Do you seen any flaws in the idea for this project? - (Jan/23/2006 )

I am about to start my project for a second time (the first time it didn't work because the culture I purchased was defective ), but before I start, I wanted to ask if anyone sees anything in my plan that looks like it may cause problems, and also, if you have any ideas that might help I would also
appreciate hearing them.

The goal of my project is to isolate a bacteriophage (cyanophage) capable of lysing anabaena flos-aquae. I plan to look for them in pond waters containing lots of algae, because it seems like the most likely place to find them. I am going to grow about 30 cultures in BG 11 medium for cyanobacteria that I prepared today from a recipe that was given to me a member of this forum (pardon me, I don't recall your SN ). The culture that I am going to use is #30.87 from SAG in Germany. Our vet kindly agreed to allow me to use his autoclave so I am going to autoclave the medium tomarrow. I'm going to put about 6 or 7 ml of BG 11 in sterile culture tubes and then add some anabaena to it. I will then let it grow in a sunny place long enough so that there is a nice amount of algae in there. After I have some good cultures going I will take sample from ponds and centrifuge them in 15 ml sterile centrifuge tubes @ 3400 RPM. I am not exactly sure what the G force is, but I calcuated something around 1200 Gs at the bottom of the tube and like 800 gs in the middle. After being centrifuged, 1 ml of the supernatant will be pipeted off and filtered through a sterile .2 μm syringe filter. Half of the supernatant will be saved in the fridge and the other half will be added to one of the algae cultures. If the algae quickly dies off it will mean that there likly is a phage (or polutant) in the sample. I can repeat it over and over again to verify that it is a microorganism killing the culture and not a pollutant. Also, I might buy myself a nice phase/darkfield microscope in the near future so I will be able to see it there is any visible CPE caused by a phage .

Thanks,
PhageMaster (9th grade)

This looks like a good plan to me. One thing you might want to think about (after you have found a phage) is to quantify the amount of phage. You could do this with serial dilutions of phage stock until you reached a 50% probability of lysing a stock solution of bacteria, or you could develop a way of producing phage plaques, similar to the methods used for counting plaque forming units of Lambda or T7 phage. Will you bacteria grow on a plate?

You could also check what things destroy the phage (alcohol? dilute bleach? exposure to UV? salt concentrations? chloroform? acids/bases?).

More aggresive would be to isolate DNA and try to guess the length of the phage DNA.

I am really having trouble getting tha anabaena grow in liquid medium, and if the anabaena is this picky with a liquid, it may not grow at all on a solid. Once I have some good cultures growing in liquid, I will try some other solid stuff. Why would I want to get the length of the phage DNA? To identify the phage? Thanks for the suggestions.

Hi

Sounds like a cool project, but don't forget that phages don't last in the environment very long, unless you can see an area of dying anabaena, you are unlikely to be able to isolate the virus from water samples (at least in quantities that will have an effect on your cultures).

Does the virus affect other types of algae? If so, could you culture those instead of anabaena?

Cheers,
Bob

I don't know if it would affect other types of algae, I have not been able to find too much material to read about the subject. I will try to find areas of alage that are dying to take samples from so I increase my chances of finding a that will do the trick. I am still trying to get my cultures up and running, I'm not at the saple collecting stage yet. Thanks for replying.