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

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)

(-) (+) (-) (+)

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

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 -