Problem with bacterial growth on agar plates

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amyCC
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Problem with bacterial growth on agar plates

Post by amyCC »

Posting on behalf of a student.

Student is doing this project: Which Acne Medication Can Really Zap That Zit? https://www.sciencebuddies.org/science- ... medication

The student says they have followed the instructions and have seen no growth on the plates after more than a week. (They also ordered and used extra bacteria and saw no growth.) Here are details provided:
 
* The agar plates, as well as the bacteria, were refrigerated upon delivery.
* The first batch were plated 11/19 – no growth.
* These were incubated at 37 degrees Celsius
* They used a microscope and confirmed there was no growth.

Student needs troubleshooting help.

(They are using a kit -- so if there is something that might be a logical problem or needs to be checked, please note that, too.)

Amy
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brandimiller610
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Re: Problem with bacterial growth on agar plates

Post by brandimiller610 »

Hi Amy,

I took a look at the experimental protocol as well as the details provided by the student. Since it seems like they aren't seeing growth of the E. coli, that means there is likely a problem with either the reconstituted culture itself or the agar plates they are using.

I would suggest to the student to check and make sure they are following the steps for reconstituting the bacteria in liquid media. Generally, E. coli grows well in nutrient broth, so I assume the protocol included with the kit would call for that. If the kit comes with a different reconstituting medium, maybe they could try using nutrient broth and see if there is a difference? The student should make sure the entire freeze-dried bacterial pellet is thoroughly resuspended in liquid medium. It is also important to ensure the nutrient broth has been prepared properly and is not expired and/or contaminated.

If they are sure the bacteria was reconstituted exactly according to the protocol, I would then have them try to inoculate nutrient broth (liquid media) as well as agar plates. That way, if the bacteria grow in the liquid media but not on the plates, we know there is something wrong with the plates.

The growth condition of 37 degrees C is fine, and it is definitely odd that they didn't see growth of E. coli after more than one week.

These are my thoughts and the troubleshooting tips that came to mind!
Hope it helps the student!

-Brandi
akulk
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Re: Problem with bacterial growth on agar plates

Post by akulk »

Hi Amy!

I agree with Brandi above; inoculating the bacterial colony in liquid broth seems like the most viable option if there continues to be no growth observed on the agar!

Relating to the solid-medium protocol, here are two troubleshooting tips that come to mind: 1) try using inoculating loops instead of cotton swabs to spread the bacterial solution onto the agar; these loops tend to deposit most of the solution onto the agar plate a bit better than cotton swabs, so perhaps this may be the source of the issue. 2) It is possible that there may be an overlap between your antibacterial zones of inhibition; try spacing out your different experimental/control groups so that each group has its own plate rather than 1 quadrant of the same plate.

Hope that this helps in some way!

Anika
amyCC
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Re: Problem with bacterial growth on agar plates

Post by amyCC »

Brandi and Anika - Thank you both! I have passed on this very helpful troubleshooting information to the student. I appreciate you both taking a look!

Amy
Science Buddies
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