Day 10 thoughts. Phagefinders.
Planned: A great idea is to attempt to use another mycobacteria, and the same soil samples. Is it possible to amplify a different phage, or the same phage? Question for Bill, is it okay to work with BCG? M. aurum is a go ahead?
Today we will also plate the samples that have incubated for 48 hours,
Did:
3 objectives: harvest D29 HT, assess our D29 dilution skills, harvest new phages for HT, plate other new phages. We will also meet with Bill.
10 AM: Counting of D29, data discussion. Examined growth of interesting organisms on hand-touched plates. Students also wanted to culture food-fungi (Rhizopus?) on 7h9-T plates.
10:30: Data recording of new phages, MP buffer on lacy plates of new phage. Steve's phage was not yet growing, no suprise this takes awhile. Decision: harvest the lacy plate we left in the fridge today. We will let the other plates grow over the weekend at 37C. Chris's phage is not yet growing, it was also decided to let these plates grow over the weekend. Today Chris's plates have a little bit of Smeg growing on them. Kristen's phage 3 A, -2 and -1 dilution, has formed a lacy plate but the smeg has not grown enough. Decision: parafilm the plate, leave over the weekend at RT and harvest Mon. Kristen's B and C phage need to grow at 37 C over the weekend so that we can harvest then. Chiara is hand-rotating the plates every 5 minutes.
10:45: We shook our soil samples to suspend any solubilized phage and let the dirt settle. Meanwhile, we prepared 100 uL M. Smegmatis aliquots in test tubes so that filtered soil samples could be added after filtration. We labeled samples and put these into our environmental sample library key. Samples were then filtered, one sample required centrifugation because even after settling, the dirt remained suspended and the syringe filters clogged. For some samples, after filtering some samples the liquid remained tinted.
11:45: Bill came in and spoke with us.
12:30: Lunch.
1:35: Students were partnered, 100 uL M. smeg aliquots from this morning were diluted 1:1 with 100 uL filtered soil sample. A few other soil samples were left to incubate over the weekend, with smegmatis having been added.
2:00: incubated samples on the block.
2:35: Harvest D29, Stephen will harvest his new phage with Kristen. Lazlo and Amanda are harvesting -4 old and new phage samples
3:00: Pick up new test tubes.
2) Bill comes in at 11:10.
3) by 11:10, have examined D29 and marked with "red" markers.
4) by 11:10, we need to know if we have lacy plates from new phages. MP buffer incubation in the AM.
5) while dilution plating in afternoon, need to harvest lacy plates. Harvest dilution plates of either D29 or new phages.
Table of D29 plaque data (# of colonies counted at the following dilutions, different student samples):
Student: 10-3 10-4 10-5 10-6 smeg: observations: Additional plaques counted today vrs. yesterday:
Lazlo can't can't lacy 69 new 0.25 dime size +8
Chris can't can't lacy 57 old 0.3 dime size +8
Stephen can't can't lacy 57 old 0.25 dime size +6
Amanda can't can't lacy 55 new 0.25 dime size +7
Kristen can't can't lacy 50 new 0.3 dime size +6
"can't," above, means that the plate did not have
Therefore, about 12% increase in number of plaques after an additional day of D29 growth. In the future, it would be interesting to note the plaque size change between day 1 and 2.
Bill requested that we keep the plates another day, as we weren't sure if it had ever been documented that new phages arise each day. We will count the plates again on Monday, and record these observations.
Today's materials:
Pointers:
Use a labbook kept on disk to display to all students and keep updated. We learned that having an excess of soil sample proved to be valuable as we recultured and thought of other potential experiments.