Bio 265L Lab Week #6 Review

York College - CUNY Bio 265L Lab Week #6: Selected Exercises and Demonstrations for Identifying Staphylococcus, Streptococcus, and Enterococcus Species

Introduction to Selected Bacteria

  • Bacteria Studied: Staphylococcus, Streptococcus, Enterococcus species

  • Clinical Significance: Understanding these species is vital for diagnosing and treating infections caused by them.

A. Staphylococcus Species

Identifying Species: Staphylococcus aureus, S. epidermidis, S. saprophyticus

  1. Mannitol Salt Agar (MSA) Plate:

    • Purpose: To determine which species ferments mannitol (positive) or does not (negative).

    • Key Points: The color change from red (neutral pH) to yellow (acidic pH) indicates mannitol fermentation.

  2. Blood Agar Plate (BAP):

    • Purpose: To identify which species is hemolytic and the type of hemolysis produced.

    • Types of Hemolysis:

      • Alpha Hemolysis: Partial lysis producing a greenish hue.

      • Beta Hemolysis: Complete lysis resulting in a clear zone around the colony.

      • Gamma Hemolysis (Non-hemolytic): No color change observed.

    • Example: Staphylococcus aureus is beta-hemolytic.

  3. Coagulase Activity:

    • Significance: Differentiates Staphylococcus aureus (coagulase positive) from other species (coagulase negative).

    • Notes: The presence of the coagulase enzyme clots plasma, indicating S. aureus.

  4. Novobiocin Resistance Test (TSA/novobiocin disk):

    • Purpose: To evaluate resistance to novobiocin.

    • Key Points: S. saprophyticus is typically resistant, whereas S. epidermidis is susceptible.

B. Selected Streptococcus Species and Their Hemolysis

Identifying Hemolytic Activity:

  1. Streptococcus salivarius:

    • Type: Alpha hemolysis.

  2. Streptococcus pyogenes and S. agalactiae:

    • Type: Beta hemolysis.

    • Differentiation: Alpha hemolysis (greenish coloration) vs. beta hemolysis (clear zone).

Beta Hemolytic Streptococci: Traditional vs. Serological Testing

  • Traditional tests have been mostly replaced with serological tests.

  1. Bacitracin Sensitivity Test (BAP/Bacitracin Disc):

    • Group A Streptococci (Sensitive): Streptococcus pyogenes.

    • Non-Group A Streptococci (Resistant): Streptococcus agalactiae.

  2. CAMP Test (BAP):

    • Group B Positive for CAMP Factor: Streptococcus agalactiae.

    • Non-Group B Negative for CAMP Factor: Streptococcus pyogenes.

C. Enterococcus Species (E. faecalis, E. faecium)

  1. Bile Esculin Test Tubes:

    • Purpose: Selective and differential medium for Enterococcus species.

    • Result: Enterococcus species are positive for bile esculin test.

    • Medium Color Change: The medium changes to dark brown or black due to hydrolysis of esculetin in the presence of bile salts.

Laboratory Experiments

  1. Catalase Test:

    • Staphylococcus epidermidis: Catalase positive.

    • Streptococcus salivarius: Catalase negative.

    • Conclusion: Enterococcus species exhibit the same result as Streptococcus species.

  2. Streaking Staphylococcus Species:

    • Plates Used: MSA and TSA with novobiocin discs.

    • Method: The plates should be divided into three sections, each streaked with one species: S. epidermidis, S. aureus, S. saprophyticus.

  3. BAP and Hemolysis Results:

    • Organisms:

      • Staphylococcus epidermidis: Gamma hemolytic.

      • Streptococcus salivarius: Alpha hemolytic.

      • Staphylococcus aureus: Beta hemolytic.

    • Comparative Purposes: S. pneumoniae, identified as alpha hemolytic.

    • Optochin Sensitivity Test for S. pneumoniae:

      • Sensitive to optochin and soluble in bile (disc P).

      • Other alpha hemolytic streptococci are resistant to optochin and insoluble in bile.

Notes on Biosafety Levels (BSL)

  • BSL-1:

    • Streptococcus salivarius (ATCC13419)

    • Staphylococcus epidermidis

    • Staphylococcus saprophyticus

  • BSL-2:

    • Staphylococcus aureus

    • Streptococcus pyogenes

    • Streptococcus agalactiae.

  • Instructor: M.Pia Di Bonaventura