Fern the Phage

As I mentioned in my last blog, at the beginning of this semester I found a phage that infects the bacterium Mycobacterium smegmatis. My journey with my phage, who I’ve named Fern, has been interesting and filled with many unexpected twists and turns.

Everything began back in March when we were collecting environmental samples. I collected a LOT of environmental samples, a grand total of… 40 samples. I was told compost bins are a good place to find them, so to compost bins I went… mine, my friend’s, my friend’s Grandma’s. Nothing. I continued trying in the garden, ponds and streams. Still my plates were free of plaques. Eventually, from a sample from the soil from on top of our old garage door that had been lying in the vegetable patch, I found a plaque, and not just one, I found 3 differently shaped plaques! The picture below shows this very plate with some plaques that have been circled. A plaque is a circular clearing that forms on plates due to a phage infecting the bacteria on the plate. If you’re interested in more information about how phages form plaques on plates click here [1].

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Photo of direct isolation plate

Unfortunately, a few labs later I was back to only one phage (the other two did not survive the phage buffer).

The plaques of the surviving phage seemed to have quite a range of different morphologies. Some were completely clear, while others had cloudy rings around the outside and some were small, some medium and some large, as you can see in the photo below. Plaques of one phage tend to have plaques with the same morphologies so I was cautious that I may have had multiple phages. However, multiple morphologies continued to be the case even after purification so I assumed that I had purified my phage.

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Photo from phage purification showing different plaque morphologies

A few weeks later we were extracting the DNA of our phage. I was very lucky and miraculously I only had to do it once. I was so surprised that something actually worked the first time as this was not my experience of phage lab so far. Some of this DNA will be sent off to the University of Pittsburgh to be sequenced!

One of my favourite parts about this journey was when I got to see my phage! Phages are way, way, way too small to see with the human eye. Fern has a head of about 62 nm and a tail of about 137nm. That’s 0.000137 mm! To see that small, we need an electron microscope. We went to the electron microscope at Auckland University and saw our phages. This is what Fern looks like…

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Fern the phage from the electron microscope

As you can see from these pictures the phage has a head and a tail. The head is covered by a capsid (a protein shell) and contains the DNA [2]. The tail is the part that attaches to the bacterium, pierces the bacterium then passes the phage DNA into the bacterium [3].

Near the end of the semester I was ready to explore more characteristics of my phage and I decided that burst size would be interesting. The burst size is the number of phages produced when a bacterium is infected by a phage. If you want to find out more about burst size click here [4]. I was modifying a protocol for a different type of bacteria using a lysate with a different concentration of phage so there was some guessing and trial and error involved. The first time I tried this experiment the concentration of my phage lysate was way too high and I couldn’t see anything. When I tried again I got the concentration right. However, I did not do the experiment for long enough.

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Plate from burst size experiment showing the two morphologies: small and large

Although I was unsuccessful in establishing the burst size I discovered something very interesting … I had two distinct plaque morphologies. Could there still be two phages? There were two distinct morphologies: small and large. So, did I ever purify my phage properly?

My first reaction was that there must be two phages. To try to figure this out I picked all the different plaques morphologies (large, small and cloudy or clear) and plated them separately. The new plates all showed small plaques, all with similar morphologies. This lead me to believe it was only one phage and why this was happening was still a mystery.

Next I thought maybe it had something to do with the age of the bacteria culture as this had only started happening later when the bacteria were older. However, after plating with both old and new bacteria I saw no difference, so it wasn’t this. Further confusing me, this time there were no small plaques. There could be so many reasons why this is happening… rapid mutations, the time at which the phage infects the bacterium and I’m sure many more things of which I’m unaware, but currently it’s still a mystery.

This course has shown me firsthand how in the real world everything doesn’t fit into the categories and rules we often try to make in science. It’s a lot more complicated than that. Currently no one knows why my phage is making two different morphologies sometimes and that’s amazing. It really reminds me of how much we don’t know and how much there is to still discover.

References:

[1] Microbial Genetics (2002). Phage Plaques. Retrieved from: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/plaques.html

[2] Orlova, E. V. (2009). How viruses infect bacteria? The EMBO Journal28(7), 797–798. http://doi.org/10.1038/emboj.2009.71

[3] Ecole Polytechnique Fédérale de Lausanne. (2016). How viruses infect bacteria: A tale of a tail. ScienceDaily. Retrieved from http://www.sciencedaily.com/releases/2016/05/160518133816.htm

[4] S. T. Abedon. (n.d) Burst Size. Retrieved from: http://www.archaealviruses.org/terms/burst_size.html

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