cell counting

[johnluc]

Hi all, does anyone on here use a microscope to count yeast cells? i've just started doing this and have some questions for the experts.
johnluc.

[killer]

Ask away anyway...

I'm far from an expert but I have counted yeast and bacteria cells using a microscope. if I can't help perhaps someone more knowledgeable can.

[johnluc]

this is what i posted on another forum:Hi all, following on from my original post i took the plunge and bought the gear to start counting cells, microscope,hemocyytometer,methalene blue and other bits and pieces and it's quite interesting, one thing i can't get my head around is the inconsistency of cell counts, did ten counts from same batch and only two came out the same and some were so vastly different (10 - 150 billion) :scratch: that it had me wondering what i had done wrong, could it be my hemocytometer, cheapish off ebay from China although the same one from the States or Europe were up to 6 times more expensive, if anyone on here counts cells any advice would be greatly appreiciated.
johnluc.

[killer]

There's a useful tutorial here

http://bitesizebio.com/13687/cell-count ... -as-1-2-3/

I would add:

Probably the main reasons for getting inconsistent numbers between counts is not homogenizing the sample. Make sure you get your cells properly suspended by agitating and take your sample straight away. If you agitate the sample and then let it settle, your cells will just fall back down to the bottom of your vessel they are in – you’ll end up taking a sample with a reduced cell count. Agitate well every time you are about to take a sample

The same thing is true of transferring the suspension to your haemocytometer – make sure your cells aren’t being lost by sticking to the glass or whatever you are using to transfer the sample. How are you transferring your cell solution ? A pipetman is best but most people won't have access to one of these.

Also make sure you pick the right grids to count in your haemocytometer – when you add your solution don’t pick outlying grids that haven’t fully filled up with liquid/ solution or you'll have a reduced cell count.

[johnluc]

thanks for your reply and link Killer, what i do is while my starter is still on stir plate, using a 5ml sterile pipette and pipette pump draw a sample out of flask, this way the yeast is in suspension so should get a uniform sample, and with the pump it's easy to dispense an accurate amount. where i think i've gone wrong is buying a cheap Hemocytometer from China on ebay, got a good one on way, only cost 10x as much
johnluc.