STAPHYLOCOCCI

• Procedure for investigating an unknown organism in microbiology laboratory

  1. Undergo screening test

  2. Perform presumptive test

  3. Perform confirmatory test

  
• Screening test


  Screens for the presence or absence of the organism in the specimen
• Presumptive test


  Presumes the identification of the unknown organism
• Confirmatory test


  Confirms the presence of the organism of interest in the sample
• Microscopic evaluation of the morphology of the organism is the first objective in the screening test
• Screening test is always done first before presumptive and confirmatory tests
• If the screening test is negative, it is impractical to perform the confirmatory test
• Presumptive tests


  1. Pigment production on potato agar

  2. Catalase production on trypticase soy agar (TSA)

  3. Hemolysis and colonial morphology on blood agar (BA)
• Confirmatory tests


  1. Mannitol fermentation test on mannitol salt agar (MSA)

  2. Coagulase test
• Staphylococci


  • Gram positive cocci arranged in clusters
  • Appear purple under the microscope
• Morphology alone cannot differentiate Staphylococcus aureus from Staphylococcus epidermidis

  
• Catalase test


  Detects the ability of the organism to produce the enzyme catalase using hydrogen peroxide

  
• Usefulness of catalase test

  Differentiates Staphylococci from Streptococci
• Performing catalase test


  1. Grow colonies on trypticase soy agar (TSA)

  2. Treat grown colonies with hydrogen peroxide (H2O2)

  3. Appearance of gas bubbles indicates a positive test

  
• Positive catalase test indicates the organism is from the Staphylococci family

  
• Blood agar


  Enrichment and differential medium
• Staphylococcus aureus


  • Produces small, circular, golden-yellow colonies

  
• Staphylococcus epidermidis


  • Produces white colonies

  
• Hemolysis/hemolytic pattern


  Indicates the ability of the organism to cause lysis of red blood cells in the blood agar medium

  
• Agar


  Medium that particular organism has to utilize substances found inside the red blood cell (a content of blood agar)
• Organism utilizing substance inside RBC

  1. Destroy RBC to get substance

  2. Cause destruction or lysis of RBCs in blood agar medium
• Hemolytic patterns


  Different patterns observed in blood agar medium depending on organism's ability to cause lysis of RBCs

  
• Hemolytic patterns


  • Beta-hemolysis
  • Alpha-hemolysis
  • Gamma-hemolysis

  
• Beta-hemolysis


  Complete hemolysis/destruction/lysis of RBCs in blood agar medium
• Alpha-hemolysis


  Incomplete destruction of RBCs
• Gamma-hemolysis


  No hemolysis of red blood cells
• Staphylococcus aureus produces beta-hemolysis, Staphylococcus epidermidis produces gamma-hemolysis
• Mannitol salt agar


  Selective and differential medium containing mannitol and 7.5% sodium chloride
• Selective characteristic of mannitol salt agar


  Only salt-loving organisms can survive and grow
• Differential characteristic of mannitol salt agar

  Organism fermenting mannitol produces acid, changing agar color from light pink to yellow
• Staphylococcus aureus is mannitol fermentation positive, Staphylococcus epidermidis is mannitol fermentation negative

  
• Coagulase


  Enzyme produced by Staphylococcus aureus that can convert fibrinogen to fibrin, causing clot formation
• Staphylococcus aureus is coagulase positive, Staphylococcus epidermidis is coagulase negative
• Coagulase tests


  • Slide coagulase
  • Tube coagulase
• Staphylococcus aureus produces yellow pigment (lipochrome), Staphylococcus epidermidis produces white pigment
• Criteria for identifying Staphylococci

  • Colonial appearance on blood agar
  • Colonial appearance on MSA
  • Gram staining reaction
  • Microscopic morphology
  • Catalase & coagulase production
  • Mannitol fermentation

  
• Catalase test


  Detects ability of bacteria to convert hydrogen peroxide to oxygen and water
• Staphylococci are catalase positive, Streptococci are catalase negative
• Coagulase test


  Detects ability of Staphylococcus aureus to produce coagulase enzyme

  
• Coagulase test types


  • Slide coagulase
  • Tube coagulase