Monitoring bacterial growth

Vorraussetzungen

Depending on the bacterium you want to analyse, you should determine beforehand:

• 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.