Download - MICROBIOLOGY DIAGNOSTIC OF MICROORGANISMS RELATED TO CARDIAC INFECTIONS Microbiology Department
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MICROBIOLOGY DIAGNOSTIC OF MICROORGANISMS RELATED TO
CARDIAC INFECTIONS
Microbiology Department
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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
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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
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PERICARDIAL FLUID
Purpose
To become familiar with :• Laboratory methods for the
isolation and identification of the etiological agent of infective pericarditis
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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
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Microscopic examination :• Gram stain smear of the
centrifuged sediment of clear slightly cloudy fluid should be examined
• Purulent material should be smeared directly
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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
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Blood sample
Bacilli Cocci Bacilli Diplococci Oviod bodies Cocci
Schema for the isolation and identification of the etiological agents of bacteremia
Gram stain
Observe for turbidity
3-to7-day trypticase soy broth culture, unvented
Gram stain
Observe for turbidity
3-to7-day trypticase soy broth culture, vented
Brucella medium
Brucella sp
Blood agar (stab and streak inoculation)
Hemolysis
Streptococcus sp (for differentiation)
Staphylococcus sp (for
differentiation)
MacConkey agar
Enteric bacteria
Lactosa fermention (-) (+)
P.Aeruginosa Salmonella sp. E.coli
H2S production (-) (+)
P.aeruginosa Salmonella sp.
Chocolate agar and CO2
Oxidase test (see Exp.30)
Neisseria sp.
Sabouraud agar
Lactophenol- cotton-blue stain
(see Exp.36)
C.albicans
Blood agar
Hemolysis
Streptococcus sp. (for differentiation)
Staphylococcus sp (for differentiation)
(-) (+) (-) (+)
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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
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Staphylococcus is :• Gram-positive cocci• Occur as irregular clusters• Non-spore-formers• Mesophilic bacteria• Resistant to drying
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Staphylococcus
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The three major species are :• S. aureus
• S. saprophyticus
avirulent strain
• S. epidermidis
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Infection associated with S. aureus :• Skin infection : boils, carbuncles,
acne, impetigo• Internal organ: pneumonia, cystitis,
tissue infection osteomyelitis,
pyelonephritis,
enteritis, septicemia,
endocarditis
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Infection associated with :• S. epidermidis : skin lesions,
endocarditis• S. saprophyticus : urinary tract
infection
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S. Aureus metabolic end product :• Coagulase :
– Bound coagulase (clumping factor)– Free coagulase
• Leukocidin• Haemolysins• Enterotoxin
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Non-toxic metabolites of S. aureus :– DNase– Lipase– Gelatinase– Staphylokinase
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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
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Coagulase test
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DNase Test
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Blood agar
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Bacteremic Pattern
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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
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Streptococcus is :• Gram-positive cocci in chains• Nutritionally fastidious• Pinpoint colonies on solid media• Requiring enriched media for growth
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The streptococci are classified
base on :• Their haemolytic activity• The serologic classification of
Lancefield
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Haemolytic activity :• Alpha-haemolysis• Beta-haemolysis• Gamma-haemolysis
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Haemolysis
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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
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Beta-haemolysis streptococci :• A complete destruction of red blood
cells• Exhibit clear zone around the colony• Producing beta-haemolysins
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Gamma-haemolytic
Gamma-haemolytic streptococci :• Absence of any haemolysis• Most commonly avirulent
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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
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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
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Group B :• Beta-haemolytic streptococci
indigenous to the vaginal mucosa• Responsible for puerperal fever,
neonatal meningitis and endocarditis
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Group C :• Beta-haemolytic streptococci• Have been implicated in erysipelas,
puerperal fever, and throat infections
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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
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Extra-cellular metabolites of
streptococci :• Haemolysin (alpha and beta)• Leukocidins• Erythrogenic toxin• Hyaluronidase (spreading factor)• Streptokinase (a fibrinolysin)• Nucleases (ribonuclease and
deoxybonuclease)
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Tabel 2. Laboratory Differentiation of Streptococci
GroupOrganisms
AS.ptogenes
BS.agalactiae
CS. equi
DEnterococciE.faecalis
Non- enterococci
S.Bovis
Hemolysis beta beta beta Alpha-gamma
Alpha-gamma
Bacitracin test
+ - - - -
CAMP test - + - - -
Bile esculin hydrolysis
- - - + +
6.5% NaCl medium
- - - Growth -
Growth at 10o C
- - - Growth -
Growth at 45oC
- - - Growth Growth or -
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