Day 12 thoughts. Phagefinders.
Planned: To make a table of the new/old smeg and D29 data, Chris is doing this. We will make new smegmatis cultures, some will be from phage D29 resistant smegmatis colonies that have arisen on plates that had excess phage. Essentially, the study will "Prove Darwin Correct, comparing to Lamarck." In this experiment, 1 very small colony of D29 resistant smeg, assumed to allow 10 x 5 bacteria into 1 mL media when vigourously resuspended with a bacterial loop, will be diluted so that 100 uL of the smeg can be added into 10 mL 7h9-T media. This allows for an innocula of 1000 Mycobacteria. In comparison, mc2155 smegmatis that has not been selected for resistance will also be diluted by the students so that 1000 Mycobacteria are added to the inkwell in 7h9-T. After growing for a few days, the D29 Resistant culture and the non-resistant cultures will be plated side-by-side on 7h9 plates, using the top-agar method. The number of phage resistant colonies that arise as a result of this experiment will be recorded. [Experiment will take a few days to complete.]
General maintenance is necessary today, we need to make stacks of each student's plates, examine for contamination, and record our findings with respect to our phages. Samples must be labeled appropriately in the incubator clean-up; sample labeling and clean-up. Sensitive plates that have plaques and will be stored are parafilmed so that they do not dehydrate.
An experiment we could in the weeks ahead: temp and shaking effects after release of phage from soil. How long does it take to get plaques if grown at RT?
math: how to dilute 1000 smeg colonies into 10 mL media.
Kristen: could replate 10-5 of her compost heap sample since she had a clear plate. How is this culture doing otherwise? How do the plates she made yesterday look?
Blake: will be digesting several phage DNA samples since he is in the regular lab.
Stephen: No colonies are expected with this particular phage, it's too early. Begin adding data to your table, however. (when plaques arise and disapear as a function of phage concentration.)
Chris: How are plates from yesterday? What is happening to plates we've left in the window?
Amanda: your new samples that incubated overnight in the shaker can be plated. Also, start 2 new samples.
Genevieve: How is it going with the new soil samples? We can start culturing these today.
Lazlo: how do plates from yesterday look
Did: Today was a bit of a lab-management day, we began the day waiting for our autclaved media to cool so we could pour plates. While waiting, we made lists of our phage sample microcentrifuge tubes. Students labeled tubes, since their are many samples that we must maintain, even after students are gone. All students were given a new smeg sample from me today since there has been a bit of contamination. The students poured 80 plates today.
Oral Lesson: Dilution for 1000 smeg in a solution. We must start new smeg samples today. Question is today, what smeg sample to use. I therefore don't let students use the smeg I have grown until alloquoting.
New samples added to List.
Did:
Student specific actions are below:
Chris: began incubating creek sample today. Sample 27. Chris also examined his plates (extensive; he has many plates). (Bill Lesson).
Lazlo: is beginning to incubate 2 new samples today. put these on list. Actually, we should have had him do this yesterday, be careful in the future...we were too harried the day before. picking of 10 colonies (Bill lesson).
Steve: we are trying to see if his phage can be purified for another round. Although he has HT of this phage, Sample 8, it has not been repeatedly purified. We therefore are monitoring what diluted HT of his new phage will do as it is incubated further. He is collecting descriptive data on his plates. His plates have contamination, which appears to have diluted with the dilution of phage. Therefore, the MP buffer and HT phage may be contaminated. Use new MP buffer, and re-filter HT phage. Today, he re-plated his phage at -3, -5, -7, -9. = 4 plates. Steve will also write up why he believes it has become contaminated.
Stephen's S5 was from yesterday, and he picked a plaque from this, also. These samples also appear contaminated. Concern about what smeg he used, he used the smeg that Lazlo and he made. Compare these data to Kristen's data. Steve's plates are now back in the incubator.
tomorrow contaminated plates get through; check for plaques first. S5 dilutions were also contaminated, leave for another day. S17, his other sample, also had grown in a bizarre manner. He is replating the S5 today.
Amanda: She is filtering from the dirt before incubating with smeg, this will allow her to plate her 2 new soil samples. Quite a bit of work, filtering, incubating with Smeg, top-agar plating.
Genevieve: Our new addition. Genevieve has mixed smeg, soil and MP buffer on 2 samples.
Kristen: is replating her 10-5. Also, replated all CH1 samples, and harvested phage A at 10-1.
Chiara: is plating one sample today, filtering from the dirt, incubating with smeg, soil and MP buffer on 1 sample. This is sample 28.
Blake: Worked in main-lab today with postdoc. He's preparing DNA digestion reagents for us, we will do the same digestion as he is doing now in future experimentation.
Reid: End of day, started independent diluted Smeg sample from my uncontaminated single colony. Also, diluted this so it would be ready for an additional sample on Thursday. Contamination objectives. Talk to EM over lunch.
New Phages at HT: Stephen, sample 8, F0.
Kristen, sample 3A, F1. = 3AF1.
Blake, sample WJ, F2. = WJF3.
Today's materials:
As always, pipettes, 50 mL conical tubes, inkwell plates, Large gloves and p200 filter tips needed replacement. We are using an excessive amount of supplies compared to other scenarios. In other conditions, pipetting can be done with glass pipettes, pipett tips can be boiled and reused. We prefer to use filter tips and disposable plastics to save time. If this isn't an affordable luxury, students should participate in dishwashing and maintenance of the lab. We assign a lab-manager for different days...students manage the lab for 2 days before
Pointers:
Although the phagefinders curricula has resulted in students conducting independent procedures on our samples, this is due to the nature of our course; we have additional resources, a dedicated instructor and a 6 hour daily timeslot for a 29 or 30 day period. Other scenarios would coordinate the student research in a more unified way. Students could all plate a collective phage, and although this curricula would narrow the number of phages found, unified procedures would allow for a more manageable classroom schedule.
75 uL turbid smeg into 10 mL 7h9-T liquid media should give a turbid smeg culture, ripe for plating, in 24 h. It is best to let the smeg set in the inkwell vial for an additional 24 h to help degrade the Tween, before using.
Aliquoting materials so that each student has their own samples is a really good idea.
Heat block and incubator temperature should be monitored carefully, even the equipment we have at AECOM seems to be finicky and otherwise fluctuates.
We tried top-agar plating C. Albicans and C. neoformans, fungal pathogens this summer, also (I did with a research fellow), top-agar plating the fungus at 3x10^3 and 1 x 10^4, very nice concentrations of fungi, however no plaques were found. We tested 6 different soil samples each, and prepped with MP buffer using 50 uL either fungi to amplify phage before filtration. No plaques were seen after growing plates at 37 C for 1 day, then letting plates remain at RT over the weekend and checking on Monday.