by Suzy Freeman
I began Monday’s lab period by examining my plates. I was examining streak plates and a spot plate. I had streaked and spotted from a direct plate and a streak plate. A previous streak had become contaminated, so I flooded the parent plate with phage buffer and filter sterilized the solution to try to plate out any existing phage and avoid the contamination. Filter sterilizing will keep out material larger than phages, but allow phages through. After some searching, I found my plates in the large stack. I wiped off the plates and peered down at the plastic covered agar. Absolutely clear! The agar was immaculate, completely clear of plaques. I looked around the room at a few of my excited peers who were happily pointing out numerous and beautiful plaques to our professors.
I knew it was time. I had to adopt a phage so I had something to work with. Of course, I would continue to try to isolate my own phage as a side project; I was going to plate out two enrichment samples today. I looked across the lab bench and asked the girl sitting across from me if she had any phages up for adoption. Her project was probably the most successful in the class. Her soil samples were from the beach, a grassy area in front of Eisenhower library near the east gate. She had a few phages she believed to be distinct which she had isolated from her samples. She passed me a plate with a gorgeous plaque pattern on it. There were vast clear patches surrounded by smaller circular plaques. The plate was labeled “plaque B.” I set aside the plate thinking that it could be the future of my experiment.
Next, I looked at my two enrichment flasks which had been sitting over the weekend. They were an awful moldy chocolate milk color with a thin brown disk of dirt around the neck of the flask. I am sure they smelled putrid. However, the contents was hopefully phage. I collected these two samples from the beach during a torrential downpour last Friday. One of the samples was partially rainwater and both had began as very murky mud. I took 25 mL of each enrichment solution and gave them to my professor to spin in the centrifuge and filter sterilize.
While my potential phages went for a ride in the centrifuge, I streaked from my adopted plate. First, I did a typical streak. I touched a lone plaque with a sterile wooden stick and dragged the stick across fresh agar in a zig zag pattern. Then I took a new stick and made another zig zag passing through the first zig zag a few times. Finally I took another stick and made a third zig zag passing through the second zig zag only once or twice. This type of streaking hopes to achieve 3 distinct levels of concentration of phage plaques on the plate. Then I mixed 4.5 mL of Top Agar with 0.5 mL of M. smegmatis, a soil bacterium. I put the layer of agar and bacteria over the streaking on my plate. Next, I flooded the parent plate with 5 mL of phage buffer and filter sterilized the solution. Another group which had adopted from the same person had discovered that their plates were contaminated. I flooded the plate to avoid such contamination. If I had to adopt, I wanted to at least be a good parent. I made another streak plate using the filter sterilized solution. I made this plate in the same way as the last streak plate, except I dipped the first sterilize wooden stick in sterilized solution instead of in a lone plaque. I also made a negative plate in the same way, except I didn’t touch the first sterile stick to anything before streaking. I put these three plates aside to solidify and turned to my spun and sterilized enrichment sample.
I was now going to make my final attempt to isolate my own phage by plating two enrichment samples. A lot of my peers in lab had had more luck with enrichment because it stimulates phages even when fewer are present. First I set up a dilution of each enrichment sample. I put 100 microliters of filter sterilized solution in a microcentrifuge tube labeled 100 and 90 microliters of phage buffer in 4 other microcentrifuge tubes labeled 10-1 ,10-2, 10-3, 10-4. Then I micropipeted 10 microliters of the 100 solution into the 10-1 tube, then vortexed the tube by dragging it along the microcentrifuge rack. I repeated this movement of 10 microliters of solution in the next tubes until each tube was diluted in the way that its name suggested. Next I added 50 microliters of each solution to a tube of .5 mL of M. smegmatis. I allowed these tubes to sit for 30 minutes so the phages could have maximal time to infect the bacteria. Next I added 4.5 mL of Top Agar to each tube of bacteria solution. I then plated the contents of each tube onto a fresh plate of agar and allowed these plates to solidify. I am really excited to examine these plates of Friday. I hope I have my own putative phage! If not, plaque B and I will become better acquainted. Fingers crossed!