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MICROPLATE READERS


is recorded continuously without any manual intervention. An example for the OD600 microplate measurement is shown in Fig. 1. Two strains of group B streptococcus (GBS) were measured in triplicate every 15 minutes for 12 hours in total. Te measurement resulted in a typical growth curve with lag-phase (0-2 h), log-phase (2-4.5 h) and stationary and death phase (>4.5 h).


FLUORESCENCE WITHOUT AUTO-FLUORESCENCE A second alternative to OD600 measurements is growth monitoring by stable fluorophore expression. To this end, a plasmid coding for a fluorescent protein needs to be inserted into the bacterial strain of interest, e.g. by electroporation. Since each cell expresses the fluorophore, fluorescence intensity directly reports on bacterial multiplication. Fig. 2A shows the increase of fluorescence intensity of streptococcus B (GBS) expressing a mutant and bright green fluorescent protein (GFP). However, measuring native (non-fluorescent) GBS in parallel revealed a significant problem: auto- fluorescence. Medium without any bacteria served as blank for both GPF- expressing and non-fluorescent GBS. Wells with medium only display higher fluorescence than samples with medium and non-fluorescent GBS, which leads to negative values in blank-corrected samples. Tis indicates the medium contains highly fluorescent components that are consumed and degraded by the bacteria.


Te issue can be solved by recording polarised emission only. Polarised fluorescence emission can be measured by


Fig.2. Fluorescent growth monitoring of GFP-expressing group B streptococcus. Medium only was used as blank and was subtracted from GFP-expressing (green) and non-fluorescent (black) GBS.A: fluorescence intensity measurement reveals high auto-fluorescence B: recording polarised fluorescence abolishes the adulteration by auto-fluorescence


exciting the fluorophore with polarised light using polarisation filters. Light emitted in the plane parallel to excitation excludes auto-fluorescence. Tis is because components responsible for medium fluorescence are small molecules that rotate faster than bigger ones. Tis means, emission is depolarised and hence not detected when detecting only the polarised emission. Fig. 2B shows the avoidance of auto-fluorescence by measuring polarised emission. Blank- corrected fluorescence values for non- fluorescent GBS stay around baseline whereas GFP-expressing bacteria show an increase in fluorescence. Te avoidance of auto-fluorescence not only enables fluorescent growth measurements, but also fluorescent assays. Tis way, growth by OD600 can be read simultaneously with any other biological parameter, e.g. gene expression using a reporter gene. In summary, a switch to microplates


provides the following advantages: higher throughput; multiplexing – measure several assays in parallel; reading without manual intervention


Fig.1. Absorbance-based growth curve of two strains of groups B streptococcus. Absorbance in a 96 well plate at 600nm was acquired every 15 min for 12 hours


20 www.scientistlive.com


THE APPROPRIATE EQUIPMENT Even though many microplate readers are capable of measuring absorbance at 600nm and/or green fluorescence, there are a few more aspects to consider for the transition from cuvettes to a


microplate. Real-time monitoring is only possible if the growth conditions for the organism of interest can be set inside the reader. Tis means that temperature needs to be adjustable. BMG Labtech microplate readers incubate up to 65°C and can accordingly be used to study thermophiles. Some organisms require specific carbon dioxide or oxygen concentrations that demand an independent regulation of both to provide suitable growth conditions. Tis can be achieved using an atmospheric control unit (ACU). For aerobic bacteria, a constant supply of oxygen needs to be guaranteed. Just as in large volume broth cultures, shaking ensures sufficient oxygen supply in microplate cultures. In order to stand continuous shaking, BMG Labtech’s Omega series and Clariostar microplate readers can be equipped with a dedicated plate carrier. It not only stands shaking but also prevents microplate abrasion inside the reader. Due to its robustness and highly sensitive polarisation measurements the Clariostar was used for the data shown above.


Dr Andrea Krumm is with BMG Labtech. www.bmglabtech.com


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