Monitoring bacterial growth
- Growth temperature
- Shaking intensity
- Generation time
- Appropriate medium
- Liquid or plate culture
Pre-culture
Streak out bacteria one to two days prior to the experiment: two days if you want to
do a selection for single clones or if you have to incubate them on antibiotics.
Addition of antibiotics usually hampers with the growth - for the determination of
growth curves, get rid of antibiotic traces. Therefore incubate them the first day with
antibiotics and the second day without.
Some bacteria do have difficulties to grow if inoculated from low density – especially
in combination of an already stationary or declining growth. Therefore some bacteria
require pre-incubation in a higher density before the growth experiment. Usually
around 2-3 hours is enough.
The time interval for monitoring the growth curves is usually about 30 min. Only slow
growing bacteria (like most Lactobacillus for example) might allow 1h in between the
measurements.
For anaerobic cultures
Bacteria should be incubated with non- or very slow shaking to generate an equal
nutrient supply (shaking is not required for oxygen supply and, for some cultures
even acts growth inhibiting).
Fill the culture vessel with medium and inoculate bacteria. Afterwards flush the flask
with nitrogen through a 0.2 μm sterile filter for several seconds. Close airtight.
Incubate at appropriate conditions.
Example: Neisseria strains
Streak out on GC plate; if necessary with antibiotics (d1), streak out further on GC
plate (d2); on the day of the growth experiment (d3) inoculate a good swab of the
overnight culture into 5mL pre warmed PPM medium and incubate for 2h at around
200 rpm and 37°C. Afterwards determine OD 550 and inoculate an OD of 0.2 into 5 mL
PPM medium. Determine growth every 30 min. For this quantity usually 50 mL falcon
tubes are used, but most other vessels can be used. Try to avoid glassware; well
plates are also not suitable as they grow poorly or not at all.
It is important not to inoculate a liquid culture overnight. If a liquid culture is started
directly from stock, bacteria might not grow and if starting from previously streaked
out plate most of the bacteria will become stationary and die overnight. In this case
there might not be enough living bacteria to start your growth culture properly and it
will take a very long time until they start growing.