2.1: Counting bacteria2.1: Counting bacteria tfriedri Thu, 10/13/2011 - 18:39
Question of the day: What concentration has the stock culture?
- Clean clear 96 well plate
- Sterile glass beads
- Sterile microfuge tubes
- Ice buckets (fill with ice at beginning of period)
- 8 LB plates per pair
- ice cold LB broth
- ~4 mL E. coli in stasis - ice cold
- spray bottle of 70% EtOH to clean tables
Program plate reader:
- A1-A12, B1-B12
Bio-hazard waste bin for
- multi-well plates
- agar plates after counting
- little tubes of E. coli
2012 new thoughts2012 new thoughts kdorfman Tue, 10/09/2012 - 21:34
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
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.
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
E coli 2013E coli 2013 kdorfman Wed, 09/11/2013 - 16:15
Week before lab:
streak out control cells on agar plate
grow overnight at 37C
cover plate and refrigerate
Two days before lab:
pick colony, put into enough LB for all students (~250 mL)
grow on shaker overnight
Day before lab:
put LB in fridge
- need ~15 mL
- 2013 - have lots of 25 mL tubes of LB. Use them up
check during day for stasis (OD = ~2.6 in previous years; growth seems to stop at OD = ~2, which is still in the linear range)
NOTE: If left too long on shaker, get too many in chains. Check culture first thing in the morning with microscope. OD = 2, still swimming. Put in fridge.
refrigerate to stop growth
Dilute or spin down and resuspend to OD = ~2.5 (around max linear correlation to conc) with ice cold LB
Aliquot ~4 mL/tube
1 tube per pair
Reagents 2012Reagents 2012 kdorfman Wed, 09/11/2013 - 16:17