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May 15, 2010 (Vol. 30, No. 10)

Improving Antibiotic Susceptibility Testing

Applying a Novel Microplate Photometer for the Assessment of Bacterial Growth

  • Results

    Click Image To Enlarge +
    Figure 1. The growth curves of Salmonella typhimurium at varying antibiotic concentrations (0.2–200 µg/mL)

    Bacterial growth. As shown in Figure 1, a difference in S. typhimurium growth speed is caused by antibiotic inhibition, which was easily detected using OD measurements. SkanIt software automatically created kinetic curves during measurement.

  • Click Image To Enlarge +

    Turbidometric performance. Table 1 presents the results of OD measurements obtained from the Multiskan FC and a cuvette spectrophotometer. The OD of the 1:10 dilution of the culture was measured at 0.319 using the cuvette spectrophotometer, which was subsequently used to calculate a range of theoretical ODs.

    The theoretical values were compared to the actual data obtained by the cuvette spectrophotometer and the microplate photometer. Since the pathlength in microplate wells is different from the 1 cm cuvette pathlength, the microplate values were corrected accordingly. The calculated difference between the theoretical and measured values shows that the data obtained using the microplate photometer correlates to the theoretical values across a wider range of the dilution series than that obtained using the cuvette spectrophotometer.

  • Click Image To Enlarge +

    Evaporation studies. The results of the evaporation study are shown in Table 2, where they are presented as both water weight loss in grams and as a percentage of the initial weight. A long incubation time clearly causes the liquid to evaporate when the plate is not covered, however the extent of evaporation is not significant for example at a seven-hour time point with either 96- or 384-well plates.

  • Click Image To Enlarge +
    Figure 2A. Evaporation on different parts of a 96-well plate during incubation at 37ºC and 50ºC without a lid. Y-axis: % absorbance at 975 nm, x-axis: plate columns, z-axis: plate rows

    As shown in Figure 2A, absorbance clearly decreases toward the edge of the plate, signifying that the wells located at the microplate edge suffer increased evaporation. Figure 2B demonstrates the difference in evaporation when a microplate is incubated with and without a lid. As expected, the use of a lid efficiently decreases the rate of evaporation, with no anomalous results caused by an accumulation of condensation.

  • Conclusion

    Click Image To Enlarge +
    Figure 2B. Evaporation on different parts of a 384-well plate during incubation at 37ºC with and without a lid. Y-axis: % absorbance at 975 nm, x-axis: plate columns, z-axis: plate rows

    These antibody susceptibility studies demonstrate that the Thermo Scientific Multiskan FC is suitable for measuring bacterial turbidity. The liquid evaporation that occurs must be taken into account when incubating at elevated temperatures over longer periods of time. However, these effects can be reduced through the use of a microplate lid, or filling the edge wells with water.

    The Multiskan FC produces accurate resulting data from a microplate with a lid, minimizing the effects of evaporation without producing any anomalous results due to a build-up of condensation.



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