Enclonar, Kimberly / MLS 3A
• Test is used to differentiate Micrococcus and
Micrococcaceae Stomatococcus (S) from Staphylococcus (R) to 0.04
November 9, 2020 units of Bacitracin
Marx P. Catalan, RMT, MSMT
• Results:
o Zones >10mm = Susceptible and typically
General Characteristics
Micrococcus
• Catalase-positive, gram positive cocci o Staphylococcus shows <10mm or no zone of
o Spherical, pairs,
inhibition
• Resident flora of the skin and mucous membranes
• Ubiquitous Oxidation/Fermentation (OF) test
• Infections are directly or indirectly transmitted • Determine the action of Micrococcus (Oxidizers) and
Staphylococcus which are facultative anaerobes
Members of the Family (Fermenters)
• All, except Planococcus have been isolated from • Two tubes of Hugh & Leifson's medium are used. In
clinically significant sources one tube, the medium is covered with vaseline.
• Note: The genus Staphylococcus - mostly associated • If the organism is an oxidizer it will produce acid only
with infections in the open tube (without vaseline - aerobic).
• Differentiate Micrococcus from Staph • If it is a fermenter it will produce acid in the Vaseline-
Genus Characteristics covered tube (closed-tube) and in the open tube
Planococcus Free living saprophyte (anaerobic).
• Some aerobic bacteria may use the peptone in the
Micrococcus Free living saprophyte, rarely cause medium, producing ammonia, with resulting alkalinity
human infections (blue) in the top part of the open tube
Stomatococcus Normal flora of primates and other • pH indicator: Bromthymol blue
mammals • Original color: Green
Staphylococcus
o Alkalinity: blue
o Acid: yellow
Differential Test for Micrococcus and Staphylococcus • CHO: 1% Glucose
• Peptone: 0.2% (end product: ammonia)
Lysostaphin Susceptibility Test • Asaccharolytic
• Antibiotic, endopeptidase o Does not utilize CHO as energy source
o It cleaves peptide linkages • Oxidizer
o Substrate: reacts only to peptides that are rich in o Organisms that utilize CHO as source of energy
glycine in the presence of oxygen
• Susceptible: Staphylococcus • Fermenter
o Cross bridge with glycine o Organisms that utilize CHO as a source of energy
o Clear tube without the presence of oxygen
• Resistant: Micrococcus • Oxidizer and ferment
o Utilize CHO in the presence or absence of
oxygen
Modified Oxidase Test
• Test to detect oxidase enzyme in aerobic organisms
with compound Cytochrome C
• Reagent:
o 6% tetramethylphenylenediamine hydrochloride
in dimethyl sulfoxide
o Or Microdase Disk (with impregnated Mod.
oxidase reagent)
• Positive: blue color
• Positive: Micrococcus
• Negative: Staphylococcus
Staphylococcus Species
Staphylococcus aureus
General characteristics
• The most significantly species, and also an important
Bacitracin Susceptibility Test cause of nosocomial infections
• 0.04 units of bacitracin inhibits of • Gram positive cocci arranged in tetrads or clusters
Micrococcus/Stomatococcus while having no effect and facultative anaerobic
on Staphylococcus • Medium sized, raised creamy colonies on BAP or CNA
with white, cream or golden yellow pigmentation;
exhibits beta hemolysis on BAP
Enclonar, Kimberly / MLS 3A
• Staphyloxanthin - yellow • Citrated plasma - not suitable for tube test because
• Ubiquitous - found everywhere other organism that utilize citrate (Pseudomonas,
• Reduces nitrates to nitrites and grows on 7.5-10% Neisseria) yield positive result
NacL (halophilic) o Binds to calcium
o Can be used because you are sure that you've
isolated gram positiive
• Staphylokinase (fibrinolysin) in S. Aureus
Virulence Factors
Cell surface Structures acting as virulence factors
• Protein A
o bind to the antibody molecule, which causes
interference with phagocytosis and fixation of
Growth on Mannitol Salt Agar complement.
• Inhibitor: 7.5% NaCl o It binds to: FC region of IgG
• CHO: mannitol • Peptidoglycan and teichoic acids
• pH indicator: phenol red o attachment to mucous membranes and enable
• Mannitol fermenter: Colonies surrounded by a yellow organism to resist unfavorable conditions
halo; Staphylococcus aureus
• NMF - Pink (CoNS) Extracellular Enzymes and Toxins
• Coagulase
Catalase Test o conversion of fibrinogen to fibrin; may coat
• Differentiates Staphylococcus from Streptococcus (or neutrophils to protect organisms from
from other gram-positive cocci resembling phagocytosis
Staphylococci) • Staphylokinase (fibrinolysis)
• Positive result - Effervescence/Bubble formation: o Dissolves fibrin clot and may enable infection to
Staphylococcus spread
• Negative result: No bubble formation/Effervescence: • Lipase
Streptococcus o Hydrolyzes lipids in plasma and skin; associated
• Blood has catalase activity, therefore colonies from with skin infections such as boils, carbuncles and
blood agar are not used furuncles
• Hyaluronidase (Spreading factor)
o Hydrolysis of hyaluronic acid present in
connective tissues, which can result in the
spread of infection
Coagulase Test
• Deoxyribonuclease (Dnase)
• Differentiates Staphylococcus aureus (Coagulase +)
o Degradation of DNA
from Staphylococcus saphrophyticus and
• Exfoliatins/Exfoliative toxins (epidermolytic toxin)
Staphylococcus epidermidis (CoNS); It is also the
o Causes sloughing off of the epidermal layer of
primary virulence factor of S. aureus
the skin. May result to SSS (Ritter disease)
• Two forms
o Cell bound coagulase or Clumping factor
o Free coagulase Cytolytic toxin
• Leukocidins
• Coagulase-reacting factor
o Lyses neutrophils and macrophages; inhibit
• Thrombin - responsible for clotting
phagocytosis
• Coagulase + CRF = coagulase-CRF complex or
• Hemolysins
thrombin-like molecule
o Group of A, B, G, O, lyse RBCs
▪ A - hemolysis can cause damage to
platelets and macrophage
▪ B - "hot-cold" hemolysin damage to RBCs
□ Aka: Sphingomyelinase/phospholipase
Slide Coagulase Test C
• Demonstrates Cell bound coagulase also referred to □ 37C and 4C
as the "clumping factor" ▪ A, G, O hemolysis may lyse WBCs
• Procedure: Rabbit plasma + organism • PVL (Panton-Valentine Leukocidin) Y-hemolysin
• Positive: Macroscopic clumping in 10s o Exotoxin and lethal to PMNs. Contributes to the
• Negative: no clumping (must be confirmed using the invasiveness of organisms
tube coagulase test) o Produced only by few strains
o Associated with "community acquired"
Tube Coagulase Test staphylococcal infection
• Confirmatory for negative slide tests; detects bound
and free coagulase Enterotoxin
• Procedure - sterile test tube with 0.5ml rabbit plasma • Group of 9 heat stable (100C, 30mins) proteins: A, B,
• Inoculate colonies to give milky suspension. Incubate C, C2, D, E, F, G, I, J
35C for 4hrs • Food poisoning: A, B, and D
• Observe 30minutes to 4hrs o heat stable and they act to stimulate neural
• Positive - coagulum/clot formation receptors in the GI tract causing pain, vomiting,
• Test can be positive for 4 hrs and then revert to and diarrhea within 6 hours of ingestion
negative after 24 hrs
Enclonar, Kimberly / MLS 3A
o Organisms are not heat stable, therefore only
toxins are transferred in food Perform AST once speciation is done
• Toxic shock syndrome: TSST-1 (Formerly F)
• Beta-lactamase (penicillinase) Culture Media for Isolation
o Hydrolysis and inactivation of penicillin • Grows easily on Sheep Blood agar (SBA)
antibiotics through breakdown of beta lactam • Selective medium for heavily contaminated
ring in penicillin molecule specimens:
o Mannitol Salt Agar with 7.5% NaCl (MSA)
Disease Association o Columbia colistin-nalidixic acid agar (CNA)
• Infection type o Phenylethyl alcohol (PEA)
o Pyogenic (pus) in nature. Accompanied by • CHROMagar S. aureus
swelling, redness, increase in temperature to the o Selective and differential medium for isolation
affected area and accumulation of leukocytes and identification of S. aureus
• Most common Staphylococcal infection • Baird-Parker Medium
o Folliculitis o For isolation of staphylococci from food
o Boils specimens
o Furuncles
o Carbuncles
o Impetigo - newborns, infants and young children
o Predominant cause of joint infection in adults
• Toxic Shock Syndrome (TSSS)
o A clinical syndrome characterized by
hypertension, fever, desquamation of the palms
and soles, chills, headache and vomiting
o Primarily affects young menstruating females
using tampons
o Caused by: toxic shock syndrome Toxin 1
• Scalded Skin syndrome (SSS)
o Ritter Lyell Disease (in newborns)
o Caused by: Exfoliative Toxins A & B
o Separation of the dermis (epidermal necrolysis)
• Food poisoning
o Ingestion of preformed enterotoxins
o Symptoms include: rapid onset of nausea,
vomiting, and diarrhea (2-6hrs after ingestion),
abdominal pain and cramping
o Caused by: Enterotoxins A, B, & D
Staphylococcus epidermidis
• Resembles S. aureus in morphology and gram stain
preparation
• Non-hemolytic in BA
• Grows, but lack of fermentation in MSA
• Coagulase negative
• Susceptible to: Novobiocin
• A normal flora of the skin and mucous membranes
(non-pathogenic)
• Has been found with increasing frequency in
immunosuppressed patients (nosocomial)
• Associated with bacterial endocarditis, frequently
following the insertion of artificial heart valves
Staphylococcus saprophyticus
• Important cause of UTI's associated pyelonephritis
and cystitis in young women and on older men who
have an indwelling catheters
• Coagulase negative; non-hemolytic
• Resistant to: novobiocin
