I’m trying to increase the frequency of my experiments using Pseudomonas aeruginosa (PAO1), but growing the volume I need from a single colony is taking too long.
I have consulted with a microbiologist a modification I plan to do and she hasn’t been able to guide me in the right direction. What I want to do is as follows:
1. Grow the bacteria in LB, inoculating from a colony that has been streak plated.
2. After mid exponential growth, I will prepare vials of 1-2 mL (20-30 in total = 20-30 experiments) and take to -20ºC freezer.
3. Use one of these vials to start a new experiment, by inoculating the LB agar volume (growth media), instead of using a single colony from the streak plate.
I know the metabolism of the bacteria might be affected by the temperature changes, but all my experiments will be performed with the same bacteria each time, at the same initial concentration, from the same parental colony. I cannot find literature that supports this and would like to know your opinion. Since I’m doing disinfection only, do you think adopting this method is feasible/reasonable?
Will this methodology work if I use a different bacteria?
Thanks in advance
The biggest problem I see with the proposed method is that freezing-thawing them (especially at -20 instead of -80) is going to reduce the number of viable bacteria, and probably less will be alive in the innoculum than expected. It also will probably take them longer than you'd think to actually grow, because they need to warm up (and probably repair themselves) from the freezing before they can actually start replicating and this will introduce a pretty noticeable lag time. I think it would probably work though.
Typically what we do in our lab is make an overnight broth culture directly from a -80 freezer stock, then just subculture from that into a larger volume of fresh media.
With few exceptions, I think streaking for isolated colonies is just to prevent contamination and due to dogma from early microbiology.
make stock of bacteria , mid-exponential phase growth, flash freeze in liquid nitrogen, store at -80 C. i published 3 papers of Staph aureus using this technique.
Thanks, I appreciate you taking time to answer this. Just to clarify a bit, off course I start from a -80 freeze stock. I'm just proposing doing streaking plate after that, inoculating growth media with a colony after, and then at mid-log phase, freezing at -20 (10-20) small volumes that I can use everyday. My theory is I will be able to reach mid-log phase in a new experiment when inoculating it with this small aliquots. Sure, the viability will be affected I agree. But, will results be feasible?
I'm only doing research on disinfection, so every experiment will start under the same conditions. Thanks again
Hi
Do you not check absorbance of the bacterial suspension? Are you able to maintain consistencies?
We do 1)-80°C 2) plate 3) directly from plate we make a bacterial suspension in saline / PBS of particular optical density / absorbance and maintain this for every experiment.
We found that bacteria from broth cultures are much more susceptible compared to plate cultures - we think on solid media they maybe more in same "age / log phase"
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