In a situation where there is a host specific phage,eg. phage specific to bacillus cereus, then chloroform is used in a step for isolating these phages. Could there be any possibility that the host killing or bacillus killing effect be mediated by chloroform in this process?
Yes indeed, if chloroform is not properly removed from the phage preparation. Chloroform is normally added to the phage isolation/purification step to get rid of bacteria. Afterwards it should be removed (e.g. by centrifugation) before using the phage preparation. Chloroform is not an essential step and it should be taken into account that it may inactivate other phages present in the bacteria. Therefore, it should be used with caution and proper controls. Also, some phages having a lipid membrane (e.g. Tectiviruses, described for the B. cereus group) are sensitive or inhibited by chloroform treatment.
Yes indeed, if chloroform is not properly removed from the phage preparation. Chloroform is normally added to the phage isolation/purification step to get rid of bacteria. Afterwards it should be removed (e.g. by centrifugation) before using the phage preparation. Chloroform is not an essential step and it should be taken into account that it may inactivate other phages present in the bacteria. Therefore, it should be used with caution and proper controls. Also, some phages having a lipid membrane (e.g. Tectiviruses, described for the B. cereus group) are sensitive or inhibited by chloroform treatment.
Annika, I want to use chloroform as a lysis agent to release my lysogenic phage from its host E. coli. Afterwards I want to concentrate the virus by ultracentrifugation. But before that I want to remove cell debris and chloroform. Will a centrifugation step do that for me? and how hard do I need to centrifuge? Will the chloroform make a pellet?
Thanks
Kristine, to remove cell debris and choloroform but not your phages then centrifuge at 3000 to 6000g during 30 min (in swing rotor if possible) and then recover the supernatant. If you're on the rush, 5 or 10 min of bench centrifuge is also working (but not at full speed !).
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology