Variables to be detailed in Materials and Methods for CLSI BMD reference method

ParameteraM07 standardData to be documented in study publication
Test medium for nonfastidious bacteriaCA-MHB manufactured according to ISO technical standard 16782:2016 (18)Brand of CA-MHB
Method for cation adjustment
Any special requirements for handling CA-MHB for specific antimicrobial agents
Test medium for fastidious bacteriaAs stipulated by M100 standard (6) or M45 guidelineBrand of alternative medium
Sources and concentrations of supplements
Any special requirements for handling alternative media for specific antimicrobial agents
Antimicrobial stock solutionsAntimicrobial powder formulation designed for AST, obtained from drug manufacturer, USP, or other appropriate commercial sources; pharmacy-grade parenteral formulations should not be usedSource of antimicrobial powder and solvent and diluent used to make stock solutions
BMD panel preparationDilution of stock solution and dispensing of 0.05 or 0.1 ml/well using pipette or dispensing deviceMethod for preparation of panels (individual or batch), volume per well, and storage conditions
Range of concentrations tested
Number of lot numbers of panels used in study
Inoculum suspension preparation and panel inoculationDirect colony suspension method or growth method to achieve 0.5 McFarland standardInoculum suspension preparation and standardization method
Dilution and inoculation of wells with pipette or inoculator device (final target of 5 × 105 CFU/ml)Method for preparation of intermediate dilutions (diluent and volumes)
Purity plate prepared (advised) (ahWG consensus is to prepare for each isolate)
Colony counts performed periodically
Method and volume for panel inoculation
Method of purity plate preparation
Targeted final organism concentration in wells
Procedure, acceptable range, and indications for colony counts
Indication of whether same inoculum is used for both BMD and cAST
Incubation procedurePrecautions taken to maintain consistent incubation temperatures and to prevent drying of panelsIncubation parameters
Temperature, atmosphere, and time varying by species (consult M07)
Reading of endpointsComplete inhibition of growth measured with naked eye (see exceptions to complete inhibition in M07b) (ahWG recommends ≥2 independent readers to determine MICs for each panel)Viewing device used to facilitate reading
Number of independent readers
Observation of any “equivocal” endpoints and how they were read (e.g., trailing) (obtaining photographs to illustrate any unique reading recommendations, if possible)
Mechanism used to arbitrate discrepant MICs between readers
QCAt minimum, following M07 QC recommendationsCLSI-recommended QC strains used
Any additional QC strains used and rationale for strain selection
  • a CA-MHB, cation-adjusted Mueller-Hinton broth; ISO, International Organization for Standardization; USP, U.S. Pharmacopeia; BMD, broth microdilution; cAST, commercial antimicrobial susceptibility test; ahWG, ad hoc working group; CLSI, Clinical and Laboratory Standards Institute; QC, quality control.

  • b Exceptions include the evaluation of trimethoprim-sulfamethoxazole, where 80% growth inhibition is read as the endpoint.