These paragraphs, due today, reflect the status of finding new phage about 3 days back (Day 13).
I so far have brought two samples into the lab. Along with plating these two samples, I also plated sample 1 from under a tree at the botanical gardens and sample 19 from under the bridge with the pigeons. I found phage in all of these apart from one of the samples from my back yard. This sample was obtained under wet leaves in my back yard. However, this was plated with many other samples of mine, and none of them worked. All of these samples, when I replated them, did indeed reveal phage. Due to this, I intend to replate this sample later. The other sample from my house came from a creek in our back yard. I took a mix of soil and water and plated that. The plaques from sample 1 were very interesting. At first, they were turbid, clear in the center with a lysogenic ring and then a thin lytic ring around that. When I picked a plaque and diluted it out, some plates mimicked that pattern, while the rest were simply lytic. Sample 19 from under the bridge had extremely lysogenic plaques. They were difficult to spot. With this being my third phage, I gave it to one of the students without phage. The creek sample from my house yielded lytic plaques after about one day. I have not yet seen this after two days of growth. In the case of isolating a new phage, I simply hope one my two phages (three if I successfully find a phage in the first sample from my house) has not yet been found. If it has been found before, that is just as good because it has never happened before.
--Chris
In the past three weeks at phage phinders, I have hunted away through many
samples only to find my plates coated with smeg. The first samples I tried were
BG2-03-2 and BG4-03-4 both from the botanical gardens. These were not given much
time to incubate or placed in the shaker room. And so the plates resulted in
having nothing but a thick coat of M. Smegmatis on them. This was no different
from everyone else's plates at the time. Next I decided to focus on BG4-03-4, as
I believed the moister darker soil where it was from would be home to more
bacteria, and therefore more phage. And so after added MP buffer and smeg to a
cap full of the BG4-03-4 soil, it was left in the shake room over the weekend.
That Monday I found my plate to be clear, despite a few specks which I believed
to be smeg. But after talking to Bill, the next day they were probably some kind
of contamination. The plate was no longer blank when Bill observed it, as the
smeg grew back. So I decided to try again using samples BG2-03-2 and BG6-03-6
again both from the botanical gardens (BG4-03-4 had run out). So I repeated the
extraction process, and placed them in the shake room over night. However, the
next day I went in to add Kristen's tubes to the rack my tubes were placed on.
We found the tubes mostly dried out and knocked on the table. I proceeded to
re-add buffer and smeg to the tubes, and place them back into the incubator.
When these tubes were plated, once again no plaques were present. I genuinely
believed there had to be phage in BG2-02-2, and I tried it one last time (using
the last of the sample), again to find only smeg. Then I decided to take matters
into my own hands and collected 3 soils from my local park. Two were from a very
boggy creek. one was sandy (SCNC-03-24) and the other was near a skunk cabbage
(SKNC-03-25.) The other was from a random patch of soil in a field where there
were many birds present, so I chased them away and took some soil from where
they were stationed (PSNC-03-26). Once again I repeated the process with SCNC
and SKNC, again only to find smeg. This is where I began to question my lack of
luck. I proposed maybe they needed to be in the shake room for more than 24
hours. However phages found by Chris and Kristen disproved this. I also
questioned my methods, perhaps I was missing important steps. I check over the
extracted procedure with my peers, and I was doing it correctly. This past
Wednesday I decided to find my own phage, one last time. I incubated PSNC-03-26
and delta TNJ-03-30 (taken from a family friends tomato garden), These were
plated on Thursday, and then taken out by Kristen to be put in room temperature.
The top agar was quite stiff when it was plating so there were some dimples. I
took a quick glance at the plates Friday afternoon, There were dimples, that I
thought to be from the agar, but they could be plaques, the smeg was a bit too
under-grown to tell. These dimples looked the same from either plate, and one
sample was from Connecticut and the other was New Jersey, which lead me to
believe they are agar dimples. very big agar dimples. They were hazy and
irregular shaped which could go either way, only time will tell. This is my last
attempt to find phage from soil. The samples I have tested seem just a prone to
phage as any other sample that as found phage. And so I blame my misfortune
hunting phage on Lady Luck. If the plates for delta TNJ and PSNC have no plaques
on Monday, I will extract the phage from the plaques made by a phage from the
PB-03-19 sample, which Chris had plated, and so graciously given to me. I will
focus on making a Hi-Titer of that phage and delve into studying it more
deeply. So far is all I can say that its plaques are very hazy and sort of
irregular shaped. There weren't too many of them. I didn't do a plaque count but
there weren't much more than 10. They were quite large and were not perfectly
round. I also will continue to help Kristen with her B1 phage, which she is
trying to make a Hi titer from as well.
--Amanda
In all of the samples I have plated at AECOM, only one of them has a possible phage. This sample, BF-03-16, after three days of incubation showed three small plaques. Two of these seemed to have been contamination, but the third was a possible plaque. The possible plaque was about an eighth the size of a dime, and was clear, leading me to believe it is a weak, lytic phage. HC-03-12 on the other hand just had a lawn M. smegmatis growing on it. I believe that HC-03-12 did not show phage’s due to the fact that Provincetown is rather clean, and sand found in most of the samples plated does not seem to produce phages that grow off of M. smegmatis. Additionally, PT-1-03-13 and PT-2-03-15, where just plated and incubating at room temperature over the weekend of the 25th, so they still might have phages. If not, Air-03-31 was placed in the shaker room, and is likely to have potential phages due to the fact that it was in a moist area with sunlight on it. Also, on the 26th, six more sample where collected from central park, which may contain phages due to the similarity in the environment and the backyards other phages have been found in, so incase BF-03-16 plaque turns out to be contamination, and PT-1-03-13, PT-2-03-15, or Air-03-31 don not show any phages, the Central Park samples can be used to propagate for phages.
--Lazlo
Mycobacteriophages attack mycobacteria. Despite their name,
mycobacteriophages do not actually _eat_ mycobacteria. They inject
theirDNA into the mycobacterial host cell, usually transforming the
bacterium into a “phage factory,” which acts only to produce phage parts and
assemble them together before releasing the newly synthesized phages into the
surrounding environment. In the process the bacteria lyses, or breaks
open, in order to release the phages. However, sometimes the phage, rather
than being lytic, behaves as a lysogenic phage. Under certain conditions,
a
phage that infects a bacterium will not immediately try to transform
the bacterium into a phage factory. Instead, the injected phage DNA either
incorporates itself into the bacteria’s DNA loop, or forms a separate
loop, either of which will remain dormant until certain stimuli are
introduced. In such a way, the bacterium is modified but not destroyed,
and every
time the bacterium replicates, the phage DNA is also passed on. Such
a
phenomenon is crucial to current research with _Mycobacterium
smegmatis_ and_Mycobacterium tuberculosis_.
Because lysogenic bacteriophages can conceivably modify bacterial DNA,
the potential is enormous for researchers to use such bacteriophages to
modify the DNA of tuberculosis-causing bacteria in order to form a less
pathogenic strain to be used for inoculations. The “phagefinders” program
works
with _M. smegmatis_, a bacteria from the same genus as tuberculosis, in
order to uncover phages to which mycobacteria such as _M. Smegmatis_ and _M.
tuberculosis_ are susceptible. Once such phages have been isolated,
great progress can be made in treating and preventing tuberculosis.
I have only recently arrived to phagefinders, and have collected three
pertinent samples thus far. The first sample I plated ('Arcturus') was
drawn from the soil of my garden, and after a day of incubation, the
plate showed a light smegmatis lawn interrupted by numerous small, clear
plaques about 1 mm in diameter. These plaques had obviously been produced
by
some sort of phage, and I decided to further analyze this phage by plating
the contents of two specific plaques at various levels of dilution. The
original plate will be left to incubate some more so I can harvest the
phage later on, as the plate is still too light to harvest at the moment.
The second sample ('Dexter') I plated was taken from the backyard of a
fellow phagefinder, Kristen, who was kind enough to let me use her sample.
At
the moment, the plate remains clear, and seems to show no sign of phage or
smegmatis. I speculate that it needs more time to grow, or that the
sample had been in the refrigerator for a period too long for the phage to
survive.
(The sample had been collected at least a week before I plated it,
whereas my garden sample was comparatively fresher.) The third pertinent
sample is not yet plated, but I hope it will yield results: it is a rainwater
sample, and I am excited at the prospect of finding phages in water, and not
just soil. There are still four plates I have not discussed, as they are
very recently plated, and they are the dilutions from the _Arcturus_ plaque,
arbitrarily described as 10-1, 10-2, 10-3, and 10-4 dilutions. I
anxiously await results from these and hope also to harvest a high titer phage
in
the coming week. In the long term, I plan to collect and plate more fresh
soil samples, as those seem the most dependable phage sources. I will
study
the _Arcturus_ phage further, utilising the current methods as well as new
ones such as the use of electron microscopy and DNA analysis.
--Genevieve
In the past three weeks I have isolated a possible 5 phages. Four of the five came from the dirt sample BG-3-03. This was isolated at the Botanical gardens as part of a collective library. Its original location was documented as under a rose bush. It was a moist, rich, and dark soil; it also contained many sticks and chips of wood. The three viruses that were pulled from the sample were labeled A, B, and C. The A phage formed large clear plaques. The A plaques were the largest ones on the plate but of the lowest abundance. Phage B formed medium sized plaques with a lytic center and a lytic ring. These were also found in a small number on the plate. Phage C formed a large number of tiny clear plaques. To understand the size range on the plate it should be noted that A plaques were about twice the size of the plaques formed by phage B, and four times the size of the plaques formed by C. I attempted to isolate the three phages only to find that Phage B was accompanied by another unidentified phage and that phage C was “piggy-backing” with A. The new unidentified plaque found with B is the possible fourth phage. It was seen in the first round of purification; the new plaques appeared to suppress phage B until they were diluted out after the 10^-3 dilution. These new plaques could be a result of two things. The first is that this new phage could be “piggy-backing” with phage B; or that phage B changes its plaque morphology based on its concentration. In order to find the answer to this puzzle we have attempted to isolate and plate the different plaque types. The newly seen plaques were labeled phage B1 and the originals were labeled phage B2. As for the problem of C attaching to A, it was most peculiar because it did not show up until the F2 generation. It could be due to clumsy lab technique or dirty equipment, but that is an unlikely scenario. I attempted to isolate phage A from C and the results of my isolation will be revealed on Monday when the plates are fully-grown. If the isolations prove unsuccessful the electron microscope will reveal how many phages are actually at work on this plate.
--Kristen
I did not find any phage in my first two samples (one from a park in Canada, one from the basement of a building at my summer camp in the Adirondacks), as they were not incubated long enough. Yet, when I re-incubated my samples, I got very faint plaques on the sample from Canada . I did dilutions and got more visible plaques on my ten to the zero sample. I picked plaques from that sample and will do dilutions today. The soil that I obtained the phage from was from a park in Montreal Canada called Place du Canada, it is dark in colour, slightly moist and loamy. My other samples were not as successful. I did not get any phage in the wet sandy sample from the CIT lodge basement, the sample from the potted mango tree in the indoor conservatory of the Bronx Botanical Gardens or the sample from the drinking fountain in the Forschheimer building. Today I will plate a sample that I took from a crack in the sidewalk near my apartment building. The sample is full of dog hair and other debris and is dark in colour.
--Chiara