M BACTE: AST

• Antimicrobial Susceptibility Testing 


  Tests to detect antimicrobial resistance, a prime important key component for the physician
• Clinical and Laboratory Standards Institute 


  Formerly known as "The National Committee for Clinical Laboratory Standards (NCCLS)", a nonprofit organization with members representing multiple disciplines, with a mission of promoting the development and use of voluntary laboratory consensus standards and guidelines
• European Committee on Antimicrobial Susceptibility Testing (EUCAST)


  Harmonize breakpoints for antimicrobial agents in Europe, act as the breakpoint committee for the European Medicines Agency (EMEA) during the registration of new antimicrobial agents
• Media in AST


  • Store the plates at 2 to 8°C
  • Use within 7 days of preparation
  • Each batch of MHA plates should be checked for sterility control
  • MHA added with 2% NaCl
  • MHA added with defibrinated sheep blood 5%
• Factors Affecting AST


  • pH
  • Moisture
  • Effects of Thymidine/Thymine
  • Divalent cations
• Antibiotic Stock Solutions


  1. Buy commercial pure source of antibiotics

  2. Don't use injectable solutions

  3. Accurate weighing of powders is must

  4. Standard strains of stock cultures should be used to evaluate the stock solution

  5. After preparing the stock solution, make 5 ml aliquots and freeze it
• Dried Filter Paper Discs

  • Whatman no.1 filter paper is made to form a disc size of 6mm
  • Keep in petri dish and sterilize in a hot air oven
  • With the help of antibiotic delivery loop which has a 20G wire with diameter of 2mm the antibiotic is delivered (0.005ml)
• Storage of discs


  • Refrigerate at 2 to 8°C
  • Beta lactam class drugs should be frozen
  • The drugs should be kept outside at room temp 1 to 2 hours before work
  • The dispensing apparatus which is used to deliver the drugs should also be refrigerated
  • Check the expiry of drugs
• Inoculum - Standard


  0.5 Mcfarland standard, prepared by 0.5ml of 0.048mol/L BaCl2 and 99.5ml of 0.18mol/L of H2SO4 added with constant stirring, turbidity standard is checked by spectrophotometer - 625nm - absorbance should be 0.008 to 0.10
• Inoculum - Standard


  1. Seal the tubes containing the Mcfarland Standards and store in dark area at RT

  2. The standard turbidity should be mixed thoroughly every time before use

  3. Check the density monthly and replace it monthly
• AST Methods


  • Disk diffusion method
  • MIC method
  • E Test
  • Automated systems
• Disk Diffusion Method


  Simplest and most convenient, widely used everywhere, developed by Kirby, Sherrris, Bauer and Turk in 1966
• Types of Disk Diffusion Method

  • Kirby Bauer method
  • Stokes method
• Kirby Bauer Method - Direct Colony Suspension

  Colonies must not be older than 18–24 hours, standardize the inoculum at the same time you prepare the suspension, suspend the colonies in saline or broth (e.g. Mueller-Hinton or Tryptic Soy Agar), adjust the inoculum to a turbidity equivalent to a 0.5 Mcfarland standard, compare the turbidity of the suspensions by placing the tubes in front of a white paper or file card with black lines, use direct colony suspension for all staphylococci and fastidious bacteria that grow unpredictably in broth: e.g., Streptococci
• Kirby Bauer Method - Log Phase

  Used for most organisms that grow rapidly except staphylococci, once you have inoculated the colonies into a broth, incubate to log phase growth, log phase growth occurs after 2–8 hours incubation, following incubation, adjust the turbidity to match that of a 0.5 Mcfarland standard
• If the suspension of organisms is too turbid


  Add more broth or saline to match the turbidity of a 0.5 McFarland standard
• If the suspension is too light for direct colony suspension

  Pick more colonies and suspend in broth
• If the suspension is too light for log phase method

  Re-incubate the suspension
• Do not use over-night cultures in liquid media nor other non-standardized inocula to inoculate the plates
• Application of Discs


  150 mm plate - 12 discs, 100 mm plate - 6 discs, drug should not be relocated, distance from the lid edge - 15mm, distance between two drug from center to center - 24mm, inoculum disc placement - incubation (only 15 minutes delay is acceptable each)
• Incubating the Plate


  Invert and incubate plates with agar side up, for nonfastidious bacteria, incubate in ambient air at 35°C for 16–18 hours, for disk diffusion testing of fastidious bacteria, use NCCLS-recommended incubation conditions
• Interpretation


  After 16 to 18 hours, confluent lawn of growth, zones of inhibition uniformly circular, read with reflected light, for MRSA read in transmitted light, when proteus is tested thin veil of swarming growth after the zone of inhibition should be ignored
• Reflected light is used for Enterobacteriaceae, such as E. coli, other gram-negative bacilli, staphylococci, and enterococci (except for oxacillin and vancomycin), reflected light also is used when measuring zones on blood MHA (BMHA)</b>
• Use transmitted light, rather than reflected light, when measuring zones for Staphylococci with oxacillin and Enterococci with vancomycin
• Double zone


  Measure the innermost zone
• Colonies within the zone


  This can be due to either a mixed culture, which usually is obvious, or a resistant subpopulation of the test bacterium
• Measure the point at which you can see an obvious demarcation between growth and no growth


  Avoid straining to see the tiniest colonies, measure the obvious zone, ignore the swarm even if it covers the zone
• Zones with trimethoprim-sulfamethoxazole (and also sulfonamides and trimethoprim alone)

  May be difficult to read because this agent may not inhibit bacteria from multiplying until the bacteria have gone through several generations of growth, you may see a light haze of growth within the zone, measure the zone at the point where there is an 80% reduction in growth
• Heterogeneous Resistance in S. aureus

  The haze around an oxacillin disk when testing S. aureus is significant and should not be ignored
• Homogenous Resistance in S. aureus

  S. aureus with homogenous resistance show confluent growth up to the disk
• Stokes Method


  Built in controls against many variables and provide dependable results, a standard sensitive strain of the bacterium is inoculated in the middle third of the culture plate, the test bacterium is inoculated in the upper and lower third of the plate, antibiotic discs are placed between the standard and test inocula so that zones of inhibition formed around each disc are composed of standard and test bacteria, the results are reported as Susceptible, Intermediate susceptible, Resistant
• Standard Strains


  • S.aureus ATCC 25923
  • E.coli ATCC 25922
  • P.aeruginosa ATCC 27853
• Dilution Methods


  The minimum concentration of antimicrobial to inhibit or to kill the microorganism is determined, MIC - Broth dilution method, Agar dilution method
• MIC


  Minimum Inhibitory Concentration - lowest concentration of antimicrobial that will inhibit the visible growth of an organism in an ideal growth condition
• MBC


  Minimum Bactericidal Concentration - least concentration of the test antibiotic which will completely kill the bacteria tested
• Broth Dilution Method


  Media: cation adjusted MH broth with a pH of 7.2 to 7.4, performed in a polystyrene panel containing approximately 96 wells, Mueller-Hinton broth is recommended as the medium of choice for susceptibility testing of commonly isolated, rapidly growing aerobic, or facultative organisms, for fastidious organisms Mueller-Hinton broth may be supplemented with 2–5% lysed horse blood
• Working Antibiotic Solution


  Take original stock solution, prepare stock dilutions of the antibiotic of concentrations 1000 and 100ug/ml, arrange two rows of 12 sterile 7.5*1.3cm capped tubes in the rack, take a 30ml universal screw capped bottle, add 8ml broth and required antibiotic concentration and mix the contents, take 2ml + 2ml and put it in first tube in both rows, remaining 4ml broth - add 4ml fresh broth, transfer 2ml+2ml and put in second tube, continue this dilution up to 11 tubes, 12th tube - control
• Inoculation


  Inoculation of 1st row with one drop of overnight broth culture 1 in 1000 dilutions, 2nd row with control with known sensitivity organisms, final inoculum = 5 * 105 cfu/ml, incubate at 37degC for 16 to 18 hours, inoculate another tube with 2ml broth and keep at 4degC in a refrigerator overnight to be used as standard for determination of complete inhibition
• Interpretation


  Positive control tube - turbidity, negative control tube - clear, MIC end point is read
• Microbroth Dilution Method


  Use double strength mueller hinton broth, 4 X strength antibiotic solutions prepared as serial 2 fold dilutions, test organism is 2X106 /ml, done in 96 well plate