Clinical Bacteriology Lesson 4: Catalase-Positive, Gram-Positive Cocci (PART 1)
Lecturer: Mr. John Kerwayne M. Villaluna, RMT
Date: August 19, 2023
GENERA TO BE CONSIDERED:
❖ Staphylococcus aureus (coagulase-positive)
● Methicillin-Resistant Staphylococcus aureus (MRSA)
○ Healthcare-Associated MRSA (HA-MRSA)
○ Community-Associated MRSA (CA-MRSA)
○ Livestock-Associated MRSA (LA-MRSA)
❖ Coagulase-negative staphylococci (most common)
● Staphylococcus epidermidis
● Staphylococcus saprophyticus
○ S. saprophyticus subspecies bovis
○ S. saprophyticus subspecies saprophyticus
● Staphylococcus haemolyticus
● Staphylococcus lugdunensis
● Staphylococcus schleiferi
○ S. schleiferi subspecies coagulans
○ S. schleiferi subspecies schleiferi
❖ Micrococcus spp.
❖ Rothia, Aerococcus, Alloiococcus spp.
OVERVIEW OF THE PRINCIPLES OF
BIOCHEMICAL TESTS FOR THE IDENTIFICATION
OF GRAM-POSITIVE COCCI:
GRAM - STAINING
● Named after Hans Christian Gram (1834)
● Differential stain
● Gram stain allows differentiation between gram-positive
and gram-negative bacteria on the basis of differential
staining with a crystal violet-iodine complex and a
safranin counterstain
● Results:
● Steps:
- heat fixation,
- crystal violet (primary stain),
- gram’s iodine (mordant),
- acetone alcohol (decolorizer),
- safranin (counterstain)
CATALASE TEST
Purpose:
○ it is used to detect the production of the
enzyme catalase;
○ it differentiates between Staphylococcus spp.
and MIcrococcus spp. (positive) and
Streptococcus spp. (negative)
● Reagent used: 3% hydrogen peroxide
○ If an organism can produce catalase, it will
produce bubbles of oxygen when 3% hydrogen
peroxide is added to it.
● Positive result: effervescence; resulting from the
production of oxygen gas from the enzymatic breakdown
of hydrogen peroxide indicates a positive catalase
result.
● Catalase-positive: Staphylococcus spp.
● Catalase-negative: Streptococcus spp.
SLIDE COAGULASE TEST
Purpose:
○ it is used to detect the production of bound
coagulase (aka clumping factor)
○ It differentiates between Staphylococcus
aureus (positive) and other staphylococci
(negative)
● Reagent used: citrated plasma (rabbit or human)
● Bound coagulase (or clumping factor) – bound to cell
wall of the bacterium and reacts directly with fibrinogen,
indicated by the formation of visible mass. It does not
require a coagulase-reacting factor (CRF).
● Facilitates in the conversion of fibrinogen to fibrin in the
immediate vicinity of the bacterium as means of self
protection fibrin capsule inhibits phagocytosis
● Result: if bound coagulase is present on the bacterial
cell wall, then the presence of plasma will cause the
bacterial cells to clump.
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 TUBE COAGULASE TEST
Purpose:
○ It is used to detect the production of free
MANNITOL SALT AGAR
coagulase
○ It is useful for differentiating Staphylococcus
aureus from other Gram-positive, Purpose:
catalase-positive cocci. ○ Mannitol salt agar is used as a selective media
○ It is also used to confirm negative slide for isolation of pathogenic staphylococci.
coagulase tests. ○ It is recommended to detect and enumerate
● Reagent used: citrated plasma (diluted 1:10 with NSS) coagulase-positive staphylococci in milk, food,
● It is an extracellular enzyme (released from the cell). It and other specimens
converts fibrinogen to fibrin by the coagulase-reacting ○ 7.5% sodium chloride inhibits the growth of
factor (CRF) activity in plasma. This is detected by the other bacteria.
appearance of a fibrin clot in the tube coagulase test. ● Formulation: 111 g/l of distilled water
● Result: a positive coag test (1-4 hrs of incubation) is rep ● Composition:
by any deg of clotting, from a loose clot suspended in ○ Proteose peptone (10g)
plasma to a solid clot. If neg, the plasma remains a ○ Beef extract (1g)
liquid. ○ Sodium chloride (75g)
○ D-Mannitol (10g)
○ Phenol red (0.025 g)
○ Agar (15g)
● Phenol red:
○ Yellow <pH 6.8
○ Red > pH 7.4
● Staphylococcus aureus (positive) (yellow)

Coagulase-positive Coagulase-negative
staphylococci (CoPS): staphylococci (CoNS):
● Staphylococcus ● Staphylococcus
aureus epidermidis
● S. pseudintermedius ● S. saprophyticus
● S. intermedius ● S. warneri
● S. schleiferi ● S. hominis
● S. delphini ● S. caprae
● S. hyicus
● S. lutrae

NUTRIENT AGAR
Purpose: 5% BLOOD AGAR
● Nutrient agar is a general-purpose medium that supports
the growth of a whole range of non-fastidious organisms
Purpose:
● It is ideally used to isolate, cultivate, and maintain
○ Blood agar is a general-purpose, enriched
non-fastidious organisms.
medium often used to grow fastidious
organisms.
● Formulation: 28 grams per liter of distilled H2O
○ It is also used to differentiate bacteria based on
● Composition:
their hemolytic properties (detecting hemolytic
○ Meat extract (1g)
enzymes).
○ Yeast extract (2g)
● Formulation: 40 g/L of distilled water + 50 ml of sheep
○ Peptone (5g)
or human blood (without anticoagulants)
○ Sodium chloride (5g)
● Composition:
○ Agar (15g)
○ Peptone (10g)
○ Tryptose (10g)
○ Sodium chloride (5g)
○ Agar (15g)
● Results:

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 ○ Beta-hemolysis (complete hemolysis)
○ Alpha-hemolysis (partial hemolysis)
○ Gamma-hemolysis (no hemolysis)

● A heat-stable staphylococcal enterotoxin causes the

CHARACTERISTICS OF STAPHYLOCOCCUS SPP. most common type of food poisoning.
● Gram-positive cocci ● Staphylococci rapidly develop resistance to many
● They produce the enzyme catalase antimicrobial agents
● Exhibit spherical shapes (0.5 to 1.5 μm) that may appear ○ Kirby-Bauer disk diffusion test (antimicrobial
singly, in pairs, and in clusters susceptibility test)
● The microscopic appearance is NOT specific or
characteristic for staphylococci
● They resemble some members of the family
Micrococcaceae, such as the genus Micrococcus
● They are non-motile
● They are non-spore-forming
● They may be aerobic or facultative anaerobic, except for
Staphylococcus saccharolyticus (obligate anaerobe)
● Colonies (after 18-24 hours of incubation at 37oC):
medium-sized (4-8 mm) and appear cream-colored,
white or rarely light gold, and buttery-looking
● Some are ß-hemolytic
● They are one of the most commonly isolated organisms

Table 14-1
ORGANISM HABITAT (Reservoir) Mode of
Transmission

Staphylococcus Normal flora: Endogenous strain:

aureus ● Anterior Sterile site by
nares traumatic
● Nasopharynx introduction
● Staphylococcus aureus is coagulase positive ● Perineal area (e.g.,surgical wound
● Staphylococci can be efficiently transmitted from ● Skin or microabrasions)
person-to-person ● Colonizer of Direct contact:
○ May become established as part of the mucosa person-to-
recipient’s normal flora person fomites
○ May be introduced to sterile sites by trauma or Indirect contact:
invasive medical procedures aerosolized
○ Infections are implicated in hospital, laboratory,
and community-acquired settings. Staphylococcus Normal flora: Endogenous strain:
● Some are members of the normal microbiota of the skin epidermidis ● Skin Sterile site by
and mucosal membranes of humans. ● Mucous implantation of
● Pathogenic staphylococci often hemolyze blood membranes medical devices
(hemolysin), coagulase plasma (coagulase), and (e.g., shunts,
produce a variety of cellular enzymes and toxins prosthetic devices)
(leucocidins, hydrolytic enzymes, exfoliatin, etc. ) Direct contact:
person-to-person

Staphylococcus Normal flora: Same as previously

haemolyticus ● Skin indicated for
● Mucous S.epidermidis
Staphylococcus membranes
lugdunensis (low nos.)

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 Staphylococcus Normal flora: Endogenous strain:
saprophyticus ● Skin Sterile unirinary
● Genitourinary tract, notably in
tract young sexually
● Mucosa active females

Micrococcus Normal flora: Endogenous strain:

spp. ● Skin uncertain. Rarely
Kocuria spp. ● Mucosa implicated infections
Kytococcus ● Oropharynx Immunocompromise
spp. d host: brain
abscess, meningitis,
pneumonia and
endocarditis

Reference: Tille, Patricia (2014). Staphylococcus, Micrococcus and similar

organism. Bailey & SCotts Diagnostic Microbiology 13th Edition (page
233). St. Louis Missouri: Elsevier-Mosby

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