Let us begin!
Just before we get into the nitty-gritty of this microbiology stuff, I’ll go through a couple of frequently asked questions for any of you new to this phantastic phage blog. Well of course the main one is; what the heck is a bacteriophage (or phage for short)?!?! My sentiments exactly. To put your troubled mind at ease, I think of them simply as a virus that infects a specific bacteria. It is also interesting to know that bacteriophage undergo a lytic or lysogenic cycle depending on the bacteriophage and it’s environment. These little organisms are the most diverse and abundant living organism in the world and to put it in perspective, my super lab leader once quoted; ‘if you were to gather up all of the phage in the entire world and weigh them, they would exceed the weight of every single elephant combined by 1,000 fold’. Wow, that really puts them into perspective, especially considering the average size of a phage is around 1-5% the size of its host bacteria (which is extremely tiny in itself!). Did I hear you ask just how abundant these little creatures are? Well studies suggest that there are around 1031 phage present in our environment and viral ecologists suggest that they infect at a rate of 1023 viral infections per second.
So what have I got in common with this super virus? Well I am one of New Zealand’s newest phage hunters! This year Massey University teamed up with the Howard Hughes Medical Institute and offered this delightful experience as a paper in the Bachelor of Natural Sciences, working with Mycobacterium smegmatis – the sweet sister of bad-boy Mycobacterium tuberculosis. So let’s get down to the business of what I have discovered so far!
After initially isolating 23 plaques (holy moly, am I right!?) from 8 soil samples collected around Auckland and completing a series of direct plating an enrichment, my initial positivity was shorted lives with the knowledge that these plaques had not further developed. It is important to note that a plaque is a clear (or sometimes cloudy) zone representing an area within the media in which the bacteria that has experienced viral lysis – in other words plaques indicate the presence of a bacteriophage that is infecting the bacterial cell that was once present. Never fear, 5 new soils samples later and I had found something! 3 little plaques on the direct plating of sample #11, from the silage plot on our farm.
Fast forward several weeks later where I examined my plates after the initial titre (a titre is the counting of the number of plaques present in order to work out the concentration of bacteriophage). There looked to be some relationship between size of the plaques and the dilution. As the dilution increased from 10-4 to 10-3, the plaques observed on the plate also increased in size. This is interesting as my phage plaques are actually decreasing in the number of plaques as the titre plates become more diluted but should in fact be remaining the same size. So why is this happening?
After discussion, a few plausible hypotheses were concocted.
- Can the bacteria detect the phage?
- Do the phage detect density and not grow to a larger size to conserve nutrients? Phage signalling
- Is the bacteria signalling to the other bacteria with the increased concentration of phage?
- Do the bacteria detect different phage and restrict growth based on that particular phage?
Any of these seemed possible. Yet elaborating on phage detection, we came to the conclusion that phage are relatively stupid and thus is it extremely unlikely that it is the phage detecting density. Yet anything is possible in science? Hmmmm back to the drawing board!
Just to conclude today’s blog, I would like to draw your attention to a second observation that I have seen. It looks as though there is a ‘halo’ morphology around these plaques. Quite interesting considering this morphology actually became present after 10 days, rather than the 24-48 hours as the guide suggested! The picture below illustrates my plate 11.5.3 on the left, 48 hours after the first streak was completed, and another 10 days looking at the same streaked plate.
Click here to read a short blog of Debbie Jacobs-Sera (University of Pittsburgh) who observed a similar phenomenon on her plate of M. smegmatis phage and the discussions of possible hypotheses below her article. In brief, some of her hypotheses include;
- After 5 days M. smegmatis runs out of nutrients and stop dividing
- Phage initially in lysogenic stage (clear plaques), then it becomes efficient to enter lytic cycle (turbid) as nutrients are depleted
- Clear plaque outline represents access to more nutrients further out
I’m hoping to keep looking into some of the possible hypotheses as to why these plaques are doing what they’re doing. There should be some interesting finds (I hope!)
In the meantime, keep phinding those phage!
We have had an interesting discussion in regards to further possible hypotheses below. 🙂