Death and destruction.Valar morghulis. “All men must die,” in high Valyrian as any Game of Thrones fan would know. An adaption of this quote is ‘everything must die’. You may be wondering what has brought on these morbid thoughts? Well I can tell you that I’m in mourning for the death of my little phages and the conclusion of an unsuccessful phage hunt.
Phages that once existed have ceased to exist in the results my latest experiment. In fact, poor results for the last few weeks had me questioning, but yesterday’s experiment confirmed it. My phages are no more.
The defining experiment that confirmed this for me was a ‘spot test’. A spot test is where the targeted bacteria is spread over an agar plate containing all the nutrients the bacteria need to grow. Next, a small sample of phage, aptly named a ‘spot’, is dotted onto the plate. So IF phage is present, then after an incubation period one would expect to see a clearing of bacteria on the part of the plate where the phage was dropped. A big IF indeed.
Way back on the 2nd of May, a spot test was performed on promising phage samples after one round of purification. Sample 10.1, isolated from the soil under a pile of decaying autumn leaves near my house, gave a very convincing result. A very large and very clear spot boldly declared the death of bacteria and the birth of my phages! In the following weeks, purification attempts yielded high results to start with, before everything gradually went downhill. Grasping at new methods, like differing agar concentrations, but frustrated at every turn, I had arrived at the moment of truth. What would happen if I did a new spot test, on the old stored 10.1 sample? Would my phages still be there or not? NOT, as it turns out. No mild clearing. Not even a glimpse of hope. No phages. At all.
Something must have destroyed my phages in storage and I’m determined to know what it is. Even Superman had a kryptonite, right? The problem, however, is the limited research out in the realm of phage literature. My very clever supervisor, the one and only Dr. Heather Hendrikson, suggested a few possible avenues to explore, in my quest to find a ‘phage kryptonite’. My phages may have been particularly sensitive to the experimental procedure, and killed off by one or more of the following; temperature fluctuations, a component in the phage buffer, the pH or somehow the introduction of a biological contaminant that destroys phages.
After perusing many sources, of little to no help, I came across a journal article where a team in Poland, isolated a giant phage, also from a Pseudomonas strain (Drulis-Kawa et al., 2014). Interestingly this phage had a latent period of 40 minutes; the time interval between infection and release of new progeny phages. On the basis of these results, the incubation periods that we used of between 20 minutes to 1 hour, were definitely long enough. They also found that the phage remained stable up to temperatures of 60oC, and within the pH range of 5-11. Such big ranges indicate that these phages were reasonably hardy, unlike mine. Maybe they don’t make things as tough in NZ as we all think?
Electron microscope pictures of the giant phage isolated by the Polish research team. (Drulis-Kawa et al., 2014).
It seems that the problem with phage forensics is that phages are just so small. The only way to visualize phages is through electron microscopy. This brutal technique involves the use of uranyl-acetate, a toxic uranium salt, as a stain and other toxic chemicals to fix and mount the phages (Bebeacua et al., 2013). While this technique does produce great images, the phage is killed in the process. Technology has not yet evolved to the place where phages can be viewed in real-time. Instead we have to settle for animations like this one, sourced by Nathan right here on the PhageHuntNZ blog. Trial and error to determine the best conditions for phage propagation is the best avenue we have. Thus identifying the phage murderer, and bringing it to justice, is difficult due to the sheer lack of evidence available.
Browsing through different journal articles for the past week, my quest for information has ended in failure just like my phage hunt. While the outcome wasn’t successful, I have learnt a lot in the process. I can only admire professional microbiologists, and their patience to work with such elusive creatures. A special thanks to Heather, and her patience, not only with the phages, but with the endless questions of our class. Good luck to the rest of the phage hunters, who still may or may not have phages.
This is Sarah, mourning phage hunter, signing out.
Drulis-Kawa, Z., Olszak, T., Danis, K., Majkowska-Skrobek, G., & Ackermann, H. (2014). A giant Pseudomonas phage from Poland. Archives Of Virology, 159(3), 567-572.
Bebeacua, C., Lai, L., Vegge, C., Brondsted, L., van Heel, M., Veesler, D., & Cambillau, C. (2013). Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1. Journal of Virology, 87(2), 1061-1068.