I like E. coli...

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I like E. coli...

Postby cjp » Tue Dec 18, 2012 5:02 pm

... Gram-positives, not so much.

This year - and last - our E. coli (K-12 strain in nutrient broth in a tube) have produced nice lawns when using it straight from the tube to inoculate nutrient agar plates with sterile swabs. However, our Gram-positive bacteria - Staphylococcus epidermidis - did not grow at all using the same technique. Now, I could really use some advice especially since there is not as much time allotted for the science fair this year and some supply companies are about to shut down for a little while for the holidays; I really need to make sure that we can make it work with the supplies that should be arriving tomorrow.

This time I ordered the S. epidermidis that is cultured on agar in a tube - as opposed to the S. epidermidis nutrient broth we just failed with. I'm a little nervous as neither my student nor I have worked with the slants, but should this work? ... She will use a sterile disposable loop (10.0 uL) to lightly scrape the top of the slant. Then she will transfer this into a tube of nutrient broth and allow it to incubate at room temperature over night. Next step is to transfer onto nutrient agar plates using sterile swabs. Does this sound reasonable? Should we do a 1:10 dilution before transfering to plates? I know we don't want to dilute too much (if at all) because we want a lawn and not be able to see individual colonies.

Last year, we never did get the Gram-positive bacteria (Micrococcus luteus) we were working with to grow a lawn and it was very disappointing. We both would really like to have a better outcome with more data for better analysis. Thanks.
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Re: I like E. coli...

Postby sarahlaugtug » Thu Dec 20, 2012 3:10 pm

Hello cjp,

Sounds like your student has a great helper! Thanks for your question. As I'm sure you know, different bacteria require different growing conditions, types of agar, different growth temperatures. Some cultures come on plates, slants, and broths--broths are for cultivating, not isolating, so they need to be plated or put onto a slant.

I'm not sure why the bacterium isn't growing on the plates. Do you get any growth at all? Any colonies? Is it the technique? It is possible that the inoculation loop hasn't been cooled and the bacteria gets singed, which would cause a plate not to have growth? Also, make sure the temperature for cultivating is correct according to your research.
What kind of agar are you using?

http://www.sciencebuddies.org/science-f ... Agar.shtml

This article may help:
http://textbookofbacteriology.net/themi ... utgro.html
I don't know if you have viewed this safety guide in regard to bacteria and pathogens. Safety is key when it comes to working with biological agents.
http://www.sciencebuddies.org/science-f ... fety.shtml

Also, see the attachment on growing bacteria and some common problems. Hope that helps. At least bacteria doesn't take a long time to grow in case something goes wrong. Please ask more questions and let us know how it progresses.
Happy Holidays!
Attachments
microbio_cultureproblems.pdf
Growing bacteria
(13.98 KiB) Downloaded 89 times
Always remain curious,
Sarah
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Re: I like E. coli...

Postby cjp » Tue Dec 25, 2012 4:55 pm

Happy Holidays everyone!

Thanks for your help Sarah; I appreciate you taking the time to provide me with all the links. I did read them and carried on with the project. Now I have a question about the results if you don't mind. We were working with green cleaners used on Staphylococcus epidermidis and Escherichia coli. One of the green cleaners used was lemon juice. Our disks dipped in lemon juice and placed on the Staphylococcus epidermidis produced no zones of inhibition. The Staphylococcus grew right up to the disk. However, the disks dipped in lemon juice and placed on the plate of E. coli did produce a light circle around the disk; however, there is light growth inside the circle right up to the disk. So for our measurements, we would record a 0.0 mm for zone of inhibition?

And if we wanted to look at the experiment in a different way - not which green cleaner is more effective against these bacteria - but if we just wanted to find out which are bactericidal and which are bacteriostatic, then the lemon juice is bacteriostatic against E. coli, since it didn't kill everything (streaks of growth can definitely be seen in the circle); the lemon juice was ineffective against the Staphylococcus since it grew right up to the disk; and the hydrogen peroxide we used was bactericidal against both kinds of bacteria since large,well-defined, and totally clear zones of inhibition grew around the disks dipped in hydrogen peroxide and placed on both types of bacteria?

Thank you so much, and thank all of you experts for the great job you do here!!! Happy New Year to all.
Last edited by cjp on Wed Dec 26, 2012 4:47 pm, edited 1 time in total.
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Re: I like E. coli...

Postby cjp » Wed Dec 26, 2012 4:45 pm

Okay, so after reading a bit, I see that the disk diffusion method alone can't indicate whether the substance tested was bactericidal or bacteriostatic. Further effort - the dilution method - must be performed to determine this. This would be an interesting project extension; unfortunately, she doesn't have time to do this now.. maybe for the spring science fair!

So we're left with the Staphylococcus epidermidis that grew right up to the disk.. no sign whatsoever of a zone of inhibition and the Escherichia coli that looked as if there were a zone upon first sight, but when it was examined more closely, light growth up to the disk could be seen within that zone. So we have to conclude that the zone of inhibition for lemon juice against both types of bacteria was 0 mm. I guess I'm wondering if it would be important for her to bring this up in analysis (the differences in appearance) or since the zone measurement was 0mm for both, it is really not significant?

Thank you so much.
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Re: I like E. coli...

Postby heatherL » Thu Dec 27, 2012 4:11 pm

Hi cjp,

Congratulations on a successful project! It sounds like you have a good idea of what's happening here. While the quantitative results are important, it can also be useful to report the qualitative observations. Do you have pictures of the plates that your daughter can present on her board? If not, perhaps she could draw the differences observed. Even a written description (like the one you gave) would be interesting to note. Although the numbers will not indicate differences, people do appreciate that visual differences can sometimes show a "biologically relevant" result where statistics do not.

Also, it is good to note that the dilution method is one way to take the project one step further in the future. Judges like to see that students are thinking about ways to improve upon their projects; that is how science progresses!

I hope that helps. Good luck, and please keep us posted with your daughter's progress as she prepares to present her project!

Heather
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Re: I like E. coli...

Postby sarahlaugtug » Wed Jan 02, 2013 3:39 pm

Hello cjp,
Sounds like your project is going well. Would you have time to repeat the experiment? This could provide another look at results to see if possibly there was an error or if indeed those are the correct results. If you don't have time, that is ok. Like Heather mentioned, you can always write this into results and explanation/ further research ideas in the student's report.

In the analysis it is important to note your observations, whether they have significance or not...maybe upon repetition you find similar or different results. You can postulate what would happen if you were to repeat the experiment, based on the research and your hypothesis. Heather's idea to include diagrams and photos is a great idea! Let us know how it goes. Happy New Year!
Always remain curious,
Sarah
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