Staph

• Gram positive cocci

  • Staphylococci
  • Micrococci
  • Streptococci
  • Pneumococci
  • Viridans Streptococci
• Staphylococci


  • Gram positive cocci in grape-like clusters
  • Non-motile
• Catalase test


  A presumptive biochemical test to identify/detect the genus Staphylococci and Micrococci
• Staphylococci


  • Able to grow in 7.5-10% NaCl
  • Resistant to 0.02-0.04 units of bacitracin
• Bacitracin


  Also referred to as Taxo A
• Pigment formation/growth on Loeffler's Serum Slant (LSS)


  • Staphylococcus aureus (golden yellow pigment)
  • Staphylococcus citreus (lemon-yellow pigment)
  • Staphylococcus albus (white pigment)
• Catalase test


  • Uses 3% hydrogen peroxide (for Staphylococci)
  • 30% H202: used in Superoxol test; presumptive test for Neisseria and Mycobacterium (heat stable catalase test)
  • 15% H202: for anaerobes
  • Can be performed on slide or in tube
  • Principle: Catalase expedites the breakdown of hydrogen peroxide into water and oxygen
• Positive catalase test


  • The organism is able to produce the enzyme catalase
  • Hydrogen peroxide, through the action of the enzyme catalase, will be broken down into water and oxygen; the release of oxygen will cause bubbling
• Catalase test results

  • Positive: Staphylococci, Coagulase Negative Staphylococcus
  • Negative: Micrococcus, Streptococci
• Staphylococcus aureus


  • Medically/Clinically significant
  • Normal flora of anterior nares and nasopharynx
  • Can cause toxin-mediated diseases and non-toxin mediated diseases
  • Facultative anaerobe
• Growth/Colony morphology of Staphylococcus aureus

  • Uniform turbidity on broth
  • On plates, they produce smooth circular and opaque colonies with oil-like or butyrous appearance
  • Jet black colonies on Tellurite agar/Vogel Johnson medium
• Tests for identification of Staphylococcus aureus


  • Catalase (+)
  • Bound & Free Coagulase (+)
  • Mannitol fermentation (+)
  • DNAse (+)
  • PYR (-)
  • VP and Nitrate (+)
  • Latex agglutination test for protein A (+)
  • Beta-hemolytic on 5% sheep blood agar
  • Growth and fermentation in MSA
• Coagulase test


  • Definitive test for S. aureus
  • Biochemical test used to differentiate the pathogenic S. aureus from the other Staphylococci
  • Reagent: Rabbit's plasma obtained using EDTA
  • Slide coagulase = detects clumping factor (bound coagulase)
  • Tube coagulase = detects free coagulase
• Coagulase test results


  • Bound coagulase: produce clot formation immediately
  • Free coagulase: produce clot formation after incubation
• Mannitol fermentation test


  • Media used: Mannitol Salt Agar (MSA)
  • Inhibitor: 7.5-10% NaCl; inhibitory agent for gram negative and other bacteria
  • Indicator: phenol red
  • Colony odor: similar to an old sock
  • Positive result: Development of yellow halo around the colonies (fermenters)
• • Fermentation: organism is capable of producing acid in the presence or absence of air
  • Oxidation: organism is capable of producing acid only in the presence of air
• DNAse test/Thermonuclease


  • Detects deoxyribonuclease
  • Used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth
  • Not specific for S. aureus; Moraxella and Serratia are also positive for DNAse
  • Dye method: Clear zone around the colonies (if methyl green is used) or Pink color (if toluidine blue is used)
  • HCl precipitation method: Clearing of agar around the colonies
• Susceptibility of Staphylococcus aureus


  • Sensitive to: Lysostaphin and Novobiocin
  • Resistant to: Polymyxin
• Cephalosporinase test


  • Used to detect the ability of the organism to produce the enzyme beta lactamase
  • Uses: Cefinase disk
  • Impregnated substrate: Nitrocefin
  • Positive result: Pink to Red (change in color) within 60 minutes
  • Negative: Yellow (no change in color)
• Virulence factors of Staphylococcus aureus


  • Enzymes: Lipase, Hyaluronidase, Beta lactamase, Staphylokinase, Coagulase, DNAse/Thermonuclease, Beta hemolysin
  • Toxins: Enterotoxins A & B, TSST-1, Exfoliatin, PVL/Panton Valentine Leukocidin
• Catalase (+): although it is an enzyme, it is not a virulence factor
• Toxin mediated diseases caused by Staphylococcus aureus


  • Food poisoning (Enterotoxin A and B)
  • Toxic shock syndrome (TSST-1)
  • Scalded skin syndrome/Ritter's disease (Exfoliatin)
• Non-toxin mediated diseases caused by Staphylococcus aureus


  • Boils, carbuncles, furuncles, cellulitis, wound infections
  • Bullous impetigo (skin infection affecting the epidermis)
  • Sty (eye infection that develops at the edge of eyelids)
• Drug of choice for treatment of Staphylococcal infections are penicillinase resistant drugs: oxacillin, cloxacillin, methicillin
• MRSA/Methicillin Resistant Staphylococcus aureus


  • Strain of S. aureus resistant to Methicillin, Nafcillin, and Oxacillin
  • Resistance is due to PBP2A (Penicillin binding protein 2A) in cell wall, encoded by mecA gene
• Methods to detect oxacillin resistance in S. aureus


  • Chromogenic test (MRSA: development of mauve-rose color)
  • Cefoxitin Disk Diffusion test
  • D-test
  • PCR (to detect the gene that codes for resistance)
• Differences between MRSA and MSSA

  • MRSA: Penicillin resistant, Oxacillin resistant, Cefoxitin resistant
  • MSSA: Penicillin sensitive, Oxacillin sensitive, Cefoxitin test negative
• Organisms that may be mistaken for S. aureus

  • S. lugdunensis
  • S. intermedius
  • S. haemolyticus
• Coagulase Negative Staphylococci (CoNS)

  • Catalase (+); Coagulase (-)
  • Includes Staphylococcus saprophyticus and Staphylococcus epidermidis
• Differences between S. aureus, S. epidermidis, and S. saprophyticus

  • Colony: S. aureus (yellow), S. epidermidis (white), S. saprophyticus (white)
  • Catalase test: All (+)
  • Coagulase test: S. aureus (+), S. epidermidis (-), S. saprophyticus (-)
  • Mannitol fermentation: S. aureus (+), S. epidermidis (-), S. saprophyticus (-)
  • Hemolysis on BAP: S. aureus (beta), S. epidermidis (gamma), S. saprophyticus (gamma)
  • Novobiocin susceptibility: S. epidermidis (susceptible), S. saprophyticus (resistant)
  • DNAse test: S. aureus (+), S. epidermidis (-), S. saprophyticus (-)
  • Phosphatase: S. aureus (+), S. epidermidis (+), S. saprophyticus (-)
  • Gelatinase: All (+)
• Coagulase Negative Staphylococci


  • S. epidermidis: Normal flora of the skin, causes UTI, stitch abscess, prosthetic heart valve infection
  • S. saprophyticus: Most common cause of UTI in young women, causes pyelonephritis and cystitis in those with indwelling catheters
• Virulence factors of Coagulase Negative Staphylococci


  Slime production or biofilm formation
• Micrococci


  • Gram positive cocci in tetrads; cuboidal packets
  • Strict aerobe
  • On BAP = gamma hemolysis
• Modified oxidase test


  • Presumptive test for Micrococci
  • Use of 6% modified oxidase reagent: Tetramethyl p – phenylenediamine dihydrochloride in DMSO (Dimethylsulfoxide)
  • Positive result: development of blue or purple-blue color change within 2 minutes
• Differences between Staphylococci and Micrococci

  • Aerobic growth: Both (+)
  • Anaerobic growth: Staphylococci (+), Micrococci (-)
  • Lysostaphin susceptibility: Staphylococci (S), Micrococci (R)
  • Modified oxidase test: Staphylococci (-), Micrococci (+)
  • Bacitracin susceptibility: Staphylococci (R), Micrococci (S)
  • Furazolidone/Furoxone susceptibility: Staphylococci (S), Micrococci (R)
  • Catalase test: Both (+)
  • Benzidine test: Staphylococci (-), Micrococci (+)
  • Glucose utilization: Staphylococci (Fermenter), Micrococci (Oxidizer)
• Stomatococcus (Now: ROTHIA)


  • An emerging pathogen of immunocompromised patients causing endocarditis and bacteremia
  • Modified oxidase test (-)
  • Bacitracin, furazolidone, and Lysostaphin resistant
  • Fermentative
  • Unable to grow in 5% sodium chloride
  • Encapsulated
  • Weakly catalase test (+)
  • Coagulase negative