Microdilution Transfer Plate Technique for Determining In Vitro Synergy of Antimicrobial Agents

Abstract

A microdilution transfer plate technique for determining in vitro synergy of antimicrobial agents is described. Combinations of gentamicin-nalidixic acid against Proteus mirabilis and rifampin-amphotericin B against Candida albicans are used as examples to demonstrate the technique. Results correlate with published data obtained by conventional methods. The technique is effective for evaluating the in vitro effects of antimicrobial agent combinations against both bacteria and fungi. The technique enables one to produce a checkerboard gradient in a fast, convenient, and reproducible way; results are easily visualized.In vitro synergy of antimicrobial agents is traditionally accomplished using a tube broth dilution "checkerboard" in which several concentrations of one drug are each combined with concentrations of a second drug (4,14). Agar dilution can be applied to this technique (11). Several agar diffusion methods are also described (7,9,12). Microdilution methods have been described for determining synergy (3, 5, 6, 15, 16). Two methods (15, 16) involve dilution of both drugs before pipetting each into the microdilution plate. The method of Kluge et al. (6) requires diluting one drug in the microdilution plate, then adding the second drug (already diluted) to the wells containing the first drug. In two other methods described (3,5) Cultures and growth media. P. mirabilis (ICN 40) was used for the nalidixic acid-gentamicin combination; brain heart infusion (Difco) was the medium used for this example. C. albicans (ICN 80) was used for the amphotericin B-rifampin combination; a medium containing a yeast-nitrogen base (Difco), Lasparagine, and dextrose was used (13).All incubations were for 18 to 24 h at 35°C. Synergy assay. The test for synergy was performed using a 96-well, flat-bottom microdilution plate (Microtest II, Falcon, Div. of Becton, Dickinson & Co., Oxnard, Calif.) and 50-Al microdiluters (Cooke) to dilute drug I. Drug II was diluted in a transfer plate (Cooke) using 25-,l microdiluters (Cooke). Wells in the transfer plate have a calibrated orifice designed to retain the material in the well by surface tension until contact is made with the fluid in the flat-bottom plate. This contact breaks the surface tension and allows all wells ofthe transfer plate to drain. The system includes a transfer plate holder and carrier, which were autoclaved before use. Pipetting into plates was accomplished using precision pipettes with sterile, disposable tips (Medical Laboratory Automation Inc., Mt. Vernon, N. Y.).The flat-bottom plate was prepared by first pipetting 50 ,l of growth media into each well. Next, 50 ,ul of drug I was pipetted into all wells of row B (12 wells across). Twofold serial dilutions were then made from row B through row G using 50-/l microdiluters.The transfer plate was prepared by first pipetting 25 ,ul of growth media into each well. Next, 25 Al of drug II was pipetted into all wells of column 2 (8 wells across). Twofold serial dilutions were made from column 2 through column 11 using 2...

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