I. Antimicrobial preparation
- Determine the concentration of a given antibiotic you would like to use for the time kill assay. Here, we selected Penicillin G, at concentrations 1 MIC and 4 MIC respectively. Hence, prior determination of MIC (minimal inhibitory concentration) is required. We recommend both E-tests and microbroth dilutions to determine MICs. For Gentamicin, we selected the following concentrations: 4mg/L and 12.5 mg/L.
- Prepare the antimicrobials. We used the same vials that are used in clinical practice. Hence, antimicrobial agents were ordered from the hospital pharmacy. Gentamicin concentrations were diluted from original vials (80 mg/2 ml), and penicillin concentrations were diluted from original vials (1 Mio IU).
- Dilute the antimicrobial agent in sterile water (or NaCl or PBS but not in Glucose) until a concentration is achieved that is 100 times higher than the one used in the experiment. Then dilute again once (i.e. 1:10) in Todd Hewitt broth (THB). Hence, you have a concentration in THB that is 10 times higher than the one used in the final experiment. In the final experimental tube the concentration will be diluted 1:10 to reach the desired concentration (See IV.2).
II. Inoculum preparation
Day 1: Plate clean bacterial stock from the -80°C on 3 blood agar plates (CSBA), and incubate over night at 37°C with 5% CO2.
Day 2: Pick 1 colony from each plate and subculture on another CSBA plate (i.e. 3 subculture plates).
Day 3: Next day, pick 1 colony from each plate and do an overnight culture in 10 ml THB at 37°C with 5% CO2 (i.e. 3 THB overnight cultures).
Day 4 (day of the experiment). Take 0.5mL of the overnight culture and add it to 9.5ml THB (i.e. total volume 10 mL). Incubate at 37°C and 5%CO2 until mid-log phase is reached.
Once mid-log phase is reached, bacteria are at a stage to be used for the experiments. Hence, dilution till the desired inoculum is required.
(To estimate the correct dilution, a growth curve prior to the experiment is necessary. On the basis of that growth curve, you should be able to estimate how often you need to dilute until you reach your desired inoculum.)
We use 1:10 dilutions, until we reach 107cfu/mL (which is 10 times higher than the cfu used in the experiment). In the final experimental tube the concentration will be diluted 1:10 to reach 106cfu/mL (See IV.2).
- Monotherapy with antibiotic 1 at a specific concentration. In our experiments, we used penicillin 1 MIC and 4 MIC.
- Monotherapy with antibiotic 2 at a specific concentration. In our experiments, we used gentamicin 4 mg/L and 12.5 mg/L.
- Combination of 2 antibiotics at a specific concentration. (e.g. penicillin 1 MIC plus 4 mg/L gentamicin, or penicillin 4 MIC plus 12.5 mg/L gentamicin, etc.). Depending on the number of combinations, the numbers of settings will increase.
- Growth control (bacteria without antibiotics).
- Negative control (antibiotics without bacteria).
- For each time point, we recommend triplicates to read out results.
IV. Time-kill assay
- Prepare three falcon tubes (15ml) per time point (triplicates). For time point 0 (starting point) and time point 24h (end point), we used the same falcon tube. In addition, we used the following time points to read out results: 0.5, 1, 2, 3, 4, 6, 8, and 12h. Hence, we used 27 falcon tubes (triplicates x 9 time points) per setting.
- Fill each tube with THB, antibiotics (see I.) and bacteria (see II.). Use volumes as illustrated below:
• Monotherapy with antibiotic 1: 4 mL THB, 0.5 mL bacteria, 0.5 mL antibiotic 1 (tot. volume = 5 mL).
• Monotherapy with antibiotic 2: 4 mL THB, 0.5 mL bacteria, 0.5 mL antibiotic 2 (tot. volume = 5 mL).
• Combination: 3.5 mL THB, 0.5 mL bacteria, 0.5 mL antibiotic 1, 0.5 mL antibiotic 2 (tot. volume = 5 mL).
• Positive control: 4.5 mL THB, 0.5 mL bacteria, no antibiotics (tot. volume = 5 mL).
• Negative control: 4.5 mL THB, 0.5 mL antibiotic 1 and/or 2, no bacteria (tot. volume = 5 mL).
- Vortex all falcons.
- From T0, obtain 100 uL, dilute accordingly, and spread out for colony counts on CSBA plates (read out next day).
- Incubate all falcon tubes at 37°C with 5% CO2.
- Remove a complete sample set (triplicates) for each time point (see IV.1.), dilute and plate (at least two dilutions per sample) accordingly (see IV. 4.).
- Count colonies the next day and plot them.