Cell Counting Question

[CDbrewer]

Hey forum folk,

I'm wondering if I need to include a dilution of methylene blue into my final dilution factor when calculating yeast density. Here is the quick and dirty procedure:

1. Dilute 1g yeast slurry with 45mL distilled water and 45mL 1%H₂SO₄ (1:10)
2. Dilute previous dilution to 1:100
3. Pipette 1mL of 1:100 dilution into 15mL vial, add 1 drop of methylene blue solution (concentration 1.5% or 1.5g/100 mL) into the 1mL of 1:100 dilution and mix until homogeneous.
4. Pipette onto hemacytometer and carry out counting for viability and density.

For calculating density, I take the number of live cells counting * 5x10[SUP]4[SUP] * dilution factor. The dilution factor here is 100, however, this is not including the methylene blue, should this be taken into account as well? Thanks.

-Patrick

[Ted Briggs]

Do the Math and see the difference. "Pharmacists have since moved to metric measurements, with a drop being rounded to exactly 0.05 mL". So your 1ml sample is actually 1.05ml, does this make a significant deviation in the ending count? I'm thinking no.

(just asking this gets you an A for anal retentive, but don't worry your not alone :-)