The following Diary includes our successes and failures, while identifying some pointers for next year's class. The daily diaries, in many ways, are too complex to read in entirety. These pages are essentially our joint lab notebook for Phagefinders 2003. However, it is worthwhile to gleam these entries if there is an area where you need additional help, and to get a sense of the content. Each daily diary entry has Planning, Action, and Pointers sections separated for expedient perusal. Pay attention to the Bold Face items that are hyperlinked below. These include student original work, significant data, Dr. Jacob's presentations, and specific instructions. I plan to improve this diary during future phagefinder sessions, but will not remove the data or methods.
Week before: Course instructor became familiar with current laboratory setting, made 7h9 plates for next week, prepared the room and made sure essential materials were in place.
July 7: Learning: what phages are, scientific research in an academic setting, who we are as learners and scientists, why does the Albert Einstein College of Medicine require us to fill out so much paperwork? Touch-and-go introduction to the phage paper, recently the cover article of the scientific journal Cell. Lab-work: half of class plated phage D29 for high-titer phage preparation, the other half of the class plated phage BXZ-1 for high titer. (Our intent: to witness phage lysis of M. smegmatis, and to help our college student prepare needed phage for her research.) Day 1 diary. A few phage biology URL's.
July 8: Trip to collect samples. We recommend getting permission to gather samples from an exciting and educational place to visit, such as the New York Botanical Gardens or the Bronx Zoo. We went to the Botanical Gardens. Day 2 diary.
July 9: Extraction of phage from newly collected samples. Learning: What objectives academic scientists have, how to isolate phage from a soil sample. Lab-work: Examination of plates set aside for high titer. Extraction of phage from soil samples and plating of samples. Day 3 diary. Note: we were using techniques different from our Isolating Phage procedure, until day 5 (see July 16 comments, below).
July 10: Isolation of phage from culture plates. Growth of M. smegmatis. Monitoring of possible new phage growth. Learning: Approaching scientific questions on our own and collaboration within the scientific lab group. Day 4 diary.
July 11: Extraction of putative phage via incubation in MP buffer over the weekend. Day 5 Diary. Student Paragraphs 1
July 14: Plating of soil samples that incubated over the weekend. Day 6 Diary.
July 15: New phage plaques might have been apparent. We collected additional samples, learned to make plates, etc. as noted in the Day 7 Diary. We also organized our samples and began recording them within our Environmental Sample Library. Dr. Jacobs visited with us; you can see what he had to say about phages today: Dr. Jacob's first talk with the Phagefinders.
July 16: We have found new phages! Next year, we'll find them sooner. Learning: It is necessary to incubate samples overnight in the presence of M. smegmatis to amplify phage in the sample. These samples did not yield phage until we did this amplification step. Therefore, these steps will be included in our Isolating Phage procedure. Other studies: Picking plaques , dilution plating and M. smegmatis culture. Day 8 Diary.
July 17: D29 phage dilution plating practice and investigation of the age of M. smegmatis culture and plaque formation. Learning: http://www.pitt.edu/~gfh/ Day 9 Diary. Some of Dr. Jacob's comments are recorded at the end of the Day 9 diary.
July 18: Day 10 Diary. Plaque-morphologies are being studied more fully.
July 21: Day 11 Diary. Student Paragraphs 2. Dr. Jacob's recorded talk # 2.
July 22: Day 12 Diary. Important Student Sample #'s. (list updated during entire course).
July 23: Day 13 Diary. Students are at a variety of stages with their phage-prospecting.
July 24: Day 14 Diary. Some phages have been re-purified and are ready for further study. Dr. Jacob's formal presentation.
July 25: Day 15 Diary. Student Paragraphs 3. Museum Attendance With Dr. Jacobs, Collection of NYC samples.
July 28: Day 16 Diary. All students have found at least one phage! Phage-prospecting continues, from new samples. Dr. Jacob's lesson: restriction enzymes.
July 29: Day 17 Diary. Electron Microscopy of several new phages. Electron Microscopy Results. Also, Genevieve composed a very nice Electron Microscopy Description.
July 30: Day 18 Diary. Propagation of phage to the F2 generation.
July 31: Day 19 Diary. We have learned how to use the clone program to program to digest phage genomic sequence, and to compare different phage genomes. Instructions for using the Clone program (PC). Dr. Jacob's recorded visit # 5.
August 1: Day 20 Diary. Most phages have been purified to F2. Some students have even made F3 because of 2 plaque morphologies in the F1. The goal has been to purify phage for 2 separate generations after the visualization of unique plaques.
August 4: Day 21 Diary. Preparation of F2 High Titer phage samples for DNA preparation. Student Paragraphs 4.
August 5: Day 22 Diary. Preparation of Phage DNA.
August 6: Day 23 Diary. Cleaning up of Phage genomic DNA and electrophoresis. Spot dilution plating of isolated, high-titer phage. Phagefinders found DNA.
August 7: Day 24 Diary. Restriction enzyme digestion of phage DNA using Bste II enzyme. Bste II restriction digestion results.
August 8: Day 25 Diary. Restriction enzyme digestion of phage DNA using Cla I enzyme. Cla I restriction digestion results. Students added vocabulary definitions to our Phagefinders vocabulary list, and composed paragraphs regarding their Bste II digestions at Student Paragraphs 5.
August 11: Day 26 Diary. Dr. Jacob's visit, EM photograph analysis, Cla I and Bste II confirmation digestions. Samples to EM for tomorrow. Students reviewed spot dilution plates.
August 12: Day 27 Diary. Electron Microscopy visit # 2. DNA from other phages.
August 13: Day 28 Diary. Analysis of Electron Micrographs, comparison. Writing and Research day.
August 14: Day 29 Diary. Clean-up day.
August 15: Day 30 Diary. Canceled due to room need. Student results are on separate web-pages, hyperlinked herein.