Picking up where my last paragraph left off, events occurred that have changed my objectives. Those two plates plated Thursday the 24th of July really did contain phage. Both the PSNC-03-26 and the delta TNJ-03-30 samples contained a phage with a very similar structure.  There were many small plaques when I checked the plates on Monday. The traits of these plaques are that they are small, hazy and irregular I then picked these plaques and diluted PSNC to the -5 and delta TNJ to the -3. These dilutions did not make a hi-titer, so I picked plaques from the F1 plates to form an F2, as well as a hi-titer. Meanwhile, I also worked on the phage Chris discovered under the bridge(PB-03-19). The plaque morphology from this phage is that they are hazy, less in number and slightly large in size than the other two phages. They are also more rounded in shape. I picked from Chris' original plate and diluted to the -3. However in trying to get an F2 or PSNC and delta TNJ, the plates had no plaques. I have two rounds of replating, one done the 31st of July (currently in room temperature) and the other set done August 1st, these are all trials for an F2 generation. The second round was started because the ones done on the 31st of July didn't look like that had plaques. The dilutions on the PB phage for F1 didn't go very well either, having no plaques. However on the -1 plate there was a clear blotch that was plaque like just enormous. The day before, Chiara had discover a large plaque on one of her plates. So that plaque had been picked and diluted to the -4. There could be a few problems that cause my struggle to form a hi-titer. The PSNC & delta TNJ plaques are small so that could mean there isn't a high concentration of phage in each plaque. Also my technique of picking plaques could be off, as supported by the lack of phage on the PB plates. When aiming for an F2 hi-titer for PSNC and delta TNJ, I made the buffer concentration, smaller, to extract the phage better. Hopefully this slight change in procedure made a difference and there will be a lacy plate on Monday.

--Amanda

Question 4 

 

Describe the location from which I got my phages. Describe the nature of their plaque morphologies. Describe what I saw under the electron microscope. Describe whether or not I think these phages will have similar genomic sequences.  

 

 

My first phage (BG1-03-1) was one collected by us as a group at the botanical gardens. It was collected from dry soil found under a Japanese tree. This was the first sample collected. It had a very interesting and unique plaque morphology. The size was not special, about ¼ of an inch in diameter. However, the actual morphology had a lytic center, with a thick, dense lysogenic ring around it and finally another lytic ring around that. They also made plaques with a lytic center and a not as dense lysogenic ring around that. This is not as unique as I have seen it multiple times before. When I examined this phage under the electron microscope, it was icosohedral tailed. The tail was about 3.5 times the length of the head.

My second phage (SHNC-03-18) came from my back yard. There is a layer of dead leaves left over from spring due to many trees. Under that layer, there is some moist soil. I collected this phage from down there. My F2 dilutions of this phage are currently growing in the incubator. The plaques that I have seen are uninteresting. They are the same size as the previous phage and seem lytic. I have not yet had a chance to examine this phage under the electron microscope.

My third phage (SHNC-03-27) was also gained from my backyard. When I was told that water based phages are rare, I went to a small creek in my backyard and collected some water and soil from beneath it. The plaques have been the same size as the previous phages as well as extremely small. They are lytic with lysogenic haloes or simply lytic. I have not seen this from under the electron microscope yet.

When studying the packet, I find that phages with similar appearances, such as D29 and L5, have similar genomic sequences. D29 is a very lytic phage. I do not know what the plaque morphology of L5 looks like. Because of this, I cannot make an educated guess on whether similar plaque morphologies yield similar genomic sequences. I have only seen one under the electron microscope, so I cannot compare them that way. I can only guess that different plaque morphologies mean different genomic sequences. Therefore, I don’t think that my phages will have similar genomic sequences.

--Chris

                                                                    Phage Finder Lab notes from Summer 2003

7/9 Collected samples at the Botanical Garden (BG)

7/10-7/11 Failed attempt at plating samples, decided to retry and incubate the BG samples with smeg all weekend in shaker room. I chose sample 8 from the conservatory. Soil was moist.

7/14 Plated F 8 (sample labeled BG8-03-08).

7/15 while some students see smeg, it appears that my phage clears the plate

7/16 I am surprised to find plaques on the F S8 plate upon inspection. I give two possible explanations for the slow growth. 1) There was smeg yesterday and I simply was unable to see it yet. 2) Reid suggests that maybe the phage produces a toxin inhibiting bacterial growth. Diluted F 8 to the 0,-1 and –2.

7/17 Chiara suggests that the lytic nature of my phage may be due to the fact that the moist area I took it from encourages bacterial growth – the phage can afford to be more lytic without a fear of killing its food source.

7/18 F 8 still shows no phage. I am not surprised due to the fact that it took time last time to grow. Decided to incubate over the weekend. Harvested F S8 from the original plate to make HT. Plated F S5 and F S17.

7/21 Checked F S8 which had been left over the weekend, saw a weird form of bacterial growth on the 0 and –1 plates, but no sign of normal looking plaques. Blamed the weird smeg on F resistant smeg (incorrectly). –2 cleared the plate. Since we don’t know what occurred over the weekend, I decided to redo the experiment with the HT. Diluted –4, -5, -6 , -7, -8 , -9 and –10. Picked plaque from F S5 (there was only one plaque on the entire plate) diluted to the –1 and –2. I used the smeg that Lazlo and I made.

7/22 Checked F S8 plates – saw signs of contamination. Contamination decreased as F became more dilute. Source of contamination is uncertain. Decided to refilter HT then use different Smeg. I was not the only person to see contamination. S5 dilutions also had contamination. Replated S8 to the –3, -5, -7 and –9. F S17 Had not grown so I decided to replate it with no dilution.

7/23 Nothing on F S17, decided to save for another day. F S5 disturbed by contamination, since there is no soil or any remnant of F S5 left, I think I will try to pick a plaque at some point, not today. Yesterdays F S8 plates are not contaminated. Despite contamination most of the plates from 7/21 still grew normally

7/24 FS17 makes a good lacey plate, almost cleared plate. Picked plaque and parafilmed for later LP in case of emergency.

F S5 contaminated but I’ll try and pick

F S8 From 7/22 clears plates at the –3 and –5 dilutions. –7 and –9 show two separate plaque morphologies= 2Fs. –9 has 2 PPU. Decided to pick hazy plaques. Labeled new “hazy” phage “8B.” dilute and plate –1, -3, -5.

(7/25 museum of Nat. Hist.)

7/28 Picked plaques from : F S17 –6 F1 from 7/24 & FS8B –5 from 7/24.

S17 –6 will be harvested tomorrow.

Harvested: FSA –3 from 7/23 and FS8B –1 & -3 from 7/24.

Dilutions: FS17 à -4, -5, -6, -7. FS8B à -1, -3, -5.

FS17 plaques have halos. As plaques get more dilute, halos become thinner and plaques get bigger.

7/29 FS17 from 7/24 – The –7 now has grown halos on its plaques. On the –6 a 3^rd ring may have appeared on some plaques. However, on yesterdays S17, smaller plaques were formed, à plaque picked on S17 less dense? Wait another day to see a thicker layer of smeg.

Picked plaque from S5 –1 and will dilute –1,-3, -5, -7.

7/30 Note: yesterdays S5 plates left at RT overnight.

Despite contamination, F S8B from plaque grew normally through with smaller plaques than in the 1^st growing.

S8A looked exactly how I expected F S8B to look in the –1 dilution and contain 2-3 different plaque morphologies in the –4. The plaques that seem more like F S8A were originally seen to have halos, unlike before, (where the plaques were clear), which may be the result of different smeg. In the –5 dilution, only clear plaques show, this may be because Amanda’s smeg was used for the –5.

F S17 grew normally except it didn’t show similar halos to the previous plating, will check tomorrow. Note: the F1 S17 plate had been heated for 24 hrs. and then left at RT, which may have produced the halos. Harvested LP of –4 dilution but also decided to pick plaque from –7 to test hypothesis on why F1 and F2 are different: Either 1) diff. temp = diff. plaque. 2) diff. smeg = diff plaque. In order to test which hypothesis is right I plated –5, -6, -7 x2 and let one grow at RT.

F S8A à picked from –1 will plate from –1 to –5.

F S8B à Picked from –5 will plate –1, -3, -4, -5

17 Tubes, 15 Plates

7/31 Note: the S17 –5 which was supposed to be incubated was accidentally left at RT over night. Not a big problem.

Yesterdays samples not ready for proper observations yet.

Made F S5 from 7/29 into HT from –3 LP.

Discovery: 7/28 on the FS17, rings formed similar to the 7/24 S17. Important: no difference between the F1 and F2 except for the time they had been growing.

F S5 forms small, clear, round plaques.

8/1 Made F S8 visual tracking sheet. Interesting thing about S8b: lower dilutions have more plaques, which stop growing when they touch each other. The plaques in lower dilutions form a clearing in the center while the more spread out plaques are entirely hazy with no bullseye in the center.

Note: The S8B plates from 7/30 seem to have been placed out of order. See tracking sheet.

Picked plaque from F S8A –5 from 7/30 & will dilute from –2 to –4 for F3.

Waiting for S8A and S8B to become lacey.

Decided to do experiment by taking some phage from the ring surrounding S17 plaques from S24 and will dilute to –1, -3 and –5.

8/4 Harvested from FS8A –3 from 8/1, even though the plaques weren’t clear in that plate, since Reid thought it was just a result of diff. smeg and overcrowding. However, to be safe, I also harvested S8B LP –3 (labeled –5) from 7/30. Even though the one labeled -4 looked more dense, we noticed some unidentified plaque/ contamination and decided to harvest the –5 instead.

F S17 RING from 8/1 doesn’t show anything but weird smeg.

F S17 left at RT from 7/30 has a halo, but at this point, it is very thin.

For FS8A info, see visual tracking sheet.

--Stephen

1. Using restriction Enzymes to determine whether a suspect committed a Crime

   By using a process called RFLP, Restriction Fragment Length polymorphism, one can cut a piece of DNA into fragments of different lengths. The lengths are determined by the areas at which the strand was cut by the restriction enzymes. Using this method, one can determine whether two substances containing genetic material, such as two samples of blood, are the same. This can be used in order to check if the blood found at a crime scene matches that of the subject. One simply has to cut both samples with the same enzyme, and then compare the fragment lengths.