In concluding my Phage Hunt with no phage discovery (*sad face*) I have been contemplating the reasons I stopped finding so many plaques. Heather linked us an interesting article about the factors influencing plaque growth so I thought I would share my view on it! First I will discuss some background information around bacteriophage plaque formation and how we achieve this in a laboratory setting. Then I will explore possible reasons for our dwindling amount of plaque formation, in attempt to answer: Where have all our plaques gone?
What are plaques?
A plaque is clear zone formed in a lawn of bacterial cells, in our case P. fluorescens SBW25 bacteria. This clearing indicates death of the bacteria in that area, due to lysis by any present bacteriophages. A single phage will infect a bacteria, replicating and releasing progeny phages that diffuse and infect neighbouring bacteria, thus giving a circular pattern of death (For more information about the action of bacteriophages have a read of Sarah’s post ‘Phage Hunt NZ Post: Fighting with phages’). Plaques may vary in size depending on the growth conditions and strain of phage present. Lytic strain phages are likely to give a clear plaque while lysogenic strains a more turbid plaque. The picture below demonstrates what typical plaques look like on a bacterial lawn, note the circular shape.
Read more about phage plaques here: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/phage/plaques.html
How do we form plaques in the lab?
To form plaques from a sample containing bacteriophages we incubate the phage sample with the bacteria (SBW25), then plate with nutrient-containing top agar on an LB plate. The plate is then left overnight to incubate, allowing plaque formation. This process was the basis of most of our phage hunt, as it allowed us to purify the phage samples we had obtained.
What influences the formation of plaques?
Getting a phage to form plaques isn’t as easy as it seems, we definitely found this out in our experimentation! One point that really caught my attention in the mentioned article was the great plaque count anomaly: “Less than 1% of the phages observed using microscopy have ever been grown in culture”. This indicates the great difficulty in forming plaques; many phages will be unable to do so and those that are able to, depend on a variety of conditions.
The concentration of agar used in the top agar will effect plaque formation. The article explains that plaque size increases as the velocity of phage diffusion increases. This may have hindered our results as small phages may be unable to form plaques in high agar concentration, as they cannot diffuse through the medium to infect bacteria. A decrease in agar concentration may therefore increase the amount of plaques forming. Another condition that affects plaque formation is incubation temperature, different phages being unable to replicate at some temperatures. In our experiments we chose an incubation temperature of 32°C, this may have prevented replication in some phages that were present.
The addition of specific cofactors may be required in some phages for plaque propagation to occur, for example calcium and magnesium ions. A cofactor is a substance necessary for certain reactions within a phage, allowing successful growth. Therefore addition of substances such as CaCl2 to the media will result in larger plaques. In some cases, for plaques to form the host bacteria must also be in a certain growth phase, the log or stationary phase, as this influences phage replication. The log phase is characterised by exponential bacterial growth, while the stationary phase is when growth has reached equilibrium. Ensuring the SBW25 bacteria are in the correct growth phase will increase formation of plaques.
With so many varying conditions that influence forming of plaques it’s no wonder we struggled to successfully do so. While we may have altered a number of these conditions in our experiments it would be difficult to get them all at the optimum level for our phages to show themselves. Even though I didn’t manage to isolate my own phage in the end, this process has definitely been very interesting and taught me a lot about bacteriophages. I hope I have better luck next time!