecahkan

eritrosit yang terdapat pada medium. Berdasar
hemolisis sel-sel darah medium disekitar koloninya,
maka bakteri di bagi atas: 1). Bakteri hemolisin ,
yang menyebabkan eritosit lisis tetapi membran sel
tidak terlepas, dan 2). Bakteri hemolisin , dimana
terjadi lisis sel eritrosit yang sempurna.

3. Koagulase yang menyebabkan penggumpalan

plasma bisa dihasilkan atau tidak dihasilkan oleh
bakteri. Berdasarkan hal tersebut bakteri dibagi atas:
1). Bakteri positif koagulase dan 2) bakteri
negatif koagulase.

ANY QUESTION????