Science Buddies: "Ask an Expert"

My science fair project this year is dealing with phages vs antibiotic resistance. I have the tryptic soy broth, T4r phages, e coli B and the lactococcus lactis cultures ready. I also have the prepoured agar plates ready as well. What do you suggest is the best method to culture the bacteria on the plates? Do you have to culture the bacteria and phages in the broth for 24 hours first then inocculate the plates? Also what is the best way to seal my petri dishes in the incubator? I have heard numerous methods and I was just wanted to know if there is a method that works best?

Hi,

This is a really great project! Here is a project from the Science Buddies website that gives a procedure for growing phage.

https://www.sciencebuddies.org/science- ... p029.shtml

One of the important points is that you need a freshly grown overnight culture of E. coli to start with. Normally, you would add the E. coli and the phage to the soft agar at the same time before you start the incubation. However, I’m not sure what you are trying to do in your experiment. The T4r phage is specific for E. coli; I don’t know what you will be using the Lactococcus lactis for in the experiment. It would be helpful if you could explain exactly what you are trying to do.

Normally you would turn the Petri dishes upside down before placing them in the incubator. You can tape each Petri dish, with clear if you want. Or you could put them back into the original plastic bag to secure them.

When trying to do unfamiliar techniques like this, it is helpful to do a small trial run before you do your definitive experiment, and I do recommend that you do it for this project. This will ensure that all of your reagents are working and will give you some valuable experience.

Donna Hardy

My science fair project is showing that phages are selective. I am perfectly aware that the phages only kill e. coli and do not kill lactococcus. What I did is I innoculated tryptic soy broth with tube cultures of lactococcus and e. coli. Then I incobated them for 24 hours at 37 degrees C. Then I used the broth and put 0.5 ml in 2 ml of soft nutrient agar per nutrient agar plate. I added 0.1 ml of coliphage bacteriophages to three of the soft agar tubes. Then I mixed these tubes and poured them in 3 of my agar plates like the others. I got my plaques in my phage plates and areas of inhibition in my antibiotic plates and growth in my control plates. Growth of the e. coli that is. None of the lactococcus grew. I even did quadrant streaks to see what each of the bacteria look like and the lactococcus didnt even grow on the streak plate. i determined that it was the agar that was the cause of the lactococcus not growing, as well as the temperature of the incubator, which i recetly found was supposed to be at 30 degrees C. I just need to know what broth and agar that lactococcus grows best in, guaranteed, because I only have one chance to get this right, no second chances.

Hi,

Lactobacillus coccus should be able to grow on Tryticase Soy Agar and it can grow at 30 degrees Centigrade, although somewhat slower compared to 37 degrees C. I think you should continue your troubleshooting. It’s possible that your culture is not viable. How old was the stock culture? When did you last grow it?

Donna Hardy