qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

No Zone of Inhibition?

Postby qqaisarr » Tue Mar 13, 2018 9:33 pm

My experiment is testing if different dilutions of disinfectants affected the development of bacterial resistance. I got the agar plates and the E. coli and all the other things that I needed for the experiment from Carolina Biology Supplier.

Afterward, I conducted the experiment. I swabbed some E. coli into an agar plate divided into four sections. I put a sterile disc dipped in different concentrations of disinfectants in each one of the sections of the agar plate and wrapped the agar plates in a blanket to keep them warm.

A day later, no zone of inhibition was produced.

What errors could I have done wrong? My project is due in less than 2 weeks so any help would be appreciated!

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Wed Mar 14, 2018 9:45 am

Hi, qqaisarr! E. coli is typically incubated at 37 degrees Celsius, or about 98 degrees Fahrenheit. A blanket isn't enough to warm the culture plates, since a blanket doesn't actually generate heat; it just helps keep in heat generated by whatever it's wrapping up. So your culture plates were probably too cold for your bacteria to grow and to allow you to see an effect of your disinfectants. You should find a way to repeat the experiment but keeping your culture plates at 37 degrees. Good luck!

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Wed Mar 14, 2018 11:45 am

nguyenmccarty wrote:Hi, qqaisarr! E. coli is typically incubated at 37 degrees Celsius, or about 98 degrees Fahrenheit. A blanket isn't enough to warm the culture plates, since a blanket doesn't actually generate heat; it just helps keep in heat generated by whatever it's wrapping up. So your culture plates were probably too cold for your bacteria to grow and to allow you to see an effect of your disinfectants. You should find a way to repeat the experiment but keeping your culture plates at 37 degrees. Good luck!

Thanks for your response! I really appreciate it! I just have another question. After I apply the E. coli to the agar plate, should I leave it at 98 degrees to culture before I apply the sterile discs with the disinfectants or should I apply the sterile discs as soon as I apply the E. coli and leave it at 98 degrees together? I don't know if that made sense but if it did, please let me know! :)

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Wed Mar 14, 2018 1:07 pm

Yes, that makes sense and is a good question! Does your experimental protocol not specify this? I would follow whatever is indicated in the protocol. If your protocol doesn't say anything about this, then I would plate the E. coli and let them grow at 37 degrees (in science we always use Celsius :D ) for about 24 hours or so, and then apply the discs with disinfectants. Good luck!

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Wed Mar 14, 2018 1:32 pm

nguyenmccarty wrote:Yes, that makes sense and is a good question! Does your experimental protocol not specify this? I would follow whatever is indicated in the protocol. If your protocol doesn't say anything about this, then I would plate the E. coli and let them grow at 37 degrees (in science we always use Celsius :D ) for about 24 hours or so, and then apply the discs with disinfectants. Good luck!

Okay, thank you so much for the help! I will repeat the experiment and let you know if it works! Thanks so much!! :D

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Thu Mar 15, 2018 6:32 pm

nguyenmccarty wrote:Yes, that makes sense and is a good question! Does your experimental protocol not specify this? I would follow whatever is indicated in the protocol. If your protocol doesn't say anything about this, then I would plate the E. coli and let them grow at 37 degrees (in science we always use Celsius :D ) for about 24 hours or so, and then apply the discs with disinfectants. Good luck!

All right, so I tried what you said. I used a sterile cotton swab to swab the liquid E. coli strain into an agar plate with 3 directions 120 degrees apart to ensure complete coverage of the plate. I took the sterile discs, dipped them into the different concentrations of the disinfectants, and placed them on the agar plate. After that, I took my heater, placed it horizontally across two chairs and put the agar plate on the floor so the heater would get even coverage of the plate. I used a thermometer and it says the agar plate was 37C, which is what it should've been. 8 hours later, no zone of inhibition is present and a lot of condensation occurred.

Any thoughts? I'd really appreciate it!

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Fri Mar 16, 2018 10:35 am

I might try culturing the bacteria as you've described here except without adding any disinfectant discs, just to make sure that you have conditions that allow your E. coli to grow at all. If they grow without any disinfectant, then we can discuss ways to troubleshoot the next step with the disinfectant discs. But if they don't even grow without adding the discs, then we know that there is a problem with the setup for culturing the bacteria in the first place.

Also, make sure you culture your plates upside-down (with the side of the plate holding the agar on top, and the empty lid face-down). This will ensure that any condensation accumulates in the lid, and not on top of your bacteria! :)

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Sun Mar 18, 2018 10:13 am

nguyenmccarty wrote:I might try culturing the bacteria as you've described here except without adding any disinfectant discs, just to make sure that you have conditions that allow your E. coli to grow at all. If they grow without any disinfectant, then we can discuss ways to troubleshoot the next step with the disinfectant discs. But if they don't even grow without adding the discs, then we know that there is a problem with the setup for culturing the bacteria in the first place.

Also, make sure you culture your plates upside-down (with the side of the plate holding the agar on top, and the empty lid face-down). This will ensure that any condensation accumulates in the lid, and not on top of your bacteria! :)

Thank you for your reply! I have always put the agar plates upside down before, so that cannot be the error. Anyways, I have done what you said which is culturing the bacteria before adding any disinfectants just to see if the E. coli is even alive. I have noticed that the agar plates are slightly more yellow, but not by much. Is this a good sign? I've talked to more people and they've told me to swab my own sample if it's my last chance.

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Mon Mar 19, 2018 7:52 am

That's good, as long as you're culturing the plates upside-down, then you don't need to be too concerned about the condensation accumulating inside the plates.

I'm not sure what the slightly more yellow agar means (I wouldn't make much of it), but are you seeing any colonies at all? If you don't see round colonies that are white / light yellow (lighter in color but less translucent than the agar), or at least streaks of white / light yellow where you swabbed your bacteria, then it means the bacteria aren't growing at all under the culture conditions you have.

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Mon Mar 19, 2018 3:20 pm

nguyenmccarty wrote:That's good, as long as you're culturing the plates upside-down, then you don't need to be too concerned about the condensation accumulating inside the plates.

I'm not sure what the slightly more yellow agar means (I wouldn't make much of it), but are you seeing any colonies at all? If you don't see round colonies that are white / light yellow (lighter in color but less translucent than the agar), or at least streaks of white / light yellow where you swabbed your bacteria, then it means the bacteria aren't growing at all under the culture conditions you have.

I did not see any colonies form so I just assumed that my E. coli sample is dead. So, I tried getting some dirty lake water and hoping it contained bacteria and did another test, but that did not work either because I didn't see any colonies form from the water.

If it is the E. coli's fault, where can I get a good amount of bacteria?

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Wed Mar 21, 2018 7:29 am

That was a resourceful idea to try using dirty lake water. Different types of bacteria require different conditions (temperature, nutrients, etc.) to live and grow, so maybe the reason that didn't work is that the bacteria that were in the lake water needed different conditions than those you had set up for culturing the E. coli.

The original E. coli from Carolina Biology Supplier were probably fine; the problem might have to do with how they were stored over the course of the several times we've tried to get this experiment to work. How did the bacteria arrive to you, and how did you store them when you weren't using them? They are very sensitive to being frozen and thawed repeatedly, so if you only received one or two vials that you took from more than once over the course of all the experiments, then they were probably dead before the experiment even started.

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Wed Mar 21, 2018 3:07 pm

nguyenmccarty wrote:That was a resourceful idea to try using dirty lake water. Different types of bacteria require different conditions (temperature, nutrients, etc.) to live and grow, so maybe the reason that didn't work is that the bacteria that were in the lake water needed different conditions than those you had set up for culturing the E. coli.

The original E. coli from Carolina Biology Supplier were probably fine; the problem might have to do with how they were stored over the course of the several times we've tried to get this experiment to work. How did the bacteria arrive to you, and how did you store them when you weren't using them? They are very sensitive to being frozen and thawed repeatedly, so if you only received one or two vials that you took from more than once over the course of all the experiments, then they were probably dead before the experiment even started.

Yes, I did mess up the E. coli a lot. For one, I accidentally stored it at 37 C because the label said so, but I later realized that it was the optimal growth health, and not for storing. After thawing it the first time, I had to freeze it and use it again because I accidently messed up a step. Anyways, what should I do now? Is there a last minute resort I can use besides E. coli?

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Wed Mar 21, 2018 4:08 pm

Ok yes, both the storage at 37C and the re-freezing and re-thawing would for sure have killed all the cells before we even got to an experiment. I'm sorry, I'm afraid I don't know of a substitute that is likely to work. :(

qqaisarr
Posts: 9
Joined: Tue Mar 13, 2018 9:30 pm
Occupation: Student

Re: No Zone of Inhibition?

Postby qqaisarr » Wed Mar 21, 2018 6:14 pm

nguyenmccarty wrote:Ok yes, both the storage at 37C and the re-freezing and re-thawing would for sure have killed all the cells before we even got to an experiment. I'm sorry, I'm afraid I don't know of a substitute that is likely to work. :(

Well thank you for your help! I really appreciate it!

nguyenmccarty
Former Expert
Posts: 53
Joined: Thu Sep 21, 2017 11:09 am
Occupation: University cell and molecular biologist

Re: No Zone of Inhibition?

Postby nguyenmccarty » Thu Mar 22, 2018 9:20 am

You're very welcome! I hope your next venture into science projects goes more smoothly!


Return to “Grades 9-12: Life, Earth, and Social Sciences”