My advisor wants me to do secondary overnights on my P. aeruginosa, S. aureus, and E.faecalis bacterial cultures before standardization and inoculation into 24-well plates. This means a primary overnight is grown for 24 hrs and then the secondary is grown for 18. He is the only one in our lab of 5 PI's that uses this method and claims that it helps set the physiology of the cells and acclimates them to the medium. If this is true, why does no other paper that I've read mention it, why don't other PI's do it? It doesn't make sense to me and just delays my work for another day. Does anyone agree with my PI and if so, can you further explain why this is a good idea? I am thinking of doing an experiment to prove myself right and grow up 1* and 2* overnights of the cultures and do plate counts of each, if they are similar then there probably isn't a big enough difference to worry about and I'll be able to just do 1* overnights. ***** For the 2* overnight, 50uL from the primary is put into a new 5mL of media.
Personally I disagree because the bacteria doubling rate is so fast and the resources in the medium are getting used up quickly. Normally, in bacterial culture, the growth curve will hit a plateau before 24 hrs at 37 degrees. That means the bacteria are acclimating to a resource-deprivation state rather than what your PI claimed. It would make more sense if you were continuously resupplying with fresh culture media.
I strongly agree with your PI, but only if the primary cultivation involved starved cells e.g lyophilized cells or cells withdrawn from other culture maintenance methods. The cells are at different levels of damage and will require different time periods for repair. The first few hours of cultivation will be used for those repairs, therefore synchrony growth and stationary phase will not set in when they are supposed to. The secondary cultivation is significant because it assumes and rightly so, that the cells have all been repaired and could go into replication and cell division synchronously (physiology) as they are supposed to. There are literature that support this. Please see these articles and please listen to your PI. He knows what he is doing. https://www.sciencedirect.com/science/article/abs/pii/S1878818116302055 andArticle Plackett-Burman Design and Response Surface Optimization of ...
Dear Alexa,
Your question is indeed very interesting and has raised already a nice discussion since the two previous answers show no consensus between them. I agree with Peter that the exponential growth phase in bacterial culture occurs fast (usually during the first 24h) to reached a plateau called stationary phase. In the stationary phase, the “physiology” of the culture begins to change as secondary metabolism (e.g. waste products, antibiotic production, antimicrobial peptides) will be favoured due to the growth itself, the depletion of nutrients and the increasing competition for resources. Of course, the time for a bacterial culture to reach the stationary phase will depend on several factors including species/strain, growth conditions, medium changes or not, etc. thus, as Maurice pointed out you can also find papers stating longer growing times for bacterial cultures. My personal recommendations are: 1) pay attention to your PI but ALSO listen to yourself, if you have doubts or you are not convinced with something then 2) run appropriate experiments and after the thorough analyses of the data you (and your PI) will have proofs of to which are the best conditions to achieve the best results according to your scientific question. Consider that if you are not particularly interested in studying the physiological changes of the stationary phase or in the production of a particular compound favoured by this stage then it is not mandatory (not recommended from my point of view) for you to keep your culture too long in the stationary phase.
Hope this helps.
Good luck!
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