Although theese bacteria should be incredibly easy to cultivate, I am experiencing some trouble when subbing/ inoculating cultures for growth experiments. An over night culture of minimal medium (modified A medium - no trace elements)is used to sub into fresh medium of the same ingredients using a 2-3% inoculum. For some reason the cells always seem shocked and don't grow for hours seem to flocculate or produce EPS (i.e. they are stressed). I can't understand why this should be as these bugs are supposed to be very hardy and don't need many thing to grow well. Plus they have originally grown well in the previous condition. The initial minimal culture was inoculated form Lb, therefore I was wondering whether the carry over of trace elements has any effect (although it shouldn't for these bugs)? Or is it simply because the cells have sat in stationary phase for a few hours?
Cheers, Paul. I think the answer lies with the last option. But inoculating at 2-3 am when I live an hour away from work is tricky unfortunately. ....
Dear Ian,
I think you're better to adapt the bacteria into the MSM (Minimal Salt Medium ).
Lag phase of the bacteria is because of bacterial adaptation to the minimal medium.
if you try to adapt the bacteria to the minimal medium before main culture, i think you are successful to remove the lag phase.
Thanks. The original culture is however the same medium. Perhaps my inoculum size (2%) is too small? Cheers, Ian
Hi!
May be overnight culture does not good for inoculating. You can try to plot a grow curve for bacteral culture in minimal medium. After that you can find out two points: period of mid-ex phase and biomass quantity (CFU is better characteristic). So when take inoculul you will sure that you have growing, not resting cells and if will find that CFU not very hight you can increase increase inoculum portion.
Good luck!
I agree the innoculum needs to be exponential growth phase cells, and watch the cleanliness of glassware (acid rinse). I think you are thinking correctly about the innoculum size, if you can also increase the volume/proportion of the innoculum, there is often some biological conditioning of the media involved, and the clumping EPS production may be related to this, but a large innoculum of stationary phase cells will not help since you are also transferring waste products. so you might try various ratios of exponential phase culture up to 50-50 and see if this helps. If you know your doubling time you can estimate when to do serial 50-50 dilutions with fresh media, (or 25%, etc.). this approach would be more vulnerable to contamination though. Would continuous culture be applicable to your work?
Yes chemostat is an option but I have 5 strains to carry out the work on which may become very laborious. But it may be the only option. Also as I am looking at nutrient limitation chemostat is probably the best. So we are in agreement that it is likely due to prolonged stationary phase prior to inoculation. Having previously worked with marine bacteria I am not used to these very short time scales.
fastest known growth rate of any bacterium is a marine bug from a salt marsh Vibrio natiregens! Continuous cultures can be difficult to set up, but once going would be less work. You may still have some population variability in cellular growth rates, especially at long generation times, but faster dilution rates near their max TD will give you better physiological and morphological uniformity.
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