BACTE

• Clinical and Laboratory Standards Institute (CLSI), formerly known as “The National Committee for Clinical Laboratory Standards (NCCLS)” nonprofit organization with members representing multiple disciplines.  
• European Committee on Antimicrobial Susceptibility Testing (EUCAST) - harmonize breakpoints for antimicrobial agents in Europe
• • To act as the breakpoint committee for the European Medicines Agency (EMEA) during the registration of new antimicrobial agents.
• • Store the plates at 2-8°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%
• →  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 2-8°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:

  • O.5 Mcfarland standard
  • Prepared by 0.5ml of 0.048mol/L of 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
• AST Methods:

  • Disk diffusion method
  • MIC method
  • E Test
  • Automated systems
• AST under diffusion

  • Kirby bauer method
  • Stokes method
• AST under Dilution

  • Tube dilution
  • Agar dilution
• Diffusion and Dilution

  • E test
• Kirby Bauer Method Types:

  1. Direct Colony Suspension Method
  2. Inoculum (Log Phase ) Method
• 1. 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 the following organisms:
  • All staphylococci
  • Fastidious bacteria that grow unpredictably in broth: e.g., Streptococci
• 2. Inoculum (Log Phase ) Method

  • 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.
• 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.
• • S.aureus ATCC 25923
  • E.coli ATCC 25922
  • P.aeruginosa ATCC 27853
• Types of MIC

  • Broth dilution method
  • Agar dilution method
• Minimum Iinhibitory Concentration – lowest concentration of antimicrobial that will inhibit the visible growth of an organism in an ideal growth condition
• Minimum Bactericial Concentration – least concentration of the test antibiotic which will completely kill the bacteria tested
• • Reflected light is used for Enterobacteriaceae, such as E. coli, other gram-negative bacilli, staphylococci, and enterococci (except for oxacillin and vancomycin).
• Use transmitted light, rather than reflected light, when measuring zones for:

  • Staphylococci with oxacillin
  • Enterococci with vancomycin
• 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.
• • Beta lactamase negative – ATCC E.coli 25922
  • Beta lactamase negative – ATCC staph.25923
• • Aminoglycosides – P. aeruginosa ATCC 27853
• • Cephalosporins – H. influenzae ATCC 49766
• • Thymidine levels – E. feacalis ATCC 29212
• • Beta lactamase positive – ATCC E.coli 35218
  • Beta lactamase positive – ATCC staph. 38591