Adapted Test Protocol
Preparation of Bacteria Culture
- Transfer 100 µL of bacteria from glycerol stock into 3 mL compatible liquid medium in a 15 mL test tube. Cultivate culture overnight at 200 rpm and 37 °C.
- Next day, inoculate 500 µL aliquot of preculture into 100 mL of a compatible medium in a 500 mL Erlenmeyer and incubate at 37 °C with 200 rpm shaking until OD600 value reaches 0.1. Prepare 9 Erlenmeyer to have 3 replicates of sample with antibacterial agent, 3 replicates of samples without antibacterial agent and 3 replicates of culture without sample.
Preparation of Samples
- Cut polymer samples into square pieces of 1 cm2 (30 pieces).
- Prepare samples of 1 cm2 without antibacterial agent treatment as control group (30 pieces).
- Remove impurities (e.g. wash with acetone if applicable).
- Sterilize samples (e.g. autoclave at 121 °C for 20 minutes if applicable).
Monitoring Bacterial Lag-Log Phases in the Presence of Antibacterial Agent
- When OD600 values of bacteria reaches 0.1, add 10 pieces of specimen with and without antibacterial treatment into 500 mL Erlenmeyer and continue shaking at 200 rpm at 37 C°.
- Measure OD600 value of bacteria in every 30 minutes until it reaches 2.0.
- Standardize relative OD600 values to evaluate influence of antibacterial polymer on the growth rate of bacteria.
- Use means of 3 cultures to create lag-log phase graphs.
- Luria-Bertani for E. coli DH5α.
- Mueller-Hinton II for S. typhimurium.
- High Salt Medium for B. subtilis BCC 6327.
- 665 mL of distilled water.
- 100 mL of 10X Spizizen’s salt.
- 25 mL of 20% w/v glucose.
- 50 mL of 0.1% w/v L-tryptophan.
- 10 mL of 2% w/v casein.
- 50 mL of 10% w/v yeast extract.
- 100 mL of 8% w/v arginine.
- 4% of w/v of histidine.
Reference: J. APPL. POLYM. SCI 2016. DOI: 10.1002/APP.43331