ID of STREPTOCOCCUS

• Streptococci


  Gram positive cocci occurring in pairs or in chains, non-motile, facultative anaerobes and catalase negative organism
• Streptococci


  • Some species cause pyogenic infection to man
  • Some species are part of the normal flora that resides in the skin, oral cavity, nasopharynx and genital track

  
• Alpha-hemolysis


  Causes partial hemolysis that develops a greenish color on blood agar
• Beta-hemolysis


  Causes complete hemolysis that makes the blood agar clear
• Gamma-hemolysis


  No hemolysis took place in blood agar
• Pre-analytical phase


  1. Sterilization of working area

  2. Considering all specimens as infectious

  3. Wearing personal protective equipment
• Materials


  • Blood agar plate
  • Bacitracin disk
  • 6.5% NaCl
  • Wire loops
  • Petri dish
  • 3% H2O2
  • Gram stain reagents
  • Alcohol lamps
  • Glass slides
  • Forceps
  • Cotton swab
  • Bacterial broth of E. faecalis, B-hemolysin-producing strain of S. aureus, S.pyogenes, S.agalactiae

  
• Cell morphology


  1. Prepare bacterial smear

  2. Stain with Gram stain

  3. Focus on oil immersion objective

  4. Observe morphology
• Cell morphology of Streptococci

  Gram-positive cocci normally arranged in chains of varying lengths, cells are about 0.5um
• Colonial morphology


  1. Collect sample from throat region

  2. Inoculate on blood agar plate

  3. Observe for hemolysis
• Colonial morphology of Streptococci

  Grayish, translucent to slightly opaque, circular, pinpoint colonies, may vary from mucoid or smooth type to matte or rough form

  
• Catalase test


  1. Emulsify colony in 3% H2O2

  2. Observe for gas bubbles
• Catalase test interpretation


  Gas bubbles - POSITIVE, No gas bubbles - NEGATIVE
• Hemolysis types


  • Beta Hemolytic Streptococcus: Group A (S. pyogenes), Group B (S. agalactiae), Group D (Enterococcal and non-enterococcal Strep)
  • Alpha Hemolytic Streptococcus: S. pneumoniae, Viridans Strep, Group D Strepcoccus
  • Gamma Hemolytic Streptococcus: Group D Streptococcus

  
• Bacitracin disk test


  1. Dip cotton swab in broth suspension

  2. Streak on blood agar plate

  3. Apply bacitracin disk and incubate

  4. Observe for zone of inhibition
• Bacitracin disk test interpretation

  Zone of inhibition around disk - POSITIVE, No zone of inhibition around disk - NEGATIVE
• CAMP test


  1. Inoculate S. aureus streak on sheep's blood agar

  2. Inoculate S. agalactiae at right angle to S. aureus streak

  3. Incubate in candle jar

  4. Observe for arrow-head shaped zone of enhanced hemolysis
• CAMP test interpretation


  Presence of arrowhead hemolysis - POSITIVE, No presence of arrowhead hemolysis - NEGATIVE
• Growth on 6.5% NaCl

  1. Inoculate Enterococci in 6.5% NaCl broth

  2. Incubate

  3. Observe for turbidity
• Growth on 6.5% NaCl interpretation


  Visible turbidity - POSITIVE, No visible turbidity - NEGATIVE
• Sodium hippurate hydrolysis


  1. Inoculate S. agalactiae in sodium hippurate substrate

  2. Incubate

  3. Add ninhydrin reagent

  4. Observe for purple color
• Sodium hippurate hydrolysis interpretation

  Formation of deep purple color - POSITIVE, No color change - NEGATIVE
• Bile-esculin hydrolysis


  1. Inoculate Group D Streptococcus on bile-esculin agar

  2. Incubate

  3. Observe for blackening
• Bile-esculin hydrolysis interpretation


  Blackening of growth or medium - POSITIVE, No blackening of growth or medium - NEGATIVE
• Post-analytical phase


  1. Sterilize all materials that have been in contact with bacteria

  2. Disinfect working station

  3. Dispose PPEs and other disposable materials properly