AST

• Test tube dilution/Broth dilution method

  1. Challenge the organisms of interest with antimicrobial agents in a broth

  2. Prepare specific amount of antibiotic in a decreasing conc. in broth by serial dilution techniques

  3. Inoculate standard numbers of the test organism

  4. Determine MIC and MLC concentration

  
• Microdilution


  • Broth volume: 0.05-0.1 ml
• Macrodilution


  • Broth volume: 10 ml or greater
• MIC


  The lowest conc. of the antibiotic without bacterial growth after 16 to 24 hours of incubation
• MLC


  The lowest conc. Of the antibiotic without bacterial growth when subcultured to a fresh medium
• MH broth with 2%NA CL- to improve detection of oxacillin-resistant staphylococci
• Agar Dilution Method


  1. Bring antimicrobial conc. and organisms to be tested together on an agar-based medium

  2. Test one or more bacterial isolated per plate

  3. Determine MIC

  
• Standard inoculum size for agar dilution: 1x10 4 CFU/ml

  
• Shelf life of agar dilution plate is only one week for most antimicrobial agents
• Agar dilution is the reference method of examining anaerobes and N. gonorrhea

  
• Anaerobes: Brucella agar with laked blood and vitamin K (wadsworth method)
• Disk diffusion Method


  Place filter paper disk impregnated with various antimicrobial agents of specific concentration on an agar plate previously inoculated with the bacterium being tested
• Standard inoculum size for disk diffusion: 1.5 x10^8 CFU/ml
• Medium for disk diffusion: MHA
• Turbidity standard


  Used to ensure a standard inoculum size by comparing the turbidity of an organism suspension with a turbidity standard

  
• 0.5% Mc. Farland Turbidity Standard

  99.5 ml of 1% H2SO4 + 0.5 ml of 1.175% BaCl2 = 1.5x10 8 colony forming units/ml

  
• Preparation of pure culture for susceptibility testing

  1. Select 4-5 colonies of the same morphology, inoculate then into broth medium and incubate for 3-5 hours to achieve a turbid suspension

  2. Select 4-5 colonies 16-24 hours of age from an agar plate and suspend in broth or 0.85%NSS to achieve a turbid suspension

  3. Match turbidity using the unaided eye by holding the bacterial suspension and Mc. Farland tubes side by side and viewing them against a black-lines background

  4. If the bacterial suspension initially does not match the standard's turbidity, the suspension may be diluted, or supplemented with more organism, as needed

  
• Within 15 minutes of inoculation, the antimicrobial agent disk are applied and the plates are inverted for incubation to avoid contamination of moisture on the agar surface that can interfere w/ interpretation of test result

  
• When disk containing known conc. Of antimicrobial agent are placed on the surface of a freshly inoculated plate, the agent immediately begin to diffuse and establish a concentration in the gradient around the paper disk
• Upon incubation, the bacteria grown on the surface of the plate except where the antibiotic concentration in the gradient around each disk is sufficiently high to inhibit growth

  
• Zone of inhibition


  The diameter of the zone around the disk where no bacterial growth occurs, measured in millimeters using a caliper
• A wide zone surrounding a disk signifies more susceptibility of the organisms to the antibiotic

  
• Zone width is related to antibiotic concentration, diffusion rate through agar and solubility

  
• Susceptibility is indicated by the presence of zone of inhibition around the antibiotic disk

  
• The zone of inhibition is inversely related to MIC - the larger the zone of inhibition, the lower the MIC
• MHA containing 5 % sheep's blood is used for testing streptococci and other fastidious organisms
• Factors affecting Zone of inhibition

  • Thickness of the susceptibilty agar plate (4-6 mm)
  • The amount of inoculum or test organism
  • The growth rate of the test organism
  • Ph of the medium (7.2-7.4)
  • The number of disk per plate
  • Concentration of divalent cations (Ca and Mg)
• Causes of false Resistance

  • Use of unduly heavy inocula of cultures or undiluted specimen materials
  • Late examination of test plates after zone have become over grown
  • Use of disc with inadequate drug concentration due to prolonged storage, failure to refrigerate as from disc container opened frequently
  • Use of wrong organism
• Etest


  1. Dilution test based on the diffusion of a continuous concentration gradient of an antimicrobial agent from a plastic strip into an agar medium

  2. Plastic strips is contains dried, predefined concentrations of stabilized drug on one side and a continuous MIC interpretative scale of the other side

  3. Place the strip on the surface of the culture medium inoculated with the desired organism