3 - Bacterial growth

3 - Bacterial growth kdorfman Fri, 08/07/2020 - 15:44

Grow bacteria

Streak frozen cells on agar plate

Pick a colony and grow overnight in liquid LB (plus antibiotic if necessary)

Concentrate ON culture in centrifuge

Make 2 dilution series: 10-fold, 2-fold

  • 2 fold

    • tubes 1A - 10A
    • 0.5 mL LB in each
    • add .5 mL culture to first, mix thoroughly
    • serially dilute by moving 0.5 mL along the series*
  • 10 fold

    • tubes 1B - 10B
    • 0.9 mL LB in each
    • add .1 mL culture to first, mix thoroughly
    • serially dilute by moving 0.1 mL along the series

Take OD measurements

  • Load 250 uL of each dilution into 96 well plate
  • samples in 1st 10 wells of 1st 2 rows
  • LB in last 2 wells
  • Read, using bug OD

Plot results

  • Put fraction of original culture next to each OD
  • Cut, paste special, values only
  • Sort data
  • Make a scatterplot
  • Find the lowest concentration just above the noise for hemocytometer

Hemocytometer

  • Load 10 uL into hemocytometer.
  • Photograph.
  • Also take photographs of too concentrated and too dilute samples

Colony Counts

  • spread 100 uL from last 5 10-fold dilutions on agar plates
  • Grow overnight
  • Photograph

Part 1 of PowerPoint from 2020 remote lab

Part 2 of PowerPoint from 2020 remote lab