BIOL5579 SP2012

Bacterial Growth Growing a bacterial strain in liquid complex medium. Determining the effect of antibiotics or glucose on bacterial growth. 1. Start a fresh culture by diluting the o/n provided to you 1:100 or 1:10 depending on your team. Start a 20ml culture in complex LB medium. 2. Incubate at 37C for the remaining of the session. 3. Measuring by OD600: take 1mL sample from the growing culture every 30min and transfer to a cuvette. If OD is over 1.0 make dilutions (e.g. or 1/5) so that the measurement is in the linear range of the spectrophotometer. Measure and record the optical density at 600nm. Remember to zero the instrument with a cuvette filled with the noninoculated liquid complex media. 4. Measuring colony forming units (CFUs): take 0.1 mL sample every hour. At each time point serially diluting the sample [0.1 mL into 0.9 mL of saline buffer; 0.85% NaCl; SB]. Repeat the dilution procedure 5 times. Plate 0.1 mL of each dilution into 1 plate of complex LB medium. Spread with sterile glass beads. Incubate. 5. Graph OD or CFUs vs time. 0.1 ml 0.1 ml 0.1 ml 0.1 ml

o/n

0.9 ml SB

Example of 1/10 serial dilutions.

Groups 1 2 3 4 5

OD600 No No Yes Yes Yes

CFUs Yes Yes No No No

(dilution) ~108 (1:10) ~108 (1:10) ~107 (1:100) ~107 (1:100) ~108 (1:10)

Antibiotic (ug/mL) /glucose (%) Yes (5g/mL) No Yes (0.2%) No Yes (50ug/mL)

BIOL5579 SP2012

Background The strain in this experiment is an E.coli K12. We will address the following questions: 1. What method is best to determine bacterial growth? Optical density or counting colony forming units (CFUs) and why. 2. Adding antibiotic or Glucose to the growth media: Does we observe an effect on growth? Have fun growing bacteria!