Fun with Phage (and TEM)

This coming week I plan to use transmission electron microscopy (TEM) to look for bacteria cells visibly infected by phage in biofilms I collect from the Crim Dell Stream on campus. This is exciting to me because I’ve always wanted to use the electron microscope and now I have a reason to!

Part of my project is looking at how phage infect bacteria biofilms in stream ecosystems, something that scientists don’t know how or even if it happens. Bacterial biofilms such as the ones I study in streams form over time, beginning with a few cells that form a colony. As the colony grows, dead cells and other biological material are combined to make a strong cuticle that protects the bacteria in the biofilms from infection. Once this cuticle exists, phage in the stream water cannot reach the bacteria and attach onto them to infect them through the lytic cycle. This process occurs when phage attach to the bacteria, insert their DNA or RNA past the bacteria’s cell membrane, and through complex reactions, force the host to make more phage.

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I hypothesize that the phage infect the bacteria through lysogenic replication to get around this problem. In lysogenic infection, the phage incorporate themselves in the bacteria DNA and can exist within the bacteria as genetic material for a long time and even over generations. Eventually, an environmental stimulus can cause the phage to be induced, which means the virus genetic material wakes up and takes over the bacteria so that it produces phage.

I have been collecting biofilms and inducing the bacteria I extract from them with mitomycin C to see if there are more phage present after induction to show that lysogenic infection may be occurring. This week I will use TEM as another source to look and see if phage are infecting bacteria in biofilms by looking for visibly infected cells which look something like this:

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I can do this both with bacteria that have been exposed to mitomycin C (remember that once induced, phage in lysogenic cycle hijack the bacteria to make more phage) or without induction to see if phage can infect the bacteria in the lytic cycle, too. Hopefully this technique will help me get closer to answering how phage infect biofilms or if they can’t get past the cuticle to infect the bacteria.

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