MICROBIOLOGY DIAGNOSTIC OF
MICROORGANISMS RELATED TO
CARDIAC INFECTIONS
Microbiology Department
BLOOD CULTURE
Purpose
To become familiar with :
• The microorganisms most frequently
associated with bacteremia
• Laboratory methods for the isolation
and presumptive identification of the
etiological agent of bacteremia
Collection of specimen :
• Blood must be drawn aseptically
• At least three blood sample from three
different veni-puncture sites, separate
from the last at least 1 hour
• 10 ml blood should be collected from
adult patient
PERICARDIAL FLUID
Purpose
To become familiar with :
• Laboratory methods for the
isolation and identification of the
etiological agent of infective
pericarditis
Collection of specimens :
• Pericardial fluid must be collected
aseptically
• Should be injected immediately into :
– Sterile tube or bottle
– Anaerobic transport medium
• Sterile heparin may be added to the
fluid
• Coagulated material should be
emulsified
Microscopic examination :
• Gram stain smear of the
centrifuged sediment of clear
slightly cloudy fluid should be
examined
• Purulent material should be
smeared directly
Culture :
The specimens should be streak
into medium such as blood agar
plate, Mc Conkey, chocolate agar,
and Sabouraud agar plate, depend
on the result of microscopic
examination
Blood sample
3-to7-day trypticase soy
broth culture, unvented
3-to7-day trypticase soy
broth culture, vented
Observe for turbidity
Observe for turbidity
(-)
Gram stain
Bacilli
Brucella
medium
Brucella sp
Cocci
Bacilli
Blood agar (stab and
streak inoculation)
Hemolysis
Streptococcus sp
(for differentiation)
Gram stain
(-)
(+)
Diplococci
Oviod bodies
Cocci
MacConkey agar Chocolate agar Sabouraud agar Blood agar
and CO2
Enteric bacteria
Lactosa fermention
(-)
(+)
LactophenolOxidase test cotton-blue stain Hemolysis
(see Exp.36)
(see Exp.30)
Neisseria sp.
Staphylococcus sp
(for differentiation)
(+)
P.Aeruginosa
Salmonella sp.
Streptococcus sp.
(for differentiation)
E.coli
H2S production
(-)
(+)
P.aeruginosa
C.albicans
Staphylococcus sp
(for differentiation)
Salmonella sp.
Schema for the isolation and identification of the etiological agents of bacteremia
IDENTIFICATION OF HUMAN
STAPHYLOCOCCAL PATHOGENS
Purpose
To become familiar with :
• The medical significance of the
staphylococci
• Selected laboratory procedures
designed to differentiate among the
mayor staphylococcal species
Staphylococcus is :
•
•
•
•
•
Gram-positive cocci
Occur as irregular clusters
Non-spore-formers
Mesophilic bacteria
Resistant to drying
Staphylococcus
The three major species are :
• S. aureus
• S. saprophyticus
avirulent strain
• S. epidermidis
Infection associated with S. aureus :
• Skin infection
: boils, carbuncles,
acne, impetigo
• Internal organ : pneumonia, cystitis,
tissue infection osteomyelitis,
pyelonephritis,
enteritis, septicemia,
endocarditis
Infection associated with :
• S. epidermidis
: skin lesions,
endocarditis
• S. saprophyticus : urinary tract infection
S. Aureus metabolic end product :
• Coagulase :
– Bound coagulase (clumping factor)
– Free coagulase
• Leukocidin
• Haemolysins
• Enterotoxin
Non-toxic metabolites of S. aureus :
– DNase
– Lipase
– Gelatinase
– Staphylokinase
Tabel 1. Laboratory test for differentiation of
Staphylococcal sp.
Test
S. aureus
S.epidermidis
S.saprophyticus
MSA :
-Growth
-Fermentation
+
+
+
-
+
-
Coagulase
+
-
-
DNase
+
-
-
Hemolysis
beta
-
-
Novobiocin
test
S
S
R
Pigmentation
Golden
yellow
white
white
Coagulase test
DNase Test
Blood agar
Bacteremic Pattern
IDENTIFICATION OF HUMAN
STREPTOCOCCAL PATHOGENS
Purpose
To become familiar with :
• The medical significance of the streptococci
• Selected laboratory procedures designed to
differentiate streptococci on the basis of
their hemolytic activity and biochemical
patterns associated with the Lancefield
group classifications
Streptococcus is :
•
•
•
•
Gram-positive cocci in chains
Nutritionally fastidious
Pinpoint colonies on solid media
Requiring enriched media for growth
The streptococci are classified
base on :
• Their haemolytic activity
• The serologic classification of
Lancefield
Haemolytic activity :
• Alpha-haemolysis
• Beta-haemolysis
• Gamma-haemolysis
Haemolysis
Alpha-haemolysis streptococci :
• Incomplete form of haemolysis
• Produce a green zone around the
colony
• Streptococcus viridans are non
pathogenic opportunist
• May produce sub-acute endocarditis
• Streptococcus pneumoniae is the
causative agent of pneumonia
Beta-haemolysis streptococci :
• A complete destruction of red blood
cells
• Exhibit clear zone around the colony
• Producing beta-haemolysins
Gamma-haemolytic
Gamma-haemolytic streptococci :
• Absence of any haemolysis
• Most commonly avirulent
Lancefield group classification :
• Classified streptococci into 20
serogroups
• Designated A through V (emitting I and
J)
• Base on the presence of C-substance,
an antigenic group-specific hapten
• Implicates the members of group A, B,
C and D in human infectious
processes
Group A :
• Beta-haemolytic streptococci in this
group referred to as streptococcus
pyogenes
• Main etiological agents of tonsillitis,
bronchopneumonia, scarlet fever,
erysipelas and cellulitis
• Responsible for glomerulonephritis
and rheumatic fever
Group B :
• Beta-haemolytic streptococci
indigenous to the vaginal mucosa
• Responsible for puerperal fever,
neonatal meningitis and endocarditis
Group C :
• Beta-haemolytic streptococci
• Have been implicated in erysipelas,
puerperal fever, and throat infections
Group D :
• Exhibit alpha or gamma-haemolysis
• Includes enterococci such as
Enterococcus faecalis
• An etiological agent of urinary tract
infections
• The non-enterococci such as S. bovis
Extra-cellular metabolites of
streptococci :
•
•
•
•
•
•
Haemolysin (alpha and beta)
Leukocidins
Erythrogenic toxin
Hyaluronidase (spreading factor)
Streptokinase (a fibrinolysin)
Nucleases (ribonuclease and
deoxybonuclease)
Tabel 2. Laboratory Differentiation of Streptococci
Group
Organisms
A
S.ptogenes
B
S.agalactiae
C
S. equi
D
Enterococci
E.faecalis
Nonenterococci
S.Bovis
Hemolysis
beta
beta
beta
Alphagamma
Alphagamma
Bacitracin
test
+
-
-
-
-
CAMP test
-
+
-
-
-
Bile esculin
hydrolysis
-
-
-
+
+
6.5% NaCl
medium
-
-
-
Growth
-
Growth at
10o C
-
-
-
Growth
-
Growth at
45oC
-
-
-
Growth
Growth or -