Link to this post | posted today, 18:12
brueschhoff	Greetings, I am trying to grow liquid cultures of lysogens to be able to complete the sensitivity assay (11.5). However, when trying to grow up liquid cultures, there is very little bacterial growth, even though I have growth on a patch plate. I am concerned that I will not be able to get a lawn on the petri dishes. Does anyone have any advice? Thanks, Beth

Link to this post | posted today, 18:27
debbie	Hi Beth, Have you colony purified the lysogen? You want to remove exogenous phage from the bacteria that you are growing in a liquid culture. If you are starting from a plate seeded (or loaded) with phage, you will get lots of killing in a liquid culture. Best, debbie

Link to this post | posted today, 18:33
brueschhoff	Hi Debbie, Thank you for your reply. I streaked for single colonies and then did three patch plates. Would it be advisable to streak more? Best, Beth

Link to this post | posted today, 19:13

debbie

Beth, Hmm. If you original colony was surrounded by phage, you may be picking up exogenous phage. So streak it 3 times on agar. Then grow a culture and plate. Is the background filled with plaques? You can perform a streak of that bacteria on a plate of wild type bacteria, but use the culture to make your lysogen plate for testing. If that is what you have done, then your lysogen is very leaky…. You may never get a culture grown, but if you do and you plate, and it is a webbed looking plate. sometimes the spot titers will still show up, even in the mess. Have you tried to grow up the lysogen using a phage seeded plate (instead of cultures the cells from a spot). if so, do you have %lysogeny numbers? Just all something to think about. debbie

Link to this post | posted 54 minutes ago

viknesh

brueschhoff Hi Debbie, Thank you for your reply. I streaked for single colonies and then did three patch plates. Would it be advisable to streak more? Best, Beth Hi Beth, Reiterating Debbie's advice, please purify colonies at least twice before preparing a liquid culture. That means streaking a colonies once, then picking a colony from the new plate and doing another streak, and finally picking a new colony to be used to inoculate a liquid culture. If you want to perform a patch test, it should also be done using these colonies that have been purified at least twice. You'll want to do this for about 5 colonies. This is described in Protocol 11.2 of the new Guide. If those cultures dont saturate, then as Debbie said you might have unstable lysogens, which has been observed for other phages. But we can only draw that kind of conclusion if we can be certain that the colonies have been properly purified.

Link to this post | posted 2 minutes ago
brueschhoff	Thank you both! I will streak out again. Best, Beth

Link to this post | posted 2 minutes ago
brueschhoff	Thank you both! I will streak out again. Best, Beth