Microalgae count problem - (May/12/2015 )

Hello everyone,
I've recently started to work with the microalgae Chlamydomonas reinhardtii and I'm trying to find the correlation between the number of cells, the optical density (600 nm) and the fluorescence of chlorophyll (ex:425nm  - em: 685nm).
I'm encountering problems in determining the number of cells in the sample. I use a counting chamber, but I rarely obtain the same number of cells when counting the same sample and when I count dilutions, the number of cells that I find is far from the expected one.
I always accurately resuspend the sample, but I found these cells precipitate really quickly and easily adhere to the pipet tip and the test tube. I'm wondering if this might the problem, any experience and/or ideas on how to solve it?

that is absolutely the problem.

you have to take sample while ensuring that the cells are evenly dispersed, maybe while vortexing.

also, if the cells stick to the tip you may want to try "slick" tips (siliconized), if they're still available or prepare your own.

Thanky you mdfenko!
I aready vortex everytime, but I'll try different tips and tubes!
;)

have you tried taking the sample while vortexing? it's difficult with a test tube, virtually impossible with an eppendorf tube. but it may be the only way to maintain even dispersion of the algae.

not while, but I always vortex and immediately take a sample and put it on the counting chamber... I can give it a try !
Anyway I haven't siliconized tips in my lab but I've found some glass test tubes... I'll use them next week to take samples of the cultures and prepare my dilutions, I hope these will work better than plastic... I'll let you know!

also, use a wide mouth tip or clip off the narrow end of the tip.

Hello, just to let you know my assays with glass tubes worked quite well (now i can count cells with an estimate error of 10%, it could be better but at least i have an idea). anyway the number of cells decreases a little if I count a sample after a while (for example after 1-2 hours) so probably I still have a problem of adhesion but it is less important. Thank you for your help!