I have recently started culturing different phytoplankton species in 24-well microplates and taking absorbance measurements using the microplate reader FLUOstar OMEGA. Surprisingly, OD data for all the species have been decreasing over the week, even though it was noticeable that the wells were getting greener and greener, which does not make sense. OD data seem fine when I use the 96-well microplate, so I supposed it had something to do with the 24-well new protocol (maybe the scanning feature, which was the only setting that was changed in the protocol). However, I tested an empty microplate under this new protocol, colouring in black some of the wells, and the results made sense, as OD data were higher in those that were coloured. I don’t know what can be causing this incongruity, can anyone come up with a possible reason??
Thank you very much,
Macarena Blanco
I am not familiar with this particular microplate reader. Can you give some information about the settings you were using? For example, at which wavelength/s did you measure absorbance?
Hi Sebastian,
I measured it at 450, 600 and 660nm, though I am mostly interested in the measurements at 660nm. Regarding scan settings: matrix scan (dimension 3x3, 15mm width and 10 flashes per scan point).
Dear Macarena,
As you did a new setting, are you still in OD units and not in transmission ?
Hi Claude,
I am still in OD units. However, I am now pretty sure that the problem was the diameter of the scanning, as I have changed it to 5 mm and the results are finally making more sense.
Thanks.
One of my recent Honours students also experienced some bizzarre OD (660-670nm) readings behaviour with a TECAN plate reader that measures in a very simialr way to the one you describe. We were growing filamentous species that adhered to the bottom of wells and it seems the actual read area in the well is very small, so at low cell density the reading area would read areas where there were no filaments and areas where where there was a filament in its gridded read pattern.
Increasing the number of read points and size of read points helped considerably- as did taking multiple replicate readings (3-5x) from each well to establish the level of read variability from the same well. If your cells dont ahere to the bottom- maybe programming some shaking before reading may help randomise cell distribution and even things out a bit.
Additonally- we also found that the cell concentration/OD curves for each species saturated at relatively low OD (roughly 0.5-0.6), but it varied with each species. So be aware that your capacity to detect increasing cell concentration using OD will have an upper limit, beyond which you cannot detect change.
Condenstation on inside of the well lid also had a significant and unpredictable effect on the readings- maybe because water absorbs increasingly strongly in the near IR range, but wouldnt think it overly dramatic through a short light path (approx 1cm) in plate well.
Hope this helps.
cheers, Chris
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