Zones of Inhibition

[iftiswasamovie]

I am doing a project called " Do Different Dilutions of Disinfectants affect the development of bacterial resistance?" located on sciencebuddies.org. I ran the experiment with common bacteria swabbed from a doorknob. However, I didn't develop any zones of inhibitions --just scattered dots of yellow, pink, and white. And a beige colored bacteria that completely surrounded some filter disks. What went wrong?

Also, I thought that as concentration of disinfectants decrease the resistance decreases, that is not the case for me. In some plates, the bacteria surrounds the highly concentrated filter disk rather than the less concentrated.

I'm very confused. Please help me.

Does anyone know how to take clear photos of petri dishes? My camera keeps distorting it and it does not come out clearly.

[donnahardy2]

Hi,

For testing dilutions of disinfectants, you normally would grow a “lawn” of bacteria on the surface of the agar plate. The following website describes two methods for making the lawn.

http://lichfield.org/ictscience_moodled ... Advantages and disadvantages

It sounds like you used a mixed culture of bacteria for your assay. Usually, microbiologists test pure cultures of single organisms. Here is information from the science buddies website on general microbiological techniques.

http://sciencebuddies.com/science-fair- ... ques.shtml

I don’t have any experience in taking pictures of agar plates. You may need to experiment with lighting and background color. You can document your results with drawings if the photographs are not clear.

Donna Hardy

[deleted-71940]

Hello!

I actually performed the same experiment in eighth grade. Like the previous expert mentioned, you will need to grow a single bacterial culture if you would like to use zone sizes to determine the effectiveness of a particular antimicrobial agent. The Kirby-Bauer Disk Diffusion method (the assay that you are using) is an assay used to test one strain of bacteria at a time.

Are you performing your experiment in a laboratory? Many bacteria need specific conditions to grow well, usually warm, moist conditions with an adequate supply of CO2. These conditions are usually simulated in an incubator. When I performed the experiment, I worked in a hospital lab and bacteria were incubated overnight between each zone measurement.

Hope this helps,
Nithin T.

[iftiswasamovie]

That seems right. However, since I have a mixed cultures, what do you believe I should do?

I put them in a shoebox over a heater.

Should I just give up on the project?

My teacher said she would give me new petri dishes, but I don't have enuf time to order a single strain of bacteria online.

I'll probably draw the pictures then.

[JaneC]

Hello,

I'm sorry that your project is not working out the way you planned. However, just because the results did not come out the way you expected, does not mean that you should just give up. For your project, you do not necessarily need to use the disk diffusion method and zones of inhibition. Just wait a couple more days for the bacteria to grow more, and compare (by eyesight) the amount of bacteria on each. For example: lets say tomorrow, the dish with the high amount of disinfection concentration has the least number of colonies; and the dish with the most diluted disinfectant has the most number of colonies. From this, you can conclude that dilution does affect the count of bacteria.

Therefore, I don't think that it is necessary for you to redo the experiment again with the petri dishes your teacher offered you. Your project is due in about two weeks, so if I were you, I would not take the petri dishes. I'm sure that if you wait a couple more days, you will have sufficient bacteria to make a valid conclusion.

Good luck,
Jane C.

(P.S. I'm studying microbiology at my university. Reply if you have any more questions)

[iftiswasamovie]

Thank you soo much for your help Jane.

My question is from earlier.

I'm having a hard time coming up with a conclusion. In some cases, the more concentration the less bacteria. In others, it is vice versa --less concentration less bacteria. For instance, for acne products the one with 12.5 % concentration killed the most. I'm very confused about this.

Thanks again!

[JaneC]

Thank you soo much for your help Jane.

My question is from earlier.

I'm having a hard time coming up with a conclusion. In some cases, the more concentration the less bacteria. In others, it is vice versa --less concentration less bacteria. For instance, for acne products the one with 12.5 % concentration killed the most. I'm very confused about this.

Thanks again!

Interesting. You said that sometimes the results were contradictory and confusing; then I think that you may have had a cross-contamination problem. Generally speaking, the more concentration of disinfectant, the smaller zone of inhibition (or in your case, the fewer amt of colonies). If the 12.5% concentration of acne product killed the most bacteria, then I'm think that there was something in the acne product that changed your results. If you tested an acne product, like Neutrogena, remember that it's mixed with other antibacterial agents. Chemicals that were mixed into the acne product (like coloring, fragrance, plant extracts) may have affected your results too. It would have been best to use just the compounds that affect acne directly, such as benzoyl peroxide and salicylic acid.

Remember, your results do not necessarily have to be what seems "right." Even though your results did not seem correct, they are still presentable.

[iftiswasamovie]

Do you believe I should state that in my Background ( my research report).

What should I conclude then?

[JaneC]

What were your complete results? And did you draw those pictures of the dishes? That will help too.

[iftiswasamovie]

Also, a bacterial colony is each type of bacteria present or each cluster of bacteria?

Do I call numerous clusters of pink bacteria spaced randomly in one section one colony? Or do I count each pink cluster as it's colony?

[JaneC]

Well, if you only have ONE single dot of bacteria on your dish, that would be a colony.

But by definition, a bacteria colony would be a cluster of bacteria

[iftiswasamovie]

Oh..I'll probably label each cluster as its own colony because there are all many differently colored clusters in one section. Then, I'll make a tally of how many of each and compare for each petri dish. Subsequently, draw my conclusion.. Does that sound right?

[JaneC]

Oh..I'll probably label each cluster as its own colony because there are all many differently colored clusters in one section. Then, I'll make a tally of how many of each and compare for each petri dish. Subsequently, draw my conclusion.. Does that sound right?

Yes, that sounds correct.

Out of curiosity, for how many days did you place the dishes on top of the heater? I remember that I performed a similar experiment in high school, except I did it in my school lab with an incubator @ 37 Celsius,

[iftiswasamovie]

Shoebox on top of a heater.. it's been about six days. Tomorrow I"ll do my final observations when it's been about a week.

[JaneC]

Okay, good luck

-Jane

[iftiswasamovie]

Actually, I analyzed the results and saw that the less concentration of disinfectants the more bacteria is killed. Any clue to why this has occurred?

[donnahardy2]

Hi,

We need more information about your experiment. Please let us know what disinfectants you were using, the concentrations, and the details of your results. It's possible that the reason is that the numbers are not significantly different, but there could be another explanation. For example, 70% ethanol is a better disinfectant compared to 100%, possibly because the lower concentration is more effective in disrupting the bacterial cell membrane. I'm very interested in knowing about your results.

Donna Hardy

[iftiswasamovie]

Oh i'm so thankful you are taking much interest into my project. It means a lot to me.
I plan on making an Excel spreadsheet sometime tonight.
Do you perhaps have an email address I can send it to?

[donnahardy2]

Hi,

Can you upload the file as an attachment to the sciencebuddies website? An Excel table of data would be excellent, and I'm sure I will have some comments. Just click on the upload attachment tab below the message window and browse for the table on your computer. It's fairly easy to do, but let me know if you have any problems.

Donna Hardy

[iftiswasamovie]

I'm sorry I've been extremely busy with much work to be done before the break. I'm attaching a sample of my results for one disinfectant. The rest may be up by Thursday.
Once again, my apologies.
Tell me what you think from this sampling.

[JaneC]

Hello,

I'm looking at the table you've attached. I'm not quite sure that I understand the rows in the table. The labels are "Distance to nearest colony/Width of colony/Orange Colonies Yellow Colonies/White Colonies/Cream # 2 Colonies/Beige" I see you have mm measurements in the chart, but I thought you were just counting the # of colonies of each dish.

[iftiswasamovie]

To be honest, I'm not quite sure how to record my results. I thought i should do it by size and number of colonies. But what do you suggest? Should I only take note of one variable? Which one?
The colors stand for the colors of the colonies.
The width of the colony refers to the one that was closest.
In the columns, the number before the arrows is how many of that kind and afterwards I show the width of them.

[JaneC]

In my opinion, I would just count the number of bacteria colonies. The chart you attached was confusing to interpret, and I'm afraid that it may be confusing too for your teacher (or judges). During your presentation, you can just throw in that the colonies were different colors as well. If you do count the colonies, AND measure the size of the bacteira, then you will have two dependent variables. You should only have one dependent variable. Thus, you should only choose one method to determine if the concentration affected the bacteria. Like I said, I think you should choose to just count the # of bacteria colonies.

If school hasn't closed for you yet, you should speak with your science instructor about having more than one dependent variable that you're measuring. He/she may be able to give you more information.

[donnahardy2]

Hi,

I’ve been looking at your results, and I understand why you are having difficulty with interpreting results. One question I have is about your experimental set-up. From your results, it looks like you may have exposed a surface to different concentrations of disinfectant and then grew the bacteria that survived at each concentration. Or, did you grow a lawn of mixed bacteria on each plate, and then added paper discs with each concentration of disinfectant and then measure the zone of inhabitation around the paper disc? If so, what was the source of the bacteria?

Your results seem to indicate a small number of colonies on each plate, however, your comments under observations seem to suggest that growth was confluent on the plates. Will you please confirm if, for example, in the 50% concentration, did you see 9 colonies total on the plate, or were you growing a lawn of mixed bacteria? Did you take any photographs of your plates?

It might be helpful if you could post your procedure section so we can understand what you did.

Do you have time and materials available to repeat your experiment? Whatever your method was, it is apparent that your results are not conclusive. If you had time to repeat the experiment, it would help resolve the questions. If you don’t have time to do it again, and then please answer the questions above so you can do the best possible job of presenting your results as they are.

Donna Hardy

[iftiswasamovie]

No, unfortunately, I do not have time to repeat the experiment.

I believe I will only depend on one variable to come to a conclusion so it will be less confusing for me.

The lawn was mixed and I had swabbed a doorknob from the entrance to my home. I swabbed at the same time period for each of them.

In a general statement, my procedure was to swab the bacteria on all the four sections of the desk. Then, within the next five minutes I placed in the filter disks. I waited about a week for it to grow and then I recorded my results.

Is there no hope for this experiment anymore? It seems to have given me WAY TOO MANY issues. School has already closed for the break and I can't get in touch with my science teacher.

For pictures, I'm having trouble taking them because they are not clear enough for a viewer.

[iftiswasamovie]

Sorry, I forgot to mention, yes at the 50 percent concentration there are nine colonies.

[JaneC]

So what dependent variable did you end up using?

Pictures are not necessary (of course unless your instructor says so). Maybe the day before your project is due, ask your teacher if you can bring in the petri dishes to show the class/judges, rather than taking pictures.

[iftiswasamovie]

I'm going to use the number of bacterial colonies.

She already said I could paste them on the board or bring them in.

[donnahardy2]

Hi,

Don't worry at all. There is lots of hope for this experiment. Some of the best project write ups are those where the results did not turn out as expected. You have a complete project and you have results. In science fair projects, the important thing is to communicate what you did and what happened in the experiment. There's no right or wrong answer. You recorded the results, and now you need to interpret them.

Here are some considerations:

1. Your source of bacteria gave you a mixed culture, so you probably had different species of bacteria in each plate.
2. You observed 0 to 1 mm zone of inhibition from each disc, which does not suggest that any concentration of disinfectant inhibited bacterial growth.
3. You only had a few colonies on each plate. Normally microbiologists count plates that have between 30 and 300 colonies per plate. So, the number of bacteria present was probably not sufficient for a valid experiment.
4. You performed the experiment one time. Results are considered more significant if they can be reproduced. Do you think your results could be reproduced?

One section of your board, which you can expand on, is the conclusion section. After you state your conclusion, you can expand on what may have gone wrong and what you would do differently if you had time to repeat the experiment. Explain how you would set up the experiment again and keep all parameters controlled with just one independent variable (the concentration of disinfectant). You can present a very high quality project by being very thorough in your presentation and make sure you have included all required sections, including the bibliography. Be sure to refer to the science buddies site for information on writing up your board:

https://www.sciencebuddies.org/science- ... oard.shtml

Please let me know what disinfectant you were using.

Donna Hardy

[iftiswasamovie]

Tonite I will post my excel spreadsheet, which I am working on now. Sorry for the delay.

Considering the fact that I am testing Dilutions do i need to include a positive control? In which, I did not change the disinfectant's concentration at all. I have a dish like that, but I'm not sure if I should include it in my results because the sciencebuddies website did not mention a positive control.
I have a negative control with filter disks only dipped in distilled water.

Thanks so much for all your help Donna and Jane.