Mod 5

• Gram-positive cocci


  • Peptococcus
  • Peptostreptococcus
  • Sarcina
  • Micrococcus
  • Staphylococcus
  • Streptococcus
• Gram-negative cocci

  • Neisseria
  • Moraxella
  • Acinetobacter
  • Kingella
• Neisseria


  • Kidney, coffee bean shaped
  • Encapsulated
  • Obligate aerobes
  • Capnophilic (requires 3-10% CO2)
  • Catalase & oxidase (+)
  • Fastidious
  • Pathogenic species grow only @ 37°C
  • Non-motile
  • Non-sporeformer
  • Non-hemolytic
  • Non-pigmented
• Neisseria gonorrhoeae


  Gonococci, agent of gonorrhea
• Neisseria meningitidis

  Meningococcus, causative organism of Meningococcal (epidemic) Meningitis
• Moraxella catarrhalis

  Formerly Branhamella catarrhalis, Neisseria catarrhalis<|>Aerobic, oxidase-positive diplococcus<|>Morphologically and biochemically resembles Neisseria<|>Will not grow on modified Thayer Martin and does not ferment any sugar<|>Differentiated on DNA-based composition
• Identification of Gram-positive cocci

  1. Catalase test

  2. Coagulase test
• Catalase test

  Staphylococci and Micrococci are capable of producing the enzyme catalase, which can be tested by mixing the bacteria with hydrogen peroxide and observing for oxygen bubbling
• Coagulase test

  Used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CoNS)<|>Coagulase is an enzyme produced by S. aureus that converts soluble fibrinogen in plasma to insoluble fibrin
• Slide test


  This strain must be tested in the tube test
• Quality Control


  To keep expenses at a minimum, it is possible to do one positive and one negative control for the class as a whole, instead of having each student or group perform them
• Clumping in the slide test

  Might not be easily observed by holding the slide on top of the workbench. It will be more visible when observed over a dark surface
• False-positive response


  If the student has allowed enough time for the drop of plasma to dry on the slide
• Positive slide test


  If the strain shows clumping only when emulsified in the plasma and there is no autoagglutination, this strain is not further tested in the tube test
• Negative slide coagulase test


  If the strain shows no clumping in either saline or plasma, it should be further tested using the tube coagulase test
• Positive control species


  Should show clumping only when emulsified in the plasma
• Negative control species

  Should not show clumping in either water (saline) or plasma
• Negative slide coagulase test

  Is not valid and should be followed with a tube test for confirmation
• Rabbit plasma

  May be obtained by centrifuging blood to which 0.1% EDTA has been added as anticoagulant. Alternatively, it may be obtained lypophilized from a commercial supplier and reconstituted by the addition of water
• Citrated human plasma

  May be obtained from a blood bank, but the blood must have been screened and found free from viral (Hepatitis B, HIV) antigens and antibodies. It must be handled with the precautions appropriate for all human body fluids
• Storing plasma

  Store the plasma in small portions at -20°C and keep a stock of in-used plasma at 4°C bringing it to room temperature before use
• Positive and negative control organisms

  • Positive control organism: Staphylococcus aureus
  • Negative control organism: Staphylococcus epidermidis
• Isolation of Gram-positive Cocci

  1. BAP

  2. GM (+) COCCI

  3. CATALASE

  4. COAGULASE

  5. NOVOBIOCIN

  6. HEMOLYSIS

  7. OPTOCHIN

  8. BACITRACIN

  9. 6.5% NaCl
• Novobiocin

  An antibiotic interfering with the unpacking and repacking of DNA during DNA replication and the bacterial cell cycle. It binds to DNA gyrase, and blocks adenosine triphosphatase (ATPase) activity
• Novobiocin Test

  1. Immerse a sterile swab into prepared suspension

  2. Inoculate into BAP or MHA plate by streaking the swab over the entire surface

  3. Allow the agar surface to dry no more than 15 minutes before applying a Novobiocin Disk (5ug)

  4. Incubate plate aerobically for 18 to 24 hours at 35 to 37 oC

  5. Measure the zone diameter using caliper or a metric ruler
• Novobiocin resistance


  A zone of inhibition less than or equal to 16mm is indicative of novobiocin resistance
• Novobiocin sensitivity


  A zone of inhibition greater than 16mm indicates that the organism is sensitive to the antibiotic
• Optochin Test


  1. Using a sterile inoculating loop, select a well-isolated colony of the alpha-hemolytic organism to be tested

  2. Streak the isolates onto 5% sheep blood agar plate

  3. Using sterile forceps, place an optochin disk onto the inoculated surface of the agar

  4. Press disk gently with the sterile forceps so that the disk adheres firmly to the agar surface

  5. Incubate the plate at 35-37°C for 18-24 hours in 5 to 10% CO2

  6. Examine the plate after 18-24 hours of incubation, measure the zone diameter
• Optochin-susceptible strains


  Can be identified as S. pneumoniae
• Optochin test result interpretation

  Positive/Susceptible: Zone diameter of ≥ 14mm

  Negative/Resistant: No zone diameter around the disk

  Zone diameter is 9-13mm, test for Bile solubility: Bile soluble strain = S. pneumoniae, NOT bile soluble = NOT S. pneumoniae

  Zone diameter ≤ 8 mm = Viridans Streptococci
• Bacitracin Test

  1. Using an inoculating loop, streak 2-3 well-isolated colonies of a pure culture onto a blood agar plate

  2. Using sterile forceps, place a bacitracin disk in the first quadrant (area of heaviest growth). Gently tap the disk to ensure adequate contact with the agar surface

  3. Incubate the plate for 18 to 24 hours at 35-37°C in 5% to 10% carbon dioxide (CO2)

  4. Look for a zone of inhibition around the disk
• Bacitracin susceptibility test

  Used to distinguish Streptococcus pyogenes (Group A) from other streptococci
• Bacitracin test result interpretation

  Positive/Susceptible: Any Zone diameter Of >10 mm

  Negative/Resistant: No Zone diameter
• Salt tolerance test


  Salt acts as a selective agent for bacteria and interferes with membrane permeability and osmotic equilibrium. A high salt concentration thus inhibits a range of bacteria but allows salt-tolerant organisms such as enterococci to grow.
• Salt Tolerance Test

  1. Inoculate one or two colonies from an 18 to 24-hour culture into 6.5% NaCl broth with or without an indicator

  2. Incubate the tube at 35-37°C in ambient air for 48 hours

  3. Examine medium for turbidity/growth
• Salt tolerance test result interpretation

  Positive: Visible turbidity in the broth, with or without a color change from purple to yellow

  Negative: No turbidity and no color change
• Bile Esculin Hydrolysis Test

  Bile-esculin hydrolysis test is based on the ability of certain bacteria, notably the group D streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile)
• Bile Esculin Hydrolysis Test

  1. Inoculate two to three colonies from an 18 to 24-hour culture onto the surface of the agar

  2. Incubate the inoculated medium at 35-37°C for 24-48 hours

  3. Observe for growth and blackening of the medium
• Bile Esculin Hydrolysis Test result interpretation

  Positive: Diffuse blackening of the more than half of the slant within 24-48 hours indicates esculin hydrolysis= GDS (Enterococcus)

  Negative: No growth; growth and no blackening of the medium
• CAMP Test

  Certain organisms (including group B Streptococcus) produce a diffusible extracellular hemolytic heat-stable protein (CAMP factor) that acts synergistically with the beta-lysin of Staphylococcus aureus to cause enhanced lysis of red blood cells