68
- Remove the PBS and repeat the wash and finally resuspend in
PBS - From the washed cell sample perform a tenfold dilution series
from neat to 10−^6 in PBS. - Perform the dilution series three times in parallel on each cell
sample. - Divide agar plates into three sections (see Note 16).
- Pipette three drops of 20 μL from each diluent onto the sur-
face of the agar, in each plate section, using a fresh pipette tip
each time. - Leave the plates level while the droplets dry before inverting
the plates - Incubate the plates at 37 °C for 2–3 weeks.
- Count the colonies.
- Check that the volume of liquid in the chemostat vessel is
constant. - Check that medium is entering the vessel and effluent is going
into the waste pot. - Check that medium and waste volumes are at the expected
levels and that pumps, stirrer, and magnetic flea are all working
correctly. - Check the waste level and swap the waste to an empty pot con-
taining neat disinfectant if required. - Fill in the chemostat run sheet for temperature pH, DOT, and
stirrer speed (rpm) and perform visual checks of the graphical
output from the Eycoferm controller data logging. - Sample 5 mL of the culture for optical density. Kill the cells by
the addition of 1/10 volume of 40% formaldehyde (v/v).
Shake the sample vigorously and leave for 16 h before the sam-
ple can be measured for optical density. Dilute each sample
fivefold in sterile, distilled water, and place the resulting cell
suspension in a plastic cuvette. Read the optical density at
540 nm against water (these readings are important for deter-
mining when the culture has passed into mid-logarithmic
growth and for monitoring steady-state) (see Note 17). - Monday: Carry out culture purity checks on agar (2× blood
agar and 2× Middlebrook agar plates) and measure the optical
density. - Friday: Check the waste levels and if necessary divert the waste
line to an empty waste bottle. Check that there is sufficient
medium supply available for the culture to use over the
weekend.
3.5 Monitoring
Chemostat Parameters
3.5.1 Daily
3.5.2 Weekly
Charlotte L. Hendon-Dunn et al.