Dr Andrea Krumm reveals how a microplate reader facilitates microbiology and overcomes auto-fluorescence
Currently, there are many scientific questions that can be answered by microbial research: physicians ask how antibiotic resistances can be overcome. Synthetic biologists study if bacteria can be engineered to digest plastic and if bacteria are suited to produce fuels. Analysis of the microbiota finds out which microbes maintain homeostasis of the human gut.
The classical microbial experiment is the acquisition of growth curves. It measures how fast microbes duplicate, if growth is impaired under specific conditions and it determines lag-phase, logarithmic growth phase and the stationary phase. In addition, growth is often determined to normalise another biological measurement. The growth is typically acquired by measuring the “optical density at 600nm” (OD600). To this end, a sample of bacterial suspension is drawn every hour, placed into a cuvette and absorbance at 600nm is measured. The measurement is based on the light scattering of bacteria: the more light is scattered, the less light reaches the detector and is expressed as increased absorbance.
However, the method has some drawbacks: it is time consuming, it requires a high sample volume, a time course requires manual intervention throughout the measurement period and it is limited in sensitivity. Here, alternatives for microbial growth measurements are outlined and how they overcome these disadvantages.
From a cuvette to a microplate
The OD600 measurement can easily be transferred to the microplate format. The 96-well format allows for measurement of 96 samples in parallel as well as reduction of the sample volume down to typically 200 µl. Most importantly, the growth 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. The 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. This indicates the medium contains highly fluorescent components that are consumed and degraded by the bacteria.
The issue can be solved by recording polarised emission only. Polarised fluorescence emission can be measured by exciting the fluorophore with polarised light using polarisation filters. Light emitted in the plane parallel to excitation excludes auto-fluorescence. This is because components responsible for medium fluorescence are small molecules that rotate faster than bigger ones. This 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. The avoidance of auto-fluorescence not only enables fluorescent growth measurements, but also fluorescent assays. This 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
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. This 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. This 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
