Identification of Staphylococcus spp.

• Staphylococcus spp. is a?

  • catalase producing
  • gram positive
  • purple
  • cocci
  • appear in singly, in pairs, and in cluster
• Staphylococcus spp. is a?

  • nonmotile
  • non – spore - forming
  • aerobic or facultatively anaerobic except for?

  - S. saccharolyticus which is an obligate anaerobe
• Staphylococcus spp. is a?

  - Toxin induced diseases such as

  1. food poisoning
  2. scalded skin syndrome (SSS)
  3. toxic shock syndrome (TSS)
• Micrococcus is a?

  • catalase producing
  • coagulase negative
  • gram positive cocci
• Micrococcus are often recovered with staphylococci and can be

  differentiated easily from coagulase-negative staphylococci (CoNS)
• SPECIMEN COLLECTION AND HANDLING

  • Clinical materials collected from infected sites should be transported to the laboratory without delay to prevent drying, maintain the proper environment, and minimize the growth of contaminating organisms.
• MICROSCOPIC EXAMINATION

  Gram positive cocci

  • Gram stains should be performed on young cultures
• MEDIA OF CHOICE

  • Grow on 5% sheep blood and chocolate agars
  • They also grow well in broth-blood culture systems and common nutrient broths
• MEDIA OF CHOICE

  Selective Media:

  • Phenylethyl alcohol (PEA)
  • Mannitol Salt Agar
  • Columbia colistin - nalidixic acid (CNA) agars
  • CHROMagar 2 - identification of Methicillin - resistant S. aureus
• Staphylococci produce round, smooth, white, creamy colonies on SBA after 18 to 24 hours of incubation at 35° C to 37° C
• S. aureus can produce hemolytic zones around the colonies and may rarely exhibit pigment production with extended incubation.
• This test differentiates catalase-positive micrococcal and staphylococcal species from catalase-negative streptococcal species

  Catalase test
• CATALASE TEST

  PRINCIPLE : The enzyme, catalase, is capable of converting

  hydrogen peroxide to oxygen and water. The presence of enzyme in bacterial isolate causes rapid elaboration of bubbles
• CATALASE TEST

  REAGENT :

  • 30% Hydrogen Peroxide (Bailey’s);
  • 3% Hydrogen Peroxide (Delost; used in the lab)
• CATALASE TEST

  RESULT:

  (+) : bubble formation/ effervescence [Staphylococci spp.]

  ( - ) : no bubble formation / non-effervescence [Streptococcus]
• CATALASE TEST

  False Positive Results:

  • Enterococci: enzyme peroxidase (slowly catalyzes the breakdown of H2O2)
  • Blood Agar
  • Platinum wire: Nichrome wire
• The test is used to differentiate Staphylococcus aureus

  (positive) from coagulase negative staphylococci (negative).

  Coagulase test
• Coagulase Negative Staphylococcus species:

  • S. schleiferi
  • S. haemolyticus
  • S. Intermedius
  • S. epidermidis
  • S. warnei
  • S. capitid
  • S. simulans
• COAGULASE TEST

  PRINCIPLE:

  1. SLIDE: Bound coagulase, or “clumping factor,” is bound to the bacterial cell wall and reacts directly with fibrinogen. [15 secs.]
  2. TUBE : The presence of bound coagulase correlates with free coagulase, an extracellular protein enzyme that causes the formation of a clot when S. aureus colonies are incubated with plasma
• COAGULASE TEST

  REAGENT: Rabbit’s Plasma [0.5 mL]

  RESULTS:

  (+) : formation of clots [S. aureus]

  ( - ) : no clot formation [CoNS]
• COAGULASE TEST

  False Positive Results:

  Citrate

  • Colonies from high salt concentration culture media
• It is used to distinguish staphylococci species (resistant) from micrococci (susceptible).

  Bacitracin susceptibility test
• BACITRACIN SUSCEPTIBILITY TEST

  PRINCIPLE:

  • The antibiotic bacitracin inhibits the synthesis of bacterial cell walls. A disk (TaxoA) impregnated with a small amount of bacitracin (0.04 units) is placed on an agar plate, allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms.
• BACITRACIN SUSCEPTIBILITY TEST

  (+): susceptible; has a zone of inhibition greater than 10mm

  ( - ): resistant: no zone of inhibition
• Used to differentiate Coagulase-Negative Staphylococci; 5ug

  Novobiocin Susceptibility Test
• NOVOBIOCIN SUSCEPTIBILITY TEST

  RESULT:

  (+) : susceptible; presence of zone of inhibition around the disk [CoNS]

  (-) : resistant; no zone of inhibition; [S. saprophyticus]