It seems to me that addition of chloroform in the post-enrichment of sample is a standardized procedure for phage isolation (correct me if I am wrong), and non-pathogenic E. coli are commonly used as host in the research that involves addition of chloroform. If I use E. coli O157:H7 that contains stx prophage, will chloroform induce the strain and release phage, and result in confounding results that the phage is not from the environment. Can anyone give me some input? thanks
If you add a drop of chloroform to the phage prep and shake it around that will kill many bacteria. Then you can add the phage prep to your indicator bacteria. But chloroform also kills the indicator bacteria so you have to be careful. Chloroform is much heavier than water. Here's what I recommend: If you have your phage prep in tube of some sort, anything from a small epindorf-type tube to an older style plating tube, screw top tube or anything else. Add a small amount of chloroform. 10-100ul (100 only for the larger volume. The amount is not critical. One drop into 10ml is fine.) and shake it for a few moments. Then let the CHCl3 settle completely to the bottom. Take samples for your assay from way above where the chloroform is.
David gives good technical advices.
In addition, I should like to note that recent data has shown that a large number of commensal E. coli strains are subjected to spontaneous induction of Stx2 prophage and with high frequency. So, if you use such strains, you could detect phages also in cultures without chloroform treatment. Other useful information you can find in this reference:
doi: 10.3389/fcimb.2015.00005
Chloroform in this context kills bacteria quickly. You could prove by titering for CFU before and after shaking with CHCl3. To get new phage from induced bacteria takes a while (the latent period from induction) and not likely from quickly-killed bacteria.
Many but not all phage are not harmed by chloroform. As an alternative you can centrifuge and/or filter away bacteria.
If there are not enough bacteria to make a mess on your indicator lawn there may not be a need to do any bacterial removal at all.
I am worried about talking past each other. Maybe there is some confusion between the idea of inducing bacteria to get more phage out of lysogens on the one hand and the completely distinct concept of wanting to get rid of bacteria that might not be as pretty on your indicator lawn?
If you want to induce bacteria hydrogen peroxide seems a better choice than CHCl3.
Adding to what David said. CHCl3 is bad choice to induce a temperate phage, but good for getting rid of bacterial cells. In addition to hydrogen peroxide , UV and mitomycin C are often used for inducing prophages from their hosts . Or any other compound that will induce an SOS response in the host
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