I’m doing a cartwheel assay to see if a bacterial strain I’ve deemed resistant from an MIC has efflux pumps. Would it be possible to use Nile red instead of EtBr for the cartwheel assays instead? the concentration is .5mg/L in the plates. I’ve tried using broth to perform the assays but only a few of the bacteria actually fluoresce.
Yes, it is possible to use Nile Red instead of EtBr for the cartwheel assay to detect efflux pumps. Nile Red is a lipophilic dye that can be used to monitor changes in the hydrophobicity of bacterial membranes, which is associated with efflux pump activity.
The concentration of Nile Red in the plates should be optimized for the particular bacterial strain being tested. A concentration of 0.5 mg/L may be appropriate for some strains, but others may require a higher or lower concentration. You may need to perform a dose-response experiment to determine the optimal concentration for your bacterial strain.
Using broth for the assay may not be the most suitable approach for all bacterial strains. The composition of the growth medium can affect the expression and activity of efflux pumps. You may want to try using solid media instead, such as Mueller-Hinton agar, which is commonly used for MIC assays. The bacterial cells can be inoculated onto the agar surface and then incubated with the Nile Red-containing media.
Additionally, you may want to consider using a positive control strain that is known to express efflux pumps, to ensure that the assay is working properly. Finally, it is important to interpret the results of the assay carefully, as there may be other factors that can affect the fluorescence of the cells, such as changes in membrane potential or alterations in membrane composition.
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