McKinney:StandardKillCurve

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This is a consensus about kill curves for M. smegmatis. Use it if you have no reason to do anything else. The method of washing before plating given here is slow, but produces the highest counts of any technique we have tried, and is in good agreement with our results from microfluidic experiments. Faster spins appear to kill otherwise viable cells after antibiotic exposure.

Materials

  • 25mL (for starter culture) + 50mL (for each kill culture) 7H9 + ADS mycobacterial medium
  • appropriate quantities of the relevant antibiotic
  • spectrophotometer and cuvettes
  • 24 LB agar plates per kill culture
  • several hundred mL of PBS + 0.1% Tween-80

Directions

  1. Inoculate a primary culture from the stock (somewhere around 100μL stock in 25mL 7H9 + ADS). Always go back to stock for doing kill curves. Inoculate and discard the vial. Do not refreeze. Do not subculture stock for kill curves. Grow primary culture to approximately A600 = 0.1-0.4 (exponential phase).
  2. From primary culture, inoculate 50mL 7H9 + ADS in a 250mL Erlenmayar flask to initial A600 ~ 0.05.
  3. Plate the first time point before adding killing agent, then add the required killing agent.
  4. Plate subsequent time points at 1h, 2h, 4h, 8h, 24h, 48h, and 96h

after the addition of antibiotics, unless you have reason to do otherwise.

To plate a time point

  1. Pipette 5mL of culture into a 15mL conical Falcon tube. Centrifugte the sample at 3500rpm for 10 minutes at room temperature.
  2. Discard the supernatant, and resuspend the pellet in 5mL PBST. Centrifuge again on the same settings.
  3. Discard the supernatant, and resuspend again in 5mL PBST. Serially dilute 100µL into 1mL PBST, and plate 100µL of each desired dilution on an agar plate (or 50µL on half plates). Spread with plastic spreaders or glass beads.
  4. Incubate plates for 2-3 days at 37°C. Count colonies on plates with between 30 and 200 colonies.
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