Science Buddies: "Ask an Expert"

Hello,

I helped my daughter with her science fair project "The End Zone : Measuring Antimicrobial Effectiveness with Zones of Inhibition." We gathered all the material from Carolina Biological. She tested hand soaps, multi-surface cleaners, and dish soaps. She had 5 different types for each category, she was able to do each category three different agar plates. She used the E.coli that was in a culture tube from Carolina Biological. We have several questions. It did not seem like the E.coli really grow on the agar plates. It was hard see the zone of inhibition. She incubated the agar plates inverted for a week in a incubation box with a heat light. Most of the agar plates had condensation in them and it was difficult to see the inhibition areas. Also I think that she had some contamination with one of her soaps disks. There seemed to be a different bacteria growing on one of her disks. Also her controls agar plates did not really look like there was anything growing on there either. My questions is do you think that she might need to repeat the experiment? Also do you think that the E.coli culture we got was not effective, because the control agar plates did not seem to grow anything? This is her first time working with bacteria. Thank you for your help.

Sincerely,

Kimberley Parker

Hi,

Sorry your experiments did not work well. It sounds like you did everything according to the book but your bacteria did not cooperate.

If the control E coli did not grow then it could be:

1. The bacteria were too old and dead. How long did you have them before using? Were they stored in the refrigerator? What did the culture look like? Were there bits floating in it or was it stringy?

2. The agar plates were bad. Were they nutrient agar? Normally E coli grows very well on nutrient agar. You should be able to see growth in 24 hours in an incubator.

3. The temperature in your 'incubator' was too high. Did you measure the temperature? It should be around 37 C. Also, E coli normally grow in the human intestine where it tends to be rather dark. If you had a light on all the time, I don't know what effect that might have. I would wrap the plates in aluminum foil to keep them dark.

4. The bacterial culture was not spread properly. How did you transfer bacteria from your culture tube to the agar surface and how did you spread them around? I like to use a sterile dropper to put a drop of culture [shake the tube first] on the agar and then use an opened paper clip, sterilized by flaming, to spread the drop evenly over the surface.

You can get a fresh culture of E coli from Carolina and try again. You can call them and ask them if there was any reported problems with the batch of E coli you received. They are very careful with their cultures and materials, but mistakes can be made.

Good luck and do let us know what you find out.

Sybee

Sybee,

Thank you for your reply. We will definitely repeat the experiment with your suggestions. The E.coli tube was cloudy and had floating things in the solution. We did order the culture tube from Carolina Biology however the culture tube did not come with directions on how to properly store the culture tube. We stored it at room temperature for about a week before she was able to preform the experiment. Do you think this was not the correct way to store the culture tube?
Thank you for time

Kimberley

Hi,

Bacterial cultures have a life cycle. If you plot number of bacterial cells against time the curve is S-shaped. The cells grow slowly at first then speed up in what is called logarithmic growth. Once they reach a high density, however, the growth slows down and eventually stops. This is called stationary phase. If the cultures are allowed to sit at room temp they may go into the next phase which is death.

Here's a reference on bacterial growth that illustrates what I said: https://en.wikipedia.org/wiki/Bacterial_growth

The best place to store the bacterial culture is in the refrigerator [NOT the freezer!]. Try to use the culture within a few days of receiving it from Carolina. Call them and ask for technical support. Find out from them at what point in the bacterial growth cycle they ship the E coli. Depending on where you live, the bacterial culture might even have been subjected to freezing temperatures in transit.

The bacteria we use in the lab are maintained in small agar tubes called slants. To start a culture i use a sterile metal loop to take a bit of the agar culture and put it into one or two milliliters of nutrient broth. I let this small culture incubate overnight [about 18 hours] at 37 C. Next morning I take 0.1 ml of the overnight culture into 10 ml of nutrient broth and continue incubating at 37 C. When this culture gets cloudy i use it to spread on an agar plate to do the inhibition assay.

You don't have to do it this way. I just wanted to tell you the procedure that a microbiologist would use. The main point is that bacteria can die if they are frozen or allowed to stand at room temp for a long time. Planning to do the experiments as soon as you receive the bacteria is best.

I hope this helps. Let us know if you need more help.

Good luck!

Sybee