Science Buddies: "Ask an Expert"

HELP Please!! we attempted the Death Rays project, followed the instructions exactly (used same amount suspension on each plate, good spread, half covered in tinfoil, used the instructed UV exposures to the 5 different sets of plates, inverted plates, 91 degree incubation). After three days of incubation, only one plate out of 15 to grew bacteria and it was only 1 colony. Carolina Biological is resending us nutrient agar plates along with a new vial of E. Coli bacterial suspension. I want to be absolutely certain we get data results this time around.

What if we FIRST grow the bacteria colonies, then apply the UV light to half the plates (other half in tinfoil), then see how many colonies are killed off? Do you think that would work? I would have thought that this would be the way the experiment should run in the first place so you have a colony count baseline. Let me know what you think as I'm really weary about repeating the original method and ending up with the same results and have nothing to use in the science fair.

thanks for your time and consideration
Kristen

Hi Kristen,

I think that sounds like a reasonable way to adjust the protocol, but make sure you will be able to identify the living versus dead colonies on the exposed and unexposed sides of the plate if you are going to let them grow out first. Check to make sure your incubator is at the correct temperature/working. This is also a decent way to make sure that your materials are good/that you are performing the procedure correctly.

Good luck!

Hi Kristen,

I am sorry that you had trouble the first time around with your cultures. It sounds like your E coli were all dead before you plated them on the agar. I know Carolina Bio says you can keep the culture tube at room temperature, but I prefer to refrigerate the bacteria. Bacteria at room temp will continue to grow and divide in the nutrient broth that they are sent in and eventually they will use up all the nutrients and begin to die. By refrigerating them at 4C [39F] you slow down their growth and division without harming them.

What volume of your E coli culture did you spread on each plate? I use 0.1 to 0.3 mL depending on how many bacteria there are in the culture. Remember that you want to have discrete colonies on the agar and not a 'lawn' of bacteria, so you need to pipet a volume that contains about 500 E coli onto each plate. Carolina Bio should tell you the number of E coli per mL in your culture and you can use that as your guide.

Be sure you shake the tube gently each time before you pipet and also be sure to spread the bacteria IMMEDIATELY. If you put the bacteria on all 15 plates and then spread them, by the time you get to the last plate, many of the bacteria would have already settled onto the agar and would not have been able to be spread.

What kind of spreader did you use? Did you flame it with alcohol or, if it was the metal paper clip spreader, did you heat it in a flame? The spreader needs to cool before you spread or the bacteria will be killed by the heat. I assume you did this correctly because you said you followed the instructions, but I am just making sure so your next experiment will succeed!

In regard to exposing spread cells to UV vs colonies, you should follow the project instructions. If you grow the bacteria first on the plates and then UV them, how will you distinguish a live colony from a dead colony? E coli is able to repair UV damage to some extent so the radiation will not kill ALL the cells in a colony. If you let the plates incubate again after you UV them then the resistant cells can grow up and you won't be able to see any difference.

This is a lot of information, so if you have more questions about the methods please ask and I will help you.

Sybee