Fermentation Contamination Issues - (Jan/27/2010 )

I am a research assistant and have recently been given the task of fermenting large amounts of E. Coli en masse (like 12 to 24 liters a week) in order to harvest their inclusion bodies. I, however, know next to nothing about fermentation, and my P.I. does not know enough to be really dangerous. We also are not a fermentation lab: we don't have a clean hood, and we use shakers and incubators from other departments. I have a protocol, including how long to shake, what temperature to shake at, which broth I should use, how much antibiotic should go in the agar, the preculture, the cultures themselves, etc. I have tried to work through it, and have grown a couple batches already, but I have been having some problems with contamination.

So, I guess I'm looking for some tips - mostly about sterile technique, but anything that anyone might think is helpful. I'll write some questions to so maybe those of you nice enough to respond would have something to aim at.

1. I use 50 mgc/mL of ampicillin in my agar and pre-culture, and 10 mgc/mL in the large cultures (I make a 100 mL preculture, then use that to inoculate 3 2 L flasks). Do I need to con someone into letting me use their clean hood when I streak, or does it really matter if I just do it at my bench, given the amount of antibiotic that is in my plates?
2. How long does antibiotic last in solution? Can I make up a stock, leave it in the refrigerator, and use it to continuously inoculate my flasks?
3. How can I make a Bunsen burner work best for me when I am inoculating flasks? I try to heat the rims of the flasks over the Bunsen burner as I inoculate, and generally work around the flame as I go, but I don't know if there's a science as to when I should flame or not.
4. Right now I think my streaked plate is actually the culprit in contamination: I have done two fermentations using colonies from that plate and both have turned out poorly. During the second fermentation, I was overly zealous about sterile technique, and I know the bacteria stock is good: hence why I am leaning to the streaked plate. How can which colony to pick off of a sterile plate and know that the colony you pick is not contamination? Can you spot it: should you run a gel to see if your protein that your bacteria should express is present? I'm sure the answer is no, there's no magic way of knowing...but I thought I would ask all the same.
5. Do I need to autoclave my pipettor? I have sterile pipet tips, falcon tubes, loops to streak bacteria, and I obviously autoclave all of my media, but is it super important to also sterilize the pipettor?
6. How can I tell if my bacteria have maxed out their production of the protein I am after? I don't want to shake them for too long or too short of time, and my protocol gives vague instructions on this point.

Thanks a lot if you have read this far. I would really appreciate anyone's help. If there is already a thread on this, feel free to re-direct me there.

Welcome to bioforum!

The first and most crucial step for your protocol is to get a pure culture of your ecoli strain of interest. You should do this in a clean hood, so you have time to work careful and avoid any contamination. If you have to move to another lab for the clean hood: you make a batch solution of your bacteria to inoculate the 2L flasks (or the preculture) i.e. you make 200 mL of bacterial culture and fill it into sterile tubes (1mL) add some glycerol and freeze them unitl use (so I would do this step aseptically too). So you just have to open the tube, pipett this frozen pre-culture to the flasks and incubate. Usually within 24-48h of incubation you have huge amounts of ecoli. With this you should have no real contamination troubles with the technique you described and need to work in the other lab only once a moth or even less.

We never autoclave our pippetts, but be careful that they are clean. If doing really crucial exp or using the pippett for other applications you might think of using filter tips.