Day 5 thoughts.  Phagefinders. 

Planned:    Teach students how to prep top agar.  Replate any virus we have, or, pick plaques for expansion of new phage, if we have plaques.  We also have other soil samples that we could plate.  We will also plate our High Titer D29 phage (method for preparing high titer phage) in order to determine the PFU that the sample we collected has.  Or, if we didn't streak M. smegmatis yesterday, streak the bacteria today.  Reserve ideas: library and pubmed (medline) searches; a medical school library visit would be timely. 

Did:  After 48 hours of growth, we discovered that we had not discovered any new mycobacteriophages.  Our plates contained confluent M. smegmatis cultures, but did not contain any phage.  The group wrote their hypothesis why they believed we had not discovered any new virus.  It was recognized that the incubator had been turned off for approximately 6 hours at the time of plating, before it was turned back on.  We questioned the validity of the one-half hour incubation of the bacteria with the M. smegmatis (the protocol has been modified on this website to indicate the preferred method as a consequence) before plating; it may be possible that the bacteria absorbed all the phage and the phage did not have time to replicate before plating.  Another suggestion: try propagating the phage at a higher or lower temperature.  Discussion: we could also incubate the soil samples without any Mycobacteria.  Student Question: if the phage is lysogenic, maybe we didn't see any plaques at 37 C.  Based on this last hypothesis, we decided to incubate our soil -MP buffer-M. smegmatis samples while shaking at 37°C over the weekend. 

Therefore, we decided to begin an incubation of our samples (same as before) with M. smegmatis for a longer period of time at 37 C, immediately.  A few samples were done this way.  We also decided to incubate the soil for the same long duration without any M. smegmatis added, under the hypothesis that the M. smegmatis could be adsorbing the phage and not allowing the virus to enter the media. 

Students seemed to be impressed that some M. smegmatis growth could be detected on the plates they prepared yesterday.  We plated the BXZ-1 at higher dilution, and planned to harvest for High Titer phage on monday, providing we have lacey plates.  We also plated the D-29 phage high titer preparation so that we could use the phage for Demi's screen, if this will work for her.

We learned how to use pubmed today, using a project connected to the internet.  The pubmed website is at www.ncbi.nlm.nih.gov ; select "pubmed" in the framed taskbar. 

Today's materials:    It is a good idea to keep Top Agar handy and ready for plate pouring by keeping the agar liquid in a 55°C water bath.  The agar is ready for plating when needed and can be maintained in the water bath for several days. 

Pointers:  We may have to replate BXZ1 for high-titer on Monday, since plates were left out overnight at 25^°C.  We had been using the same M. smegmatis samples. 

Thoughts:  Students generated the following ideas:  in the future, we could use our second incubator for a hotter temperature of plaque formation.  We can also attempt to get plaque formation at room temperature.  We could use a different Mycobacteria in order to attempt propagation of phage on a different bacterial host.  (M. aurum?)