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A preliminar study was done on the instrument TECAN Infinite F200, a multifunctional microplate filter-based reader described in Instruments, to understand the behaviour of the measurements of absorbance and fluorescence intensity in different situations.
The results of this experimental setup, grouped by arguments, are described here. If you need more information about a single experiment, you can find the detailed protocol at Download Protocols.
Studies:
ABSORBANCE:
FLUORESCENCE:
EVAPORATION:
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CONCLUSIONS: EXPERIMENTAL SET UP AND MEASUREMENT METHODS
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• In long experiments do not use the wells on the frame, but fill them with water or growth medium to maintain the temperature homogenous in the plate;
• in long experiments cover the plate with the lid to limit the evaporation phenomenon;
• use the O.D. measurements to quantify bacterial growth, regardless of the volume in the well;
• do not dispense water if you perform fluorescence measurements;
• work with volumes not larger than 200 μl and prefer the M9 medium if you perform fluorescence measurements;
• in long experiments, if you perform fluorescence measurements on fluorescent cultures, always subtract the blank value individually on every sampling to correct the descending evolution of the fluorescence during the incubation.
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