2.1: Counting bacteria

2.1: Counting bacteria tfriedri Thu, 10/13/2011 - 18:39

Counting Bacteria

Question of the day: What concentration has the stock culture?

Materials

  • 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
  • hemocytometer

Program plate reader:

  • Bug_OD
  • A1-A12, B1-B12

Bio-hazard waste bin for

  • multi-well plates
  • agar plates after counting
  • little tubes of E. coli
  • hemocytometers
disposable_hemocytometer.pdf

2012 new thoughts

2012 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

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

E coli 2013

E 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

test-onculture18_ps1.xlsx

Reagents 2012

Reagents 2012 kdorfman Wed, 09/11/2013 - 16:17

Reagents