Why is my experiment going wrong?

[mthai]

Hi, I'm in the 7th grade and doing a science project on the effects of different dilutions of disinfectants on bacterial resistance. I know exactly what I'm doing and the procedure of the experiment, but all my problems are occurring during the experiment. It seems that when after I incubate by plates, I see no zones of inhibition around my disinfectant-impregnated filter disks though I see my bacterial colonies, M. luteus, growing close enough to the disks that it seems there's no disinfectants on the disks (I'm using the Kirby-Bauer disk diffusiong method). Help!

[WJClancey]

Hi,

I'm not an expert in this kind of experiment, so I can only ask some basic questions that perhaps you have already considered.

The first thought that comes to mind is whether the disinfectants are known to be good? For example, this means you've taken they from a known source and they haven't degraded (e.g., changed chemically because of heat). Second, I'd wonder whether the disinfectants were too diluted to show an effect. Is the range of solutions wide enough to include one that is known to be sufficiently strong? In this case, your background reading might have revealed a known result for a certain dilution. Third, is it possible that these organisms aren't affected by the disinfectants you are using? For example, are there resistant varieties?

You described the problem well, especially in mentioning that you see the colonies growing. That shows that the bacteria are alive. It's given that information that I posed the three hypotheses above.

Perhaps someone more familiar with this experiment can provide additional help.

Bill

[mthai]

Thanks for your time for answering my question.

You posed some really good points on your reply, and actually I haven't considered some of them while doing the experiment, but I found this project on Science Buddies (https://www.sciencebuddies.org/science- ... ?from=Home) and I followed exactly as the directions told me. As for the disinfectants I used, I used 70% isopropyl alcohol, mouthwash (Listerine) and pine oil. The last two disinfectants mentioned was found on Science Buddies under my specific project, and I got the idea of using alcohol and the bacteria M. luteus from a microbiology book that contained a project idea very similar to mine ("Do bacteria become resistant to household disinfectants?", (Dashefsky, 1995)), so the disinfectants used should be effective, even with a 12.5% dilution.

I tried a couple things that may have impacted my experiment. I incubated just an agar plate to see if possibly the agar was contaminated, and it wasn't. Another thing that I was suspecting that may have affected the results was the amount of time the bacteria was exposed to the air while I was inoculating the plates. It took me about 5 minutes per plate. Do you think this may have affected the results? Also, since I used a box, a thermometer, and a lamp as my incubator, could the disinfectants evaporate under the lamp? You told me you weren't an expert, but just in case you knew, because you probably know more than I do.

Lastly, I don't quite understand the first thing you pointed out to me on your reply when you said "known to be good". If possible, can you please explain to me again what you mean?

Thanks,
mthai

[WJClancey]

Thank you for the great explanation and pointer -- I learned a lot from it. You have an interesting experiment, and I was impressed by the level of detail Science Buddies provides.

First, I note that the procedure mentions that if you don't find an effect, "suspect either your impregnation technique, or poor contact of the filter paper with the agar." You probably should verify these steps first.

Regarding the disinfectant being good, now that I see the choices (alcohol, mouthwash, and pine oil), I believe that degradation is probably not a concern. Presumably you used all new, fresh products and not something that's been opened and used over a long period for other purposes?

I am wondering whether the combination of bacterial species and disinfectant might require higher concentrations than were suggested by the procedure. Again, proceeding as a non-expert here, my first thought would be to use 100% solutions for a round of tests. This would establish efficacy, that is, that the bacteria are sensitive to these disinfectants. I see that the report you cite indicates that even at 12.5% you should see a result. Maybe you could find a clue in procedure they followed, looking for a difference from what you did (I don't have a copy so can't help directly here).

You mention in particular the heat, and certainly one (again non-expert) idea that comes to mind is that the lamp indeed caused evaporation of the alcohol and mouthwash (which probably contains alcohol). The disinfectant therefore diffused throughout the airspace in the dish, causing no specific effect at the edges of the disks. Presumably you followed some information about how to do the incubation so you didn't overheat the plates.

As for the 5 minute delay, the procedure does say to wait 5 minutes and your issue appears to be growth, rather than the bacteria being weakened. So that's probably not it.

Did you try the control of dipping the disks just in sterile water?

I'm curious to find out more, so keep me posted.

Bill

[mthai]

Again, you pose good points that I haven't considered deeply. I remember that when I placed the disks on the agar, I didn't press on the disks - just let them fall onto the agar from my tweezers in a similar way someone were to place a cover slip on a microscope slide - so maybe that may have had some effect to the overall result. Also, the alcohol I used has been used in my house for quite a while, so the next time I experiment, I'll use new, unopened alcohol. As for the evaporation from the lamp, there is not much I can do. In order to have an incubator in my home, I must have the lamp as a heat source, and it would be impractical for my dad and I to drive to the high school every day to use their incubator. If you have any ideas as an altenate heat source, please feel free to post me any new ideas you may have. I have searched all over the Internet for ideas, but all of them require a lamp. Continuing on with this statement, I believe that I wasn't overheating the plates because the ideal temperature for growing my type of bacteria is 30 degrees Celsius, and the incubator temperature was exactly that. In addition, you mentioned before about using higher concentrations for this different bacteria I was using (and I will), and it says in Science Buddies to use the E. coli bacteria, and when I told my parents I had to use that bacteria, they totally freaked out and said I had to get a bacteria that wasn't pathogenic (that's how I ended up with using M. luteus). And, oh yes, I have been dipping my control disks in distilled water the same way as I have been dipping my other disks.

mthai

P.S. In case you may have been wondering on how I incubated my plates - and I had no previous knowledge on how to do it and had to rely on the not-so-reliable Internet - right after I inoculated my plates (with a 5 minute waiting period prior to doing so) I placed them in the incubator upside-down and began incubating them. Also, should I add some more agar to the plates? I was thinking that maybe the disinfectants weren't able to diffuse to the agar because I only put 8 mL of agar into each plate. I hope this knowledge is helpful!

[WJClancey]

Hi,

I tried to reply several times last night, but there was a problem with the web site.

I posted an email on the Expert Forum for someone with more expertise to help us here.

I think as long as you don't have the lamp shining directly on the culture dishes, but have instead placed them in some kind of relatively closed container (a box) with the lamp acting as an indirect heat source you're okay. Of course, you would use a thermometer to establish and maintain the 30c. I might be more concerned than is necessary, but this is how I'd set it up.

I'm guessing the placement of the disks is the main issue here.

Incidentally, I found a fascinating article about the antimicrobial effect of spices, such as cumin and cloves, particularly against M. luteus. Although you probably don't want to change your experiment now, this article might interest you -- http://bulletin.piwet.pulawy.pl/archive ... gaoglu.pdf

Bill

[adance]

Hi mthai,

I taught a college-level class where students did the same experiment you are--and they got some similar results, that alcohol did nothing! I think that is because it evaporated too quickly.

You are doing a great job thinking through your experiment and explaining your problems. Some thoughts:

I think there is a difference between an "antibiotic" and a "disinfectant." You could try finding this on the internet--one will just kill cells and the other will prevent growth. Ethanol, for example, will kill on contact but won't keep bacteria from growing after it's dried.

Are you spreading liquid cultures on your plates? If so, keep them rightside-up for a few minutes to let the liquid soak in, otherwise things can get smeared when you turn them over.

As others have said, use fresh disinfectants. For ethanol, the best disinfectant concentration is supposed to be 70%, isopropanol I would guess is similar.

I do not think exposing the bacteria to the air is the problem, as you suggested--but, it could be possible that tough, disinfectant-resistant bacteria are getting into the culture at that point.

On amount of agar--it should be around a quarter to half-inch thick.

I would try your experiment again using full-strength disinfectants as a positive control.

Good luck! Let us know how it goes.

[mthai]

Thanks, I'll remember to apply these corrections from you guys when I re-experiment.

Also, I read the article about the antimicrobial effects on spices, and it was indeed rather interesting! And to answer adance's question on the difference between an antibiotic and a disinfectant - antibiotics kill bacteria and disinfectants prevent growth (right? The internet wasn't very helpful because they had different explanations on the differences so I had to guess a little)

I will experiment again soon, when I receive my M. luteus through the mail. If I have any more questions or if I come across some more problems during the experiment, I'll be posting. I'll let you know how the results turned out. Thanks again! (It's probably a little too early to start celebrating, though)

mthai

[Lise Byrd]

mthai,

You've done a great job examining what could be going wrong in your experiment! Here's another suggestion, if you would like to take it: You might consider using bleach as a positive control. In my experience, bleach is extremely effective at killing bacteria, so using filters with bleach on them would let you know whether you're doing your procedures correctly.

I also agree with the other Experts who suggested using a higher concentration of your disinfectants.

Good luck with your next run of experiments!
Sonia