While growing my bacterial culture in liquid media for protein expression, I have been observing that the bacterial cells (E. coli) grow well for some till OD600 reaches ~0.4-0.6, then suddenly disappears. The turbid culture having grown cells turned clear, and I could not see significant cell debris precipitated. But I do see some particles that make the media look hazy. Seems like the cells are getting lysed. I have tried changing the media components, resources, temperature, cell lines, genes of interest, etc. BUt nothing seems to work. The pH of the media seems to be the same before and after inoculation and lysis as well. The behavior is quite random and doesn't follow any pattern. Any suggestions on possible causes or similar experiences would be really helpful. Thanks.
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That sounds like phage. The pattern of initial growth followed by catastrophic death with some cell debris is classic for induction and lysis.
Have you observed anything strange on plates? You could just do a simple plaque assay using some of your liquid culture to confirm.
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$\begingroup$ I thought of that possibility as well. The transformed colonies on plates with antibiotic looks normal. A few times I have observed wierd colony morphology (not quite circular, but distorted). I did try plaque assay, but couldn't notice any area of clearance on LB-agar plate with bacterial lawn. We fumigated (formaldehyde-permanganate) the lab and all equipment, washed all glassware with bleach, and alcohol followed by autoclaving before preparing fresh media. Still the problem persists. Do you know of any other method to detect/ get rid of phage contamination? $\endgroup$– JunoCommented Jan 10 at 7:05
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2$\begingroup$ That's tough. I was in a lab that struggled with recurrent phage contamination before, and there isn't a great solution. Fumigation sounds like a good idea - we weren't able to do that. We found a few things seemed to work well enough to keep things under control: this might not work for your combination of phage and cells. -Virkon is better than bleach. Wash all flasks and regularly wipe down counters, pipettes, and common instruments and spaces. -High temperature is better than autoclaving. See if there's an oven you can use to sterilize flasks. $\endgroup$– ksdjnfCommented Jan 10 at 16:03
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2$\begingroup$ (cont) -Filter pipet tips may prevent transferring aerosolized phage. -Try to prevent aerosols in general. Be gentle with your cells and don't leave plates open. -You mentioned trying alternative strains, but you might look into this a bit more. Some common phage receptors differ between B and K strains, for instance. -You could try low temperature or cold inductions to potentially reduce induction. -Regular maintenance: once phage has established itself, you probably want to have regularly-scheduled deep cleaning (especially surfaces where it can linger) to keep it down. $\endgroup$– ksdjnfCommented Jan 10 at 16:03
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2$\begingroup$ (end) -Vigilance: you will never remove all the phage, you'll just keep them under control. Make sure everyone watches for signs of lysis and communicates them. $\endgroup$– ksdjnfCommented Jan 10 at 16:03
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2$\begingroup$ Is it possible that your E. coli strain is a lysogen? Prophage induction at high cell density could explain why your culture is crashing $\endgroup$– acvillCommented Jan 11 at 12:48