Start with ice cold culture in stationary phase, OD = ~1.5

Make a 2-fold dilution series (in microtubes?)

Make a 10-fold dilution series (in microtubes?)

Load 96 well plate

Get OD’s

Keep cells on ice!

Make OD vs dilution curve (combine data from 10-fold and 2-fold dilution series)

Count cells from some well (should have theoretical OD = ~ 10-6).

Too many? Use a more dilute one. Too few? Go up one.

Make agar plate from 8 wells

Calculate OD (from Od vs dilution factor) for the wells used in hemocytometer

Calculate OD for the wells used in the agar plate

Get cells/mL/OD conversion factors by both methods. Compare.

OR: make a table: if OD of first well is __, then use well# __ for the hemocytometer.

**Important observations**

A stationary phase culture (left at RT for several days) has OD = 2.6

Relationship between dilution and OD is linear through about OD = 1.5

By hemocytometer counts, there are ~5 x 10^8 cells/mL/OD

Cells are countable (19 cells/0.004 µL and 27 cells/0.005 µL) in the hemocytometer at 5 x 10^6 cells/mL, or blank corrected OD = ~0.01 (about where it slips into the noise)

So recommend either

- to start with well #9 in the 2-fold dilution series, or
- if they have done the OD's first, use the first well with a blank-corrected OD below 0.03