Science Buddies: "Ask an Expert"

Hi All,

I am thinking of trying an experiment using bacteriophages, antibiotics and E Coli for my science project but I need a little help with diluting my phage solution. I want to dilute it similar to the dilution of my antibiotic(ampicillin) of 1mg added to 10ml of water. I am starting with a 10^9 titer solution of coliphage t4 but confused on how to proceed. I am unsure whether to do a serial dilution and not sure how it works. Can anyone explain how to find an accurate concentration of coliophage to have a similar impact on the E .coli as my ampicillin solution?

Thanks!

Hi,

That's a good question and a reasonable one to ask, but not so easy to answer. The killing of E coli by a bacteriophage is measured by a test called a plaque assay, which requires a pretty large dilution of the phage followed by spreading a drop of it onto a 'lawn' of E coli grown on agar. Wherever the phage have infected the bacteria and destroyed them, you will see clear areas on the agar surface that are called plaques. You count the number of plaques and that gives you a measure of how active the phage is. Here's one youtube video that explains how to do the dilutions but there are several more that you can watch if you still need help: https://www.youtube.com/watch?v=F-L74qIoChA

As for the plaque assay itself, there are a couple of different procedures depending on what kind of phage you have. This one seems like it describes the kind of plaque assay I had in mind: https://www.youtube.com/watch?v=W8nOklhhEw4

I'm sure you will have questions, so take a look at these videos and any others that look interesting. You can learn a lot by watching and listening to these speakers. Seeing a procedure demonstrated is always better than just reading a description of it.

Good luck!

Sybee

Hi,

Thank you so much for responding. I watched the videos but I can’t find a way to integrate them into my experiment. I wanted to use a filtered disk soaked in ampicillin for one trial and measure the inhibition zone to measure resistance over time. How could I generate a similar trial for the bacteriophage to measure resistance? Could I just dip a filtered disk into the bacteriophage solution to match my ampicillin trial or should I try and find a way to match the ampicillin trial to this one where I spread the solution over the plate and then measure the plaques to find resistance?

Thank you so much again

Hello again,

I understand why you are confused. The experiment has to be done in two different ways--one for ampicillin and one for phage.

The phage assay is actually a way to measure the number of active viruses in the virus solution. What you need is a way to measure the numbers of E coli bacteria before and after exposure to the virus.

In the same way, you want to measure how many E coli bacteria are left after incubating them with ampicillin.

Your original goal was to find the amount of phage that would kill the same number of bacteria as a dose of ampicillin--is that correct?

Can you help me out by explaining your logic here. What is the hypothesis of your project? Why do you want to determine the number of virus that would have an equivalent killing effect on E coli as ampicillin? In order to use the phage in place of ampicillin to treat an E coli infection?

The way you would do this would be put a measured amount of ampicillin on the surface of the agar in a Petri dish, spread it around well with a glass spreader, then put on a drop of E coli culture and spread that around. As a control, you would spread the same amount of E coli with no amp. Put the plates in a warm place [90 F or 32C] and leave them in the dark until the bacterial colonies are large enough to see and count easily. The difference in the colony count between the plates with amp and those without amp tells you how effective that dose of amp was at preventing the growth of E coli.

One thing that you need to remember about ampicillin is that it is a bacterio-static chemical. That means it prevents the bacteria from growing and dividing. It does not kill them outright like some chemicals.

OK. Now for measuring how well the phage kills E coli, you would do a similar experiment but this time you would add a measured amount of phage to a certain amount of E coli culture and then spread a drop of this on the agar in a Petri dish. Again, you use E coli alone (no phage) as the control. The phage will attach to bacterial cells in the culture and cause them to break open and die. You will see fewer bacterial colonies on the agar when you plate E coli + phage.

Are you understanding this ok? There are lots more youtube videos about this and you should choose some to watch until this all begins to make sense and you start to see how these experiments are done and what sort of equipment and solutions you need.

I'll be here to help you when you get stuck.

Good luck!

Sybee

Hi,

I wanted to try and find the rate of anit biotic resistance between ampicillin and bacteriophage against ecoli. This is why I wanted to use dilutions of both substances to be equal in “strength”. I wanted to conduct the first trial with just the starter culture of ecoli and then use the ecoli around the edges of the plaques and inhibition zones for consecutive trials. Would this be possible using the method of spreading the ampicillin or phages in the plate?

Thanks!

Hmm...those are hard questions to answer. E coli can become resistant to amp but I don't know how it would become resistant to coliphage. Is that what you are trying to compare? Are you thinking of using phage as a way to kill human pathogens like E coli or MRSA?

You can work out a dose of amp that would kill the same number of bacteria as a certain number of phage, but that would take some pretty involved methods--not impossible but pretty tedious and time-consuming.

I did a search for "can e coli become resistant to bacteriophage" and found a lot of interesting articles:

https://www.tandfonline.com/doi/full/10.4161/bact.24219

Some are rather technical, so if you get stuck on some point just ask us. The conclusion I got from the first paper I read was that E coli can become resistant to phage but that this is not a problem for using phage to kill bacteria. I'm not sure how they know that. You need to take a look at the paper and other sources.

I hope this helps. We can talk some more about this and see how it works as a project. It's a good idea, but as always, the problem is in the details.

Good luck!

Sybee