Methodology- Bacteriophages

[claudiamodica]

hello, so I am conducting an experiment testing the effect of bacteriophages at varying temperatures on bacteria. After looking at multiple different experiments, I am unclear on the methodology to do this experiment. All different experiments used different types of agar and solutions to plate the bacteria and to grow the bacteriophages, so how should bacteriophages be grown, and any advice on how to conduct this experiment, specifically how to plate the bacteriophage in relation to plating the bacteria. Much appreciated !

[brandimiller610]

Hi,

I hope you're having a great day and thank you for your question!

For the most part, there is a standard procedure for plating bacteria and bacteriophages -- there will be slight differences in the type of media used based on the bacterial species and the specific phages used. What bacteria and bacteriophages do you plan to use for your experiments? I would reference a method that uses the same bacteria/phages you plan to use because that would indicate what media you should use.

Bacteria can be isolated on solid agar using the quadrant method or by streak plating -- again, the kind of agar will depend on the bacteria you plan to use. LB (Luria-Bertani) agar might be a good place to start. It is widely used to cultivate E. coli strains, but is generally considered non-selective (meaning many strains of bacteria can grow on it). Agar plates streaked with bacteria can be incubated at 37 degrees Celsius until colonies are seen (generally 24-48 hours is sufficient for most bacteria). Please see this link for additional details on plating bacteria on agar: https://asm.org/Protocols/The-Streak-Plate-Protocol

To plate bacteria and phages together, soft agar and the "double layer agar method" are commonly used. Please see this paper attached for the method: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515400/. You can find it under the subheading "Double layer agar method". Briefly, bacterial culture and your phage filtrate will be mixed together in soft agar, poured into petri dishes, and then allowed to incubate. You would perform the Plaque assay then to determine the effect of the phage on your lawn of bacteria. Please see more information about plaque assay and protocols here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278645/

Again, these protocols are general and there may be slight modifications depending on the type of bacteria and phages you use. I suggest trying to find a paper that uses the specific bacteria and phages you plan to use.

Hope this helps! Please feel free to reach out on this forum if you have any questions or concerns about your project, we are happy to help!

--Brandi