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The responses of 20 ampicillin-susceptible Haemophilus influenzae type b clinical isolates to the bactericidal action of ampicillin were studied by using a modified agar dilution plate count method. A well-defined paradoxical effect was observed in each of the 24-h

The bactericidal dynamics of oxacillin against four Staphylococcus aureus isolates with known 24-h "persister" percentages were studied by using the agar dilution plate count method. Isolates were selected to provide a representative spectrum whose individual 24-h trough intrinsic persister percentages ranged from greater than 1 to less than 0.01%. Resultant agar dilution plate count method killing curve patterns were found to be reproducible and served to characterize each isolate. The paradoxical effect was observed for each isolate, with paradoxical peaks tending to develop and diminish sequentially during the course of oxacillin action. The observed strain-dependent dynamics of oxacillin killing underscore the artifactual nature of the so-called tolerance phenomenon and negate the usefulness of MBCs and MBC/MIC ratios for the characterization of S. aureus isolates.

The effect of increased temperature on Staphylococcus aureus during the inoculation step of the agar dilution plate count method was investigated as a possible cause of artificially high persister counts. For some isolates, exposure of the inoculum to increased temperature resulted in higher persister counts and diminution or loss of the paradoxical effect. The persister patterns for three representative S. aureus isolates are presented to illustrate the strain-and temperature-dependent nature of the phenomenon. For any isolate, the net effect appears to be caused by an interplay of temperature-induced inoculum loss and temperature-induced cell division cycle blockage. A modification of the agar dilution plate count inoculation step to circumvent such problems is described.We previously introduced the agar dilution plate count (ADPC) method as a procedure for the quantitative measurement of bacterial inhibition and killing by P-lactam agents (32,33). From continued work with Staphylococcus aureus, we have now found that occasional strains may be affected by increased temperature exposure during the ADPC inoculation step, resulting in variable and falsely high persister measurements. We present here the persister patterns determined for three S. aureus isolates selected to represent the temperature-related effects that we observed. Based on an analysis of the patterns for these isolates, the probable operative mechanisms are presented and discussed and modification of the ADPC inoculation step to avoid such problems is described.MATERIALS AND METHODS S. aureus strains. S. aureus C13, R4, and SPRMC 9942 were selected as representative strains. Isolates were retrieved from stock storage at -70°C in defibrinated sheep blood by two 24-h passages on sheep blood agar incubated in air at 35°C. ADPC inoculation methods. For each isolate, a log-phase inoculum suspension was prepared as previously described (32, 34) to provide a common source for the inoculation methods to be compared. The methods were designed to represent conditions of increasing temperature exposure ranging from those which should not cause inoculum damage to those for which inoculum damage might be expected. The conditions compared were as follows.(i) Method 1. Approximately 3 x 105 CFU contained in a 0.1-ml sample of an appropriate Mueller-Hinton broth (MHB; Difco Laboratories, Detroit, Mich.) dilution of the common source were pipetted onto the surface of a prepared ADPC plate containing oxacillin (Bristol Laboratories, Syracuse, N.Y.). The plate was then streaked with a bacteriologic loop in an overlapping fashion to thoroughly distribute the inoculum over the entire surface.(ii) Method 2. Plates were prepared as described for method 1 followed by overlaying the streaked surface with * Corresponding author. 10 ml of 48°C molten Mueller-Hinton agar II (MHA; BBL Microbiology Systems, Cockeysville, Md.) having the same oxacillin concentration as that of the ADPC plate.(iii) Method 3. Approximately 3 x 105 CFU contained in 0.1 ml of an appropriate MHB d...

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