PENJELASAN PRAKTIKUM

MIKROBIOLOGI BLOK KARDIOVASKULER

 STAPHYLOCOCCUS

Frequent Human Infection Rare Human Infection

S. aureus S. capitis
S. epidermidis S. caprae
S. hemolyticus S. saccharolyticus
S. lugdunensis S. warneri
S. saprophyticus S. pateuri
S. hominis
S. auricularis
 STAPHYLOCOCCUS SP
 Bulat dengan diameter
kira 1 µm
 Bersusun berkelompok
secara tidak beraturan
 Gram positif
 Non-motil
 Non spora
 Katalase +
 STAPHYLOCOCCUS
AUREUS
 Bakteri gram +
 Menghasilkan pigmen lipochrom
 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;
Slide coagulase test. Rapid method for
observe agglutination
identifying S. aureus. Saline control on
(+ve)
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 ‘coagulase-
reacting factor’ in
plasma  form a
complex
 Complex will react with
fibrinogen to form fibrin
(‘clot formation’)
MEDIUM MSA

Beef extract                                                     1.0 g
Peptone (Difco) or Polypeptone (BBL)        10.0 g
NaCl                                                              75.0 g
Mannitol                                                         10.0 g
Phenol red                                                       0.025 g
Agar                                                              15.0 g
Distilled water                                             1,000 ml 
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

use DNA as a source of carbon and energy for
growth. Use of DNA is accomplished by an
enzyme called DNase.
NOVOBIOCIN SENSITIVITY

 Useful for presumptively distinguishing

Staphylococcus saprophyticus from other CoNS
CHARACTERISTIC S. aureus S. epidermidis S. saprophyticus
Pigment Yellow to White White to pale
white gray

Hemolysis + ± -

Coagulase production Yes No No

Mannitol Yes No No
fermentation
Novobiocin sensitivity Sensitive Sensitive Resistance
 STREPTOCOCCUS
PHYLOGENETIC SPECIES LANCEFIELD GROUP TYPE OF
GROUP HAEMOLYSISA
Pyogenic group Str pyogenes A ß
Str. agalactiae B ß
Str. equisimilis C ß
Mitis group Str. pneumoniae O α
Str mitis O α
Str. oralis Not identified α
Str. sanguis H α
Str. gordonii H α
Anginosus group Str. anginosus G, F (and A) α
Str. intermedius α
Salivarius group Str. salivarius K None
Bovis group Str. bovis D Α or none
Mutans group Str. mutans Not designated None
Str. sabrinus Not designated None
 GRAM-STAIN
 Gram-positive
spherical or ovoid
cocci
 Diplococci to long
chains (in broth
cultures)
 old cultures or dead
bacteria may appear
gram-negative
STREPTOCOCCUS PYOGENES
 Gram +
 Berbentuk rantai yang
panjangnya bervariasi
 Ada yang berkapsul
hialuronik
 Bersifat fakultatif anaerob
 Tumbuh subur pada keadaan
mikroaerofilik, dengan
temperatur optimum 37°C
 Faktor Virulensi
Hemolisin
Protein M,F,dan G
Kapsul hialuronik
Substansi C dan antigen
membran sitoplasma
Eksotoksin
Spreading factor ( proteinase,
streptokinase, nuklease, dll)
 HEMOLYSIS

 ß-hemolysis – complete hemolyis of rbc

Hemolysin induced
Marker for pathogenicity

 α-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
ß-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.
aureus
 IDENTIFICATION OF NON-
BETA-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-BETA-
HEMOLYTIC STREPTOCOCCI
Streptococcus Optochin Bile Bile esculin
suscept soluble
S. pneumoniae + + -

S. bovis - - +

Other viridans group - - -a

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
 Blood agar
Optochin resistant

Bile Soluble - Tes Katalase

Mannitol Salt
Agar

DNAse Test
Bacitrasin
Blood Agar
Resistant

Tes Katalase
Novobiocin Resistant

Mannitol Salt Agar

Katalase Test
Koagulase Test
Bile Optochin
Soluble + Blood agar
Sensitive

Tes Katalase
 Blood Agar

Bacitrasin
Sensitive

Tes Katalase CAMP Test

 V

Blood agar Mannitol Salt Agar

Novobiocin Oxacilin
Sensitive DNAse Test Resistant
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????