MT 3A (LECTURE) CLINICAL BACTERIOLOGY
• Novobiocin-resistant CoNS – Examples are
CATALASE-POSITIVE, GRAM-POSITIVE COCCI
Staphylococcus, Micrococcus, and Other Similar Organisms
Catalase (+), gram (+) cocci
GENERAL CHARACTERISTICS
• Heterogeneous group of organisms
• Previously, genus Staphylococcus was included
with the genus Micrococcus in the family
Micrococcaceae
• molecular phylogenetic and chemical analysis,
Staphylococcus has now been combined with
Bacillaceae, Planacoccaceae, Listeriaceae into the
order Bacillales
• Several of the Micrococcus spp. are now
reclassified into the genera Kocuria, Nesterenkonia,
Kytococcus, and Dermacoccus – these genera are
reorganized into Micrococcaeae and
Dermacoccaceae
• Alloiococcus otitidis – biochemically similar to the
families included in this chapter; belongs to the
family Carnobacteriaceae
• All organisms discussed in this chapter are catalase
positive, gram positive, aerobic or facultatively
anaerobic
• Exceptions (obligate anaerobes and maybe gram
negative):
✓ Staphylococcus aureus subsp. Anaerobius
✓ Staphylococcus saccharolyticus
• Genus Staphylococcus are non-motile and non-spore
forming
• Coagulase-negative staphylococci (CoNS) or non-
Staphylococcus aureus
• Divided into two groups based on novobiocin
susceptibility pattern
• Novobiocin-susceptible CoNS – Examples are
Staphylococcus capitis, Staphylococcus haemolyticus,
Staphylococcus hominis subsp. hominis,
Staphylococcus lugdunensis, Staphylococcus
saccharolyticus, and Staphylococcus warneri
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CHAPTER 7
Staphylococcus cohnii, Staphylococcus kloosii,
Staphylococcus saprophyticus, and Staphylococcus
xylosus
• The skin colonizers – Micrococcus spp., Kocuria spp.,
and Kytococcus spp.
✓ Easily confused with staphylococci
✓ Usually associated with skin lesions
✓ isolated from immuno-compromised
patients
• Clinically significant organisms in this chapter belong
to genus Staphylococcus
Epidemiology
• Staphylococci associated with infections in humans
are colonizers of various skin and mucosal surfaces
• Three types of nasal carrier states associated with
Staphylococcus aureus:
✓ Persistent carriers - harbor a single strain for an
extended period of time
✓ Intermittent carriers – harbor different strains
over time
✓ Individuals that do not harbor any organisms or
non-carriers
• Because the carrier state is common, infections are
frequently acquired when the colonizing strain gains
entry to a normally sterile site due to trauma or
abrasion to the skin or mucosal surface
• High Incidence of Carrier State
✓ Health care workers
✓ Immuno-compromised individuals,
including those with insulin-dependent
diabetes mellitus
✓ Long-term haemodialysis patients
✓ IV drug users
✓ Vaginal carriage may be seen in
premenopausal women
• Staphylococci are also transmitted from person to
person
✓ Upon transmission, organisms become
established as part of recipient’s normal
flora
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✓ Later introduced to sterile sites by trauma

or invasive medical procedures
✓ Person-to-person spread of antimicrobial-
resistant staphylococci strains occurs in
hospitals and presents substantial infection
control problems
✓ More recently, Staphylococcus aureus
infections have been encountered in the
community setting as well
APPROACH TO IDENTIFICATION
➢ Microdase disk (modified oxidase test)
▪ Differentiate Micrococcus spp. from
Staphylococcus spp.
▪ Colony from an 18- to 24-hour-old culture is
smeared on the disk
▪ Micrococcus spp. turn blue within 2 minutes
(positive)
▪ Species identification testing include
formation of acid from carbohydrates,
followed by tests for glycosidases,
hydrolases, and peptidases

MICROCOCCACEAE
Micrococcus

• Catalase – positive
• Coagulase – negative
• Normally found in:
✓ Environment
✓ Skin
✓ Maybe isolated as contaminant or as part of
the normal of the skin and respiratory tract
• Opportunistic pathogen found in immuno-
compromised patients
• Often recovered with staphylococci
• Low pathogenic significance

DIFFERENTIATION BETWEEN STAPHYLOCOCCI AND

MICROCOCCI IN ROUTINE LABORATORY
➢ Bacitracin and Furazolidone Resistance
• Oxidation-Fermentation of Glucose (OF Tube)
▪ Disk tests are used
o Staphylococcus spp. are capable of oxidizing
▪ 5% sheep BA is streaked in three directions
and fermenting glucose
with a cotton-tipped swab that has been
o However, Micrococcus spp. fail to produce
dipped in a bacterial suspension prepared
acid (hence, fail to ferment) from glucose
to match the turbidity of the 0.5 McFarland
under anaerobic conditions
standard
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▪ A 0.04-U bacitracin-impregnated disk and a
100-μg furazolidone-impregnated disk are
then placed on the surface of the streaked
5% sheep BAP
▪ Interpreted based on inhibition or
sensitivity of bacteria by measuring the
zone of inhibition present around the disk
▪ For bacitracin, susceptible ≥10 mm; for
furazolidone, susceptible ≥15 mm
GENERAL CHARACTERISTICS OF STAPHYLOCOCCACEAE
COLONIAL CHARACTERISTICS
➢ Lysostaphin Susceptibility Test
▪ Lysostaphin is an endopeptidase that
cleaves the glycine-rich pentapeptide
cross bridges in the staphylococcal cell
wall peptidoglycan
▪ The susceptibility of Staphylococcus to
lysostaphin is used to differentiate them
from Micrococcus
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CHAPTER 7
▪ Staphylococcus spp. are susceptible and
show a 10-16 mm zone of inhibition, while
Micrococcus spp. are not inhibited
FAMILY STAPHYLOCOCCACEAE
• Gram positive cocci (spherical cells)
• Arranged in singly, in pairs and in “grape-like”
clusters
• Catalase positive
• Non -motile, non-spore-forming
• Aerobic or facultative anaerobe except for
Staphylococcus saccharolyticus (obligate anaerobe)
• Resembles Micrococci
• Part of the normal flora of the skin
✓ Cream-colored, off - white or rarely light gold
and “buttery-looking”
✓ Oil-paint like, pin head colonies
✓ Jet black in tellurite lysine agar
• Common isolates in clinical laboratory
• Responsible for supurative type of infections
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• Rare strains staphylococci: • Enterotoxin B

✓ Fastidious
✓ Requires CO2 , hemin or menadione for growth
• Small colony variants (SCV) of Staphylococcus
aureus
✓ Grow on media containing blood
✓ Colonies appear as pinpoint to small
✓ non- hemolytic, some are β hemolytic
✓ non – pigmented colonies on BA
✓ SCV may result from limited nutrient or other
selective conditions and may revert to normal
Staphylococcus aureus phenotype following
subculture
• Coagulase test
✓ differentiates staphylococcal species
✓ Staphylocoagulase
• Staphylocoagulase-producing staphylococci
✓ Staphylococcus aureus
✓ Staphylococcus intermedius
✓ Staphylococcus delphini
✓ Staphylococcus lutrae
✓ some strains of Staphylococcus hyicus
• Mistaken for coagulase-positive staphylococci due
to the presence of clumping factor:
✓ Staphylococcus lugdunensis
✓ Staphylococcus schleiferi
• Coagulase-positive isolates from human sources are
considered to be Staphylococcus aureus
Coagulase – negative Staphylococci (CoNS)
• Most clinically significant and commonly recovered
species:
✓ Staphylococcus epidermidis
✓ Staphylococcus saprophyticus
✓ Staphylococcus haemolyticus
✓ Staphylococcus lugdunensis
✓ Staphylococcus schleiferi
✓ Results in vomiting and diarrhea
✓ Linked to staphylococcal pseudomembranous
enterocolitis
➢ TSST – 1 (Toxic Shock Syndrome Toxin-1)
• former Enterotoxin F
• causes menstruating-associated TSS – tampon
users
• super antigen stimulating T – cell proliferation
• production of large amount of cytokines
• systemic effects such as fever, multisystem
involvement including desquamative skin rash
and hypotension potentially leading to shock
and death
➢ EXFOLIATIVE TOXIN
• epidermolytic toxin or exfoliatin
• Epidermolytic toxin A – heat stable, resist
boiling for 20 minutes
• Epidermolytic toxin B – heat labile
• causes generalized desquamation of the
staphylococcal SSS (scalded skin syndrome)
also known as Ritter’s disease
• causes bullous impetigo
➢ CYTOLYTIC TOXIN
• acts on host membrane and mediate destruction
• Extracellular proteins that affect RBC and WBC
• Four hemolysins produced by Staphylococcus
aureus
✓ Alpha (α) hemolysin
✓ Beta (β) hemolysin
✓ Delta (δ) hemolysin
✓ Gamma (γ) hemolysin

Staphylococcus aureus

VIRULENCE FACTORS
Toxins

➢ ENTEROTOXINS – causes food poisoning

• Stable at 100˚C for 30 minutes
- Heat stable and resistant to action of gut
enzymes
- Produced when S. aureus grows in
carbohydrates and protein foods
• Nine serologically distinct enterotoxins
✓ Group A – E
✓ Group G – J
• Staphylococcal food poisoning
✓ enterotoxin A, B and D
• Enterotoxin B and C ; sometimes Enterotoxin G & I
✓ associated with toxic shock syndrome (TSS)

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Extracellular enzymes – tissue destroying Infections caused by Staphylococcus aureus

• staphylocoagulase ➢ Skin and wound infections

o can lead to fibrin formation around ✓ Folliculitis
bacteria, protecting it from phagocytosis ✓ Furuncles
o produce mainly by Staphylococcus aureus ✓ Carbuncles
• hyaluronidase – (spreading factor) ✓ Bullous impetigo – highly contagious
o enhance the spread of infection and Spread by:
survival in tissues, tunnels through tissues • direct contact
o also called Duran – Reynal factor • fomites
• Staphylokinase – (Fibrinolysin) lysis fibrin or clot • autoinoculation
o used in medicine to treat patients suffering
from coronary thrombosis
o tunnels through tissues
• Gelatinase – degrades gelatin
• DNase – degrades DNA
• Protease – breaks down protein
• Penicillinase – inactivate penicillin
• Lipase
o degrades fats and oils, which often
accumulates on the surface of our body
o facilitates Staphylococcus aureus ➢ Scalded skin syndrome – usually afflicts neonates
colonization of sebaceous glands ✓ caused by staphylococcal exfoliative or
o tunnels through tissues epidermolytic toxin
• phosphatase ✓ Extensive sloughing of epidermis to
• thermostable deoxyribonuclease produce a burn-like effect on the patient
➢ TSS
✓ rare but potentially fatal
Antigenic structure
✓ multisystem disease
• Teichoic acid protects the organism from lysis
➢ Septic arthritis
• Peptidoglycan and probably aids in adherence
• in children
• Clumping factor – a component on the cell wall of
✓ trauma to extremities
Staphylococcus aureus
✓ with history of rheumatoid arthritis
✓ responsible for the clumping of the whole
✓ intravenous drug abuse
staphylococci in the presence of plasma
➢ Toxic Epidermal Necrolysis (TEN)
• Protein A - this is a group specific antigen unique to
✓ multiple causes
strains
✓ drug induced
✓ prevents antibody-mediated phagocytosis
✓ hypersensitivity reaction
by PMN’s
➢ Food poisoning
✓ protect organism from opsonization
✓ intoxication resulting from ingestion of a
✓ unique to Staphylococcus aureus strain
toxin formed outside the body
✓ ability to bind the Fc portion of IgG
✓ 2 – 8 hours after ingestion of the food
✓ negate the protective effect of IgG
✓ Resolve within 24 – 48 hours
• Capsular polysaccharide – protects the organism
➢ Staphylococcal pneumonia
from phagocytosis
o secondary to influenza virus infection
➢ Staphylococcal bacteremia
Reservoir or habitat
o leading to secondary pneumonia and
• Anterior human nares
endocarditis
• Nasopharynx
➢ Staphylococcal osteomyelitis
• Perineal area
✓ secondary to bacteremia
• Skin surfaces
➢ Endocarditis
• Colonizer of mucosa
➢ UTI
• Axillae

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Other Staphylococcus species • Septic arthriris

• Vascular catheter infections
Staphylococcus epidermidis • UTI
• Normal flora of skin and mucous membranes • Cause community-associated and hospital-
• Skin contaminant in blood culture associated acquired infections
Virulence factors
✓ Exopolysaccharide “slime” or biofilm Other Coagulase-negative Staphylococci (CoNS)
✓ Antiphagocytic
✓ Exotoxins: delta toxin ➢ Commonly isolated CoNS
Predisposing factors: predeliction to foreign bodies • Staphylococcus haemolyticus
✓ catheterization ✓ commonly isolated
✓ implantation of medical devices (shunts, prosthetic ✓ reported in:
devices) • wounds
✓ immunosuppressive therapy • bacteremia
✓ direct contact: person to person • endocarditis
• UTI
✓ some are vancomycin resistant
Infection caused by Staphylococcus epidermidis
➢ Bacteremia associated with indwelling vascular Other bacteria recovered with Staphylococci
catheters
✓ in neonates under intensive care • Aerococcus
➢ Endocarditis involving prosthetic cardiac valves • Rothia
➢ Infection associated with CSF shunts, prosthetic • Alloiococcus otitidis
joints, vascular grafts, post surgical ocular
infections Laboratory Diagnosis of catalase (+), gram (+) cocci
➢ Predominantly hospital acquired Specimen collection and handling
➢ Causes health care acquired UTIs ➢ Handling and transport
✓ Transported in laboratory without delay
✓ Prevent from drying
Staphylococcus saprophyticus ✓ Maintain proper environment
• normal flora of skin, genitourinary tract ✓ Minimize growth of contaminating organisms
• adhere more effectively to the epithelial cells lining ➢ Collection
the urogenital tract than other CoNS ✓ No special procedures
• should be consider significant even in low number ✓ Proper cleansing of surrounding area to avoid
in urine culture contamination of skin microbiota
• Urine isolates that are CoNS are tested further ✓ Aspirate - best sample
Infections caused by Staphylococcus saprophyticus
• associated with UTIs in young sexually active Microscopic examination
women ▪ Majority are gram-positive cocci
Isolation and Identification ▪ Some species within the Micrococcaceae and
➢ Presumptive identification - Novobiocin Dermacoccaceae exhibit rod-shaped cells and are
susceptibility test (R) motile
➢ Obligate aerobe ▪ Pairs, tetrads, and ultimately, irregular clusters are
➢ Mannitol fermenter observed
➢ Phosphatase (-) ▪ Gram stains should be performed in young cultures
– very old cells may lose ability to retain crystal
Staphylococcus lugdunensis violet and may appear gram variable or gram
• CoNS negative
• Positive clumping factor test (slide coagulase) ▪ Staphylococci appear as gram-positive cocci,
• Negative tube coagulase reaction usually in clusters
• Contains the gene mecA that encodes oxacillin ▪ Micrococci typically appear as gram-positive cocci
resistance in tetrads
Infection caused by Staphylococcus lugdunensis ▪ Culture should be done regardless of results of
microscopic examination
• Bacteremia
• Wound infections
• Endocarditis
• Endothalmitis
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ISOLATION AND ID
➢ Media of Choice
▪ Grow on 5% sheep BA and CHOC (CA)
▪ Grow well in broth-blood culture systems
and common nutrient broths, such as
thioglycollate broth, dextrose broth, and
brain-heart infusion
▪ PEA and Columbia CNA – eliminate
contamination from gram-negative
organisms in heavily contaminated
specimens such as feces
▪ Mannitol Salt Agar (MSA)
✓ Contains high concentration of
NaCl (7.5% to 10%), mannitol, &
phenol red
✓ Staphylococcus aureus ferments
mannitol and produces a yellow
halo on the media as a result of
acid production, altering the pH
• CHROMagar for MRSA
✓ Selective and differential media for ID of
MRSA
✓ More widely used for the direct detection
of nasal colonization
✓ Selective agent is cefoxitin – MRSA is
resistant to this antibiotic
✓ Mauve-colored colonies indicates MRSA
✓ Other colonies form white to blue to green
colonies
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CHAPTER 7
➢ Incubation Conditions and Duration
▪ 5% sheep BA and CHOC are incubated at
35oC in CO2 or ambient air
▪ Growth is usually observed within 24 hours
of incubation
▪ MSA and other selective media (PEA and
Columbia CNA) may require incubation for
at least 48-72 hours before growth is
detected
✓ Gram stain is used as the initial presumptive
identification method for all gram-positive cocci
✓ Microscopic along with macroscopic colonial
morphology provides a presumptive identification
✓ Staphylococcus spp. and Micrococcus spp. are
distinguishable from the related family
Streptococcaceae by the catalase test
✓ Aerococcus, Enterococcus, and Rothia (formerly
Stomatococcus) may show pseudocatalase reaction
– i.e. they may appear to be catalase positive
• Once an organism has been characterized as a
gram-positive, catalase-positive cocci, complete
identification may involve a series of tests:
✓ Atmospheric requirements
✓ Resistance to 0.04 U of bacitracin
(Taxo A disk) and furazolidone
✓ Possession of cytochrome C as
determined by the microdase
(modified oxidase) test
• However, most microbiologists proceed
immediately to a coagulase test based on
recognition of a staphylococcal-like colony and a
positive catalase test
➢ Coagulase test
• Once an isolate is identified as, or strongly
suspected to be, a Staphylococcus sp., a
coagulase test is performed to
differentiate Staphylococcus aureus from
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other species collectively referred to as ✓ Usually done when isolates suspected to be

coagulase-negative staphylococci (CoNS)
• Coagulase, an enzyme produced by
Staphylococcus aureus, binds plasma
fibrinogen and activates a cascade of
reactions causing plasma to clot
• Two types of coagulases:
✓ Bound coagulase
✓ Free coagulase
➢ Bound coagulase or clumping factor
✓ rapid screening test
✓ Presumptive identification for Staphylococcus
aureus
✓ demonstrates the presence of cell-bound
coagulase (clumping factor)
✓ clumping factor converts fibrinogen to fibrin =
clot
✓ Other species (+) for clumping factor (slide
test)
o Some strains of Staphylococcus
lugdunensis
o Staphylococcus schleiferi
Staphylococcus aureus fail to give a positive
slide coagulation test
✓ Performed by inoculating a tube containing
plasma and incubating at 35oC
✓ Production of coagulase result in clot formation
within 1 to 4 hours of inoculation
✓ Some strains produce fibrinolysin – dissolve
the clot after 4 hours of incubation, and yield
false negative results
✓ Plasma (preferably rabbit’s plasma) containing
EDTA rather than citrate should be used since
citrate-utilizing organisms may yield false-
positive results

✓ Detected using a rapid slide test (i.e. slide

coagulase test)
✓ A positive test is indicated when organisms
agglutinate on a glass slide mixed with plasma
✓ Most, but not all, stains of Staphylococcus
aureus produce clumping factor and are readily
detected by the slide test
✓ 10% to 15% of strains may give a negative slide
coagulase test due to masking of bound
coagulase by capsular polysaccharides
✓ False positives may occur as a result of auto
agglutination when colonies are grown on
media with high salt concentrations

➢ Free coagulase
✓ Tube coagulase test (detects both free and
bound)
✓ Definitive test for Staphylococcus aureus
✓ used to confirm all slide test negative results on
clinically significant isolates
✓ demonstrates the presence of extracellular
staphylocoagulase (free coagulase)

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➢ PYR – pyrrolidonyl arylamidase
Differentiates Staphylococcus aureus (-) from:
✓ Staphylococcus lugdunensis
✓ Staphylococcus schleiferi
✓ Staphylococcus intermedius
Other tests used to confirm Staphylococcus aureus
➢ Latex agglutination tests (detect clumping factor
and protein A), and passive hemagglutination test
(detect clumping factor) – no longer used
extensively because it often fail to detect MRSA
➢ Monoclonal antibodies to capsular polysaccharide
serotypes 5 and 8
– Higher sensitivity but not specific
– False positive reactions occur in the
presence of CoNS species such as
Staphylococcus haemolyticus,
Staphylococcus hominis, and
Staphylococcus saprophyticus
➢ Nucleic Acid Testing
• PCR amplification assays detecting methicillin-
resistant Staphylococcus aureus (MRSA) from
clinical swabs
✓ Detect the mecA gene (encodes methicillin
resistance) in conjunction with a species-
specific target gene
✓ Staphylo Resist and StaphPlex Panel
✓ Caution in interpretation of results as
several species of staphylococci may reside
in the normal flora including methicillin-
resistant CoNS causing false positive
results
• Single-locus amplification
✓ Utilize a set of oligonucleotide primers
which bind to downstream sequence of
staphylococcal cassette chromosome
region encoding the mecA gene (SSCmec)
and the flanking open reading frame
(orfX)
✓ Allows the amplification of the nucleic acid
region that indicates antibiotic resistance
coupled with a species-specific marker
✓ BD Gene OHM MRSA assay, Genotype
MRSA Direct, Geno-Quick MRSA, and the
Roche Light-Cycler MRSA
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CHAPTER 7
➢ Molecular testing techniques
✓ Accuprobe – commercially available DNA probe
assay for confirmatory ID
✓ nuc gene amplification – gene which encodes
thermostable nuclease
✓ MRSA may be identified using staphylococcal
insertion sequence IS431
✓ Amplification assays to detect species-specific
genes or chromosomal sequences
✓ Qualitative nucleic acid hybridization assay that
targets rRNA sequences in Staphylococcus
aureus and CoNS
ANTIMICROBIAL SUSCEPTIBILITY TEST
• CLSI do not require routine antimicrobial
susceptibility testing of Staphylococcus
saprophyticus from urine because isolates typically
are sensitive to agents commonly used to treat
UTIs
• Most Staphylococcus aureus - resistance to
penicillin due to the production of β lactamases
Methicillin Resistant Staphylococcus aureus MRSA
▪ MRSA – methicillin-resistant Staphylococcus aureus
▪ Penicillin-resistant strains
• Requires treatment with penicillinase-
resistant penicillin
• Nafcillin or oxacillin
TYPES OF MRSA
• HACO-MRSA – health care-associated community-
onset methicillin-resistant Staphylococcus aureus
• HA-MRSA – hospital-associated methicillin-resistant
Staphylococcus aureus
CONTROL OF MRSA
• requires strict adherence to infection control
practices including:
✓ Barrier protection
✓ Contact isolation
✓ Handwashing compliance
• Rapid test aids in the control of these agents
• Vancomycin – treatment of choice
• Heterogeneous in resistance to β lactams
➢ Cefoxitin – used to detect methicillin resistance
✓ better inducer of mecA-mediated
resistance
➢ Growth
✓ enhance at neutral pH
✓ NaCl concentration of 2% - 4%
✓ cooler incubation temperature (30-32˚C)
✓ prolong incubation (up to 48 hours)
➢ Oxacillin-salt agar plate
✓ Screen for MRSA in clinical sample
✓ Differentiate MRSA from isolates that are
hyperproducers of β-lactamases or
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borderline oxacillin-resistant APPENDIX

Staphylococcus aureus (BORSA)
✓ Not recommended to screen CoNS SELECTED CULTURE MEDIA
➢ “Gold standard” for detection of mecA gene –
molecular nucleic acid probes or PCR amplification ➢ Columbia CNA with 5% Sheep’s Blood Agar
➢ Chromogenic selective differential media: Purpose:
• MRSA select ▪ Used to isolate and
• Spectra MRSA differentiate
• CHROMagar MRSA staphylococci,
✓ Antimicrobial compound – cefoxitin streptococci, and
inhibits non-MRSA isolates enterococci, from
➢ Oxacillin resistance – due to the gene mecA, carried clinical specimens
on a cassette known as SCCmec ▪ Undefined, differential,
➢ SCCmec – Staphylococcal chromosome cassette and selective medium
✓ gene codes for an altered penicillin- that allows growth of
binding protein(PBP) gram-positive organisms
✓ PBP2a or also known as PBP2’ and stops or inhibits
growth of most
Vancomycin Resistant Staphylococcus aureus or VRSA gram-negative
organisms
• Vancomycin – drug of choice for serious Principle:
staphylococcal infections ▪ Casein, digest of animal tissue, beef extract, yeast
• VRSA – vancomycin resistant Staphylococcus aureus extract, corn starch, and defibrinated sheep’s
✓ isolated from patients undergoing long-term blood provide carbon and energy
vancomycin treatment ▪ Sheep’s blood supplies the X factor (heme) and
• VISA – vancomycin intermediate Staphylococcus makes possible differentiation of gram-positive
aureus organisms based on hemolytic reaction
▪ Yeast extract provides B-vitamins
Isolation and ID of gram-positive cocci ▪ Colistin and nalidixic acid (CNA) act as selective
agents – inhibit gram-negative organisms
▪ Particularly effective against Klebsiella, Proteus, and
Pseudomonas spp

➢ Phenylethyl Alcohol (PEA) Agar

Purpose:
▪ Undefined, selective medium
▪ Used to isolate
staphylococci and
streptococci from
specimens containing
mixtures of bacterial
flora
▪ Typically used for
specimens thought
to also contain
Escherichia coli or
strains of Proteus
▪ Allows growth of gram-positive
organisms
▪ Stops or inhibits growth
of most gram-negative organisms
▪ Most gram-positive rods will also grow on this
medium, except Bacillus anthracis, which is unique
among the Bacillus spp. in its lack of growth on this
medium

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Principle:
▪ active ingredient, phenylethyl alcohol, functions by
interfering with DNA synthesis in gram-negative
organisms
▪ PEA also inhibits facultative gram-negative rods,
especially swarming Proteus spp., but permits the
growth of gram-positive cocci
▪ When prepared with 5% sheep blood,
it is used for cultivation of
gram-positive anaerobes

➢ Mannitol Salt Agar (MSA)

Purpose:
▪ Used for isolation and
differentiation of
pathogenic
staphylococci, principally
Staphylococcus aureus
Principle:
▪ Mannitol - carbohydrate
source
▪ 7.5% NaCl – medium
selective for Staphylococcus aureus since most
bacteria cannot survive this level of salinity
▪ Phenol red (pH indicator) is yellow below pH 6.8,
red at pH 7.4 to 8.4, and
pink above 8.4
▪ Staphylococcus aureus
ferments mannitol and
produce acid, which turns
the pH indicator yellow
▪ Development of yellow
halos around bacterial
growth is presumptive
evidence that the organism
is Staphylococcus aureus\
▪ Good growth that
produces no color change is presumptive
evidence for nonpathogenic Staphylococcus and
Micrococcus spp.
▪ With few exceptions, organisms that grow poorly
on the medium are not staphylococci
▪ Enterococcus may be able to grow on mannitol
salt agar and weakly ferment mannitol
▪ Differentiation is possible through a catalase test
▪ Some strains of Staphylococcus aureus may be
slow in fermenting mannitol, so plates should not
be discarded until after 48 hours of incubation

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