Help!!

[TigTigger]

Hello!!

 

Right we have a leetle problem in our lab and I was wondering if any wise folks out there may have an idea or solution...any thoughts at all most welcome....

 

Something is killing our E.coli...about 2 hours after inoculation the E.coli start to lyse. Initially this happened infrequently so it wasn't really a cause for concern, but now it happens almost every prep. Sometimes 1 or 2 flasks will survive, so I think can safely rule out problems with reagents. We have tried using new flasks, new stocks of antibiotic, new medium, transformants etc etc. I know that this sounds like a phage infection, serial dilutions of the lysed culture onto plates of confluent E.coli showed no bands of clearing one would expect in the presence of phage??!!!!

 

Help!!!!!

 

xx

[ecoli]

Have you tried cleaning out/sterilizing your incubators? It's possible that that's the source of the infection.

[TigTigger]

Yup and tried using alternative incubators in other locations...it's most puzzling that the cell lysis doesn't occur in every flask...have a suspicion it's something in the lab environment rather than just isolated to an incubator?

[CharonY]

That is odd. If there is really a phage infection (and sudden lysis of colis usually indicates that). Though I wonder, do you inoculate massively? I mean if you inoculate with a single colony you would not observe lysis within two hours, would you? So do they simply don't grow or does it really clear up?

Does this lysis only occur in liquid medium (standard LB?)? That, is you don't have lysis on your plates at all?

Have you tried to mix inoculated, grown E. coli cells with lysed cultures?

Phage contamination is a rather serious matter, though.

[TigTigger]

The overnight inoculations grow fine i.e. inoculating with a single transformant, we then inoculate LB 1/100 with overnight...the cells grow normally for 2+ hours then just as OD595 0.4-0.5 is reached clearing starts and what looks like protein clumps appear in the medium. Earlier today I did a small scale prep (2x500ml flasks), 1 flask lysed before optimal OD reached, other a couple of hours into induction...so something obviously got into that one when I added IPTG!!! The flasks aren't opened at all in the incubator and everything is done using sterile technique.

 

I inoculated the remaining overnight culture with 1/100 dilution of lysed culture, but no lysis apparent!!! verrrrrry odd!!!!

[CharonY]

Well, unless you cultivate anerobically you will always have a slight exchange of your flask with the surrounding air, which is sufficent for phage infection, for instance. But then you would see lysis after inoculation with infected cultures. Odd indeed. So if I understand you correctly you only have trouble when inoculating from liquid, but not if directly from plate (as your o/n cultures)? This would also speak against phage infection in the incubator as these cultures should be equally affected. Have you tried new pipets?

[TigTigger]

We all use different pipettes....but maybe should try new ones.

 

Any thoughts about what it could be if not phage??!!! It's obviously immensely frustrating!!

[CharonY]

Actually the only thing that springs to my mind are the media components and anything that comes into direct contact with the medium (glass wares, pipettes etc.). Unless of course that for some reason you started to incubate them at 50° that is

BTW have you looked under the microscope what these clumps really are? That is if they possibly are cell clumps?

[KAZU]

Try a different water supply. We had a contamination issue a while back and we sterilized everything, but contamination was do to the corrupted Milli Q system. Take your water sample streak it, pH it, whatever.