Hello,
I have used mitomycin C (0.5 μg/mL) to induce prophages in Escherichia coli K-12. After 24 h exposure, I filtrated an aliquot of the suspension and placed a drop of the filtrate on a colony of fresh K-12. The idea was, the induced bacteria would have caused lysis on the growing bacteria.
However, there was no inhibition of the growth.
Assuming that the mitomycin was working and that 24 h exposure are the right time (which are not given), I have been thinking: if I induce phages in K-12, wouldn't be K-12 immune to exposure to K-12 derived phages?
In that case, what reporter bacteria can I use for demonstrating phage induction in K-12?
Essentially I need a strain that is susceptible to the phages present in K-12 -- any tip?
Thank you
You need to provide some necessary details in order to better diagnose and give advice. E. coli K12 is not one single strain, there are many many many derivatives of E. coli K12. It is true that the original K12 strain that was studied 60-80 years ago was a lysogen of bacteriophage lambda, but most K12 strains currently used in labs are no longer lysogens (except for some cryptic elements that do not produce phage).
You are also correct that if you have a lysogen and it releases phage, that you need to use a different non-lysogenic strain to see that phage.
If you generate an induced lysate from the original K12 strain you would only see plaques on a different K12 strain that was no longer a lysogen.
So which strains are you using to induce and which strains are you using to show the phage?
Regarding how to know if it worked, if you add mitomycin C to a growing culture (mid-log phase and not saturated) then after about 45 minutes you should see the culture lysing in a very noticeable way. Not 100% of the cells but a really large fraction of them and there should be all sorts of swirly debris floating in the flask or tube.
You should NOT continue to grow overnight, mitomycin C action is quite fast and any lysis that is going to occur will be done within 1 hour. So you should harvest the culture supernatant at that time by centrifugation. If you keep growing then the culture will overgrow with various mutants.
MItomycin C is soluble in water at 1mg/ml but there should not be a problem if you dissolved in ethanol so long as you are only adding a very small amount (1% or less) to your cultures.
You need to go to the old literature to find protocols, like from the 1950s-1970s when a lot of phage was being done. You might also find some protocols in microbiology teaching lab manuals.
Do you know what strain you have and what other strains you have available to you?
I checked, but the strain I am using is only registered as 'K-12', no serovar. I think I'll have to buy a well-defined strain to be sure... Thanks again for your tips.
If you can provide more details about the strain and where you got it from, I might be able to figure out if it is a lambda lysogen or not. It may also be that your strain is not a lysogen depending upon what it is.
There are numerous non-lysogenic versions of E. coli K12 that can serve as an indicator strain, but I would suggest MG1655 should be generally available from many sources and is well characterized. Other common strains used for phage work include W3350 or W3110. But if you have a bacterial genetics lab at your university, they are likely to have strains available.
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