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

Improving Antibiotic Susceptibility Testing

Applying a Novel Microplate Photometer for the Assessment of Bacterial Growth
  • Reija-riitta Harinen

As commonly prescribed treatments for bacterial infection, antibiotics are substances or compounds that kill (bactericidal) or inhibit the growth (bacteriostatic) of various bacterial pathogens. Many factors impact the efficacy of an antibiotic, such as the location of infection, the bioavailability of the antibiotic at this site, and the ability of the bacteria to resist or inactivate the antibiotic. As a result, antibiotic susceptibility testing has become an extremely important process to accurately assess the activity of specific antibiotics against a target infection.

Usually carried out to determine which antibiotic will be most successful in vivo, antibiotic susceptibility testing can be conducted using a nephelometer or a photometer to assess the turbidity of a liquid sample.

The measure of turbidity, or optical density (OD), refers to the scattering of light through the measured sample. Therefore, when measuring the OD of a bacterial culture, the amount of scattered light is directly proportional to the number of bacterial cells in the sample.

If the antibiotic has been successful, the bacterial growth is inhibited and a low OD value will therefore be obtained. If the antibiotic has been ineffective, the bacteria will continue to proliferate and the OD will consequently be higher.

In this study, we investigate the effectiveness of a filter-based microplate photometer, the Thermo Scientific Multiskan® FC from Thermo Fisher Scientific, in producing reliable and accurate bacterial turbidity measurements in antibiotic susceptibility studies. Growth curve measurements, turbidometric measurements, and liquid evaporation were all studied.

Methods

Three different methods were carried out—bacterial growth, turbidometric performance, and evaporation studies. In two of these, Salmonella typhimurium were cultured in 96-well microplates, which enabled the simultaneous measurement of a large number of samples. As such, different growth conditions could easily be tested.

Bacterial growth curves. S. typhimurium were cultured for 16 hours in the presence of varying concentrations of the antibiotic erythromycin (a bacteriostatic macrolide). The Multiskan FC was programmed using its accompanying SkanIt software to automatically measure the OD every 15 minutes, shaking the plates during each interval. The instrument temperature was maintained at 37ºC throughout each measurement session.

Turbidometric performance. S. typhimurium were cultured to their late logarithmic phase, with an OD of 3.19. A 1:2 dilution series was subsequently prepared from the culture, and 300 µL of each dilution added to separate wells of a 96-well plate, with seven replicates. The OD of the dilution series was measured at 595 nm using the Multiskan FC. A cuvette spectrophotometer was used to calculate theoretical ODs for the dilution series as a point of comparison, based on a 1:10 dilution of the culture.

Evaporation studies. Two 384-well microplates, one with a lid and one without, were filled with 50 µL of water per well and incubated at 37ºC in the Multiskan FC for 24 hours. Evaporation was also measured using a 96-well plate filled with 300 µL of water per well, where the plate was incubated at 37ºC and 50ºC without a lid. Each plate was weighed and the change in water absorbance at 975 nm measured at 0, 1, 3, 7, and 24 hours.

Results

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.

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.

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.

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

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.