Hi everybody!
I was making a in vivo absorption spectrum from cultured filamentous brown algae and the spectrum that I have obtained is really similar with previous reports in this kind of algae. However, I have a baseline shift of 0.029 (when I use samples with low OD = 0.020) and 0.073 (when i use samples with higher OD=0.6). I m not sure if there is a way to correct these shifts or if I just simply can present the data like that. As a blank, I m using autoclaved culture medium.
Also, I have one more question: is it good a 7 nm intervals for making an absorption spectrum? When I made it with 2 nm I obtained too much noise (even with Savitzky-Golay method to smooth the data). With 7 nm intervales I obtain a spectrum really similar to that it is know for brown algae.
Many thanks in advance for your answers
Have you tried suspending your cells in 60% sucrose? Works well on most unicellular algae and photosynthetic bacteria. Other approach is to use a spectrophotometer with a Taylor Integrating Sphere. Such equipment is rare. Just a matter of luck if you can find someone who has one. The integrating sphere attachment costs about as much as the spectrophotometer itself. I had access to two (at Uni of Sydney and UTS) in Australia but I cannot find one in Thailand.
If you do not happen to have an IS, there is still a workaround. Basically, you deposite the cells on a GF/F glass-fiber filter and cover with it the output (detector) window of the spectrophotometer cuvette compartment (cells facing the detector!), another (empty) filter is used as the blanc. Be sure that both filters are wet but not dripping. See also the file attached. Good luck!
Thanks so much for your answers! Actually, I m using a microplate reader. I will try your suggestions! Thanks so much again
with most plate readers, the OD=0.020 is not interpretable. Basically, you cannot use such low densities in plate-based assays. Do you have a calibration curve covering this range?
Solovchenko's suggestion of using a near Lambertian material like a glass fibre filter is a good one. Filter your beast onto a glass fibre filter and then use a cork borer to make disks small enough to fit in your plate wells. White surface down.
My suggestion of 60% sucrose probably will work as well. See my PS bacteria papers on Research Gate.
Thanks so much for ur suggestions. I will check the paper u recommend me Raymond :)
Some additions relevant to measurements of a turbid samples with a plate reader:
- use a white-walled, transparent flat bottom plate if available
- consider using a multiple read feature (if supported by your reader) overing as much sample area as possible
- double-chec that the readings are in range.
P.S. 50% glycerol also works well.
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