BACTERIOLOGY

• Staphylococci are normally found on skin and mucous membrane as well as in our environment
• It is difficult to determine whether the given staphylococci isolate is pathogenic or not
• Characteristics of pathogenic staphylococci strains

  • Hemolyze red blood cells
  • Produce catalase
  • Produce plasma coagulase
  • Ferment mannitol
• Sterilization of working area is very necessary by using Lysol or bleach to wipe the tabletops
• The laboratory scientist should consider all specimen as infectious and must wear personal protective equipment at all times inside the laboratory
• Pre-analytical phase


  1. Prepare Mannitol Salt Agar

  2. Prepare Nutrient Agar Blood plate

  3. Collect skin and nasal swabs
• Materials


  • Bacterial suspension of Staphylococcus aureus
  • Cedarwood Oil
  • Glass slide
  • Gram stain reagents
  • Microscope
  • 3% H2o2
  • 0.5 mL 1:4 rabbit's plasma
  • Test tubes
  • Cotton swabs
  • Wire loops and alcohol lamp
• Procedure


  1. Collect bacterial sample in skin/nasal cavity

  2. Inoculate sample in Mannitol Salt Agar

  3. Inoculate S. aureus and S. saprophyticus in Mannitol Salt Agar

  4. Incubate plates at 35°C for 18 hours

  5. Observe for growth and color development
• Mannitol Fermentation


  Yellow halo around colonies = Positive (Mannitol fermenter)

  No yellow halo around colonies = Negative (Mannitol non-fermenter)
• Cell Morphology


  1. Prepare bacterial smear

  2. Stain with Gram stain

  3. Focus on oil immersion objective

  4. Observe morphology
• Colonial Morphology


  1. Get inoculum from S. aureus culture

  2. Streak on BAP using multiple-interrupted streak method

  3. Incubate at 35°C for 18 hours

  4. Examine colonies
• Catalase Test


  1. Pour 3% H2O2 solution over 24-hour nutrient agar

  2. Emulsify colony in 3% H2O2 on glass slide

  3. Observe for gas bubbles

  
• Catalase Test Interpretation


  Gas bubbles = Positive

  No gas bubbles = Negative

  
• Coagulase Test (Slide or clumping factor test)

  1. Place drop of rabbit's plasma and saline on slide

  2. Emulsify S.aureus inoculum in each drop

  3. Mix well for 5 seconds

  4. Observe for clumping
• Coagulase Test (Slide or clumping factor test) Interpretation

  Clot formation = Positive

  No clot formation = Negative
• Coagulase Test (Tube Method)

  1. Inoculate S. aureus to 0.5 ml of 1:4 rabbit's plasma

  2. Incubate at 35°C for 4 hours

  3. Observe for coagulation formation

  4. If no coagulation, incubate further up to 18 hours
• Coagulase Test (Tube Method) Interpretation

  Coagulation formation = Positive

  No coagulation formation = Negative
• After the experiment, all materials that have been in contact with bacteria should be sterilized properly
• PPEs should be disposed in infectious trash bag and should not be worn outside the laboratory
• Streptococci are gram positive occurring in pairs or in chains, non-motile, facultative anaerobes and catalase negative organism
• Streptococcal hemolytic properties


  • Alpha-hemolysis (partial hemolysis, greenish color)
  • Beta-hemolysis (complete hemolysis, clear agar)
  • Gamma-hemolysis (no hemolysis)
• 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 S. aureus, S.pyogenes, S.agalactiae
• Streptococcal cell morphology


  Gram-positive cocci normally arranged in chains, cells about 0.5um
• Colonial Morphology


  Collect sample from throat region

  Inoculate swab and bacterial suspensions on blood agar plate

  Observe for hemolysis after incubation
• Catalase Test


  Emulsify colony in 3% H2O2 on glass slide

  Observe for gas bubbles

  
• Hemolytic properties of Streptococci

  • Beta Hemolytic: Group A (S. pyogenes), Group B (S. agalactiae), Group D (Enterococcal and non-enterococcal Strep)

  Alpha Hemolytic: S. pneumoniae, Viridans Strep, Group D Streptococcus

  Gamma Hemolytic: Group D Streptococcus
• Bacitracin Disk Test


  Used to differentiate group A streptococci from other groups of beta-hemolytic streptococci, based on sensitivity of S.pyogenes to low concentration of Bacitracin
• Bacitracin Disk Test Procedure

  Dip cotton swab in broth suspension

  Streak surface of BAP

  Apply Bacitracin disk and incubate

  Observe for zone of inhibition
• Bacitracin Disk Test Interpretation

  Zone of inhibition around disk = Positive

  No zone of inhibition around disk = Negative
• CAMP Test


  S. agalactiae produces CAMP factor which enhances hemolysis of sheep RBC by Staphylococcal beta lysine
• CAMP Test Procedure


  Inoculate S. pyogenes and beta-toxin producing S. aureus on sheep's BAP

  Incubate in candle jar

  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


  Enterococci can withstand higher salt concentration than non-enterococci
• Growth on 6.5% NaCl Procedure


  Inoculate Enterococci broth suspension to 6.5% NaCl broth

  Incubate and observe for turbidity
• Growth on 6.5% NaCl Interpretation

  Visible turbidity = Positive

  No visible turbidity = Negative
• Sodium Hippurate Hydrolysis

  Based on presence of hippuricase enzyme in S. agalactiae which hydrolyzes hippuric acid to glycine and benzoic acid. Glycine reacts with ninhydrin to give purple color.
• Sodium Hippurate Hydrolysis Procedure

  Inoculate S. agalactiae suspension in sodium hippurate substrate

  Incubate and add ninhydrin reagent

  Observe for deep purple color

  
• Sodium Hippurate Hydrolysis Interpretation

  Formation of deep purple color = Positive

  No color change = Negative

  
• Bile-Esculin Hydrolysis


  Group D streptococci can grow in 40% bile and hydrolyze esculin to esculetin and glucose. Esculetin combines with ferric citrate to give black complex.
• Bile-Esculin Hydrolysis Procedure


  Inoculate Group D Streptococcus colonies on bile-esculin agar

  Incubate and observe for blackening of agar or growth