Please HELP! Dilutions of Disinfectants Questions

[godelbor]

Greetings,
I am trying to assist my daughter on her science project.
The link below is the project I want to work on however I am confused on several parts of the instructions. If you open the link, I will point out which steps I do not understand but note I am numbering my own questions here.
https://www.sciencebuddies.org/science- ... 0&from=TSW

1. Looking over the materials needed and the wording, it appears the number of plates indicated is for testing of one disinfectant but am I correct when I say it really is set up for testing 4 different disinfectants?


Do Different Dilutions of Disinfectants Affect the Development of Bacterial Resistance?



1. Materials needed page 2-3 of 5: 20 agar plates, 4 plates per disinfectant, per round (3 dilutions + 1 control) x 5 rounds of selection = 20 plates total per disinfectant.
2 Experimental Procedure page 3 of 5: Step 4 Use a permanent marker divide the bottom of the three test plates into four equal sections. Label each plate with the disinfectant and dilution to be tested.
This can not be right because each plate will contain 4 different disinfectants at a certain dilution correct?[/color]
Experimental Procedure page 3 of 5: Step 5 Use a permanent marker to divide the control plate into four equal sections. Label the plate "no disinfectant."
Again, the only purpose I see for dividing the control plate into 4 is because this is written to test 4 disinfectants correct?
3. Measuring Zones of Inhibition page 3 to 4: Step 2 You will get four separate measurements for each dilution of each disinfectant—one from each quarter section of the test plate.
Four separate measurements for each dilution? How if I only prepared 3 dilutions? Is the sample of the control plate dipped in sterile water being counted as a diluted sample? If it is, I thought it was in a separate plate not in the quarter of the test plate?
4. Selecting for Resistant Bacteria page 4 of 5: Step 1 For each plate, use a sterile swab to pick up bacterial colonies growing closest to the disinfectant-impregnated disk.
Since I am assuming there are 4 disinfectants in each plate, I must swipe the most resistant colony regardless of the disinfectant and apply it to the entire plate of the 2nd round,same dilution correct?

Again my best guess is that the experiment is setup for testing 4 different disinfectants. So if I wanted to simply test two, I can divide each plate in half correct? If I wanted to decrease the number of plates, can I use the 3 different levels of dilution on a single plate?

Also, what is the purpose of repeating the control plate?

I am not sure why 5 rounds is necessary unless in addition to testing if different dilutions affect bacterial resistance, it is also testing bacterial’s immunity development to the disinfectant since we are using the most resistant over and over again on each round. Is this correct?

Is the K12 strain E coli safe to use? I've been warned of the dangers from our local clinic and they have refused to provide me with some but I believe they are thinking of the non K12 type. They did offer the use of their lab if I wish.

Any help with these questions would be greatly appreciated since the project date has been moved up and am now short of time.
Thanks in advance.

[donnahardy2]

Hi,

Here are answers to your questions. This is a good project for a middle school student; you will both learn a lot:

1. The project idea gives a guideline for setting up an experiment. Each agar plate is inoculated with a lawn of one bacterium, and each disk contains a dilution of the antimicrobial compound to be tested. A control disk does not contain any added compound.

Since you will be selecting a resistant colony from the first plate to make a plate for the next generation, you really do need one plate per disinfectant. If you do 2 disinfectants, you will need 10 plates for the entire experiment.

Do Different Dilutions of Disinfectants Affect the Development of Bacterial Resistance?


3. You would have the control (no disinfectant) plus the 3 dilutions for each sample. The control could be just a plain sterile disk with nothing added. You could do more than 3 dilutions for each plate.


4. To see a trend in resistance, you would need to select a resistant colony from one disinfectant and keep using the same disinfectant in order to see a change in results

It would be difficult to use just half a plate for this experiment, because after the first round your plate will contain organisms that are developing resistance to one of the disinfectants. You need a control that shows expected results so that you can confirm that experimental results are due to the independent variable. If there are no controls, your results will not be valid.

For this experiment, 5 rounds would be necessary to show a quantitative change in ability to grow in the presence of a particular concentration of disinfectant.

The K12 E coli would be ideal for this project because it is a non-pathogenic organism. The offer of the lab to work in is the best possible solution to getting a source of the organisms, and also presumable the expertise of microbiologists who can show your daughter the techniques. I would encourage your daughter to take advantage of this offer if at all possible.

One more comment. Bacteria do not develop resistance to some chemicals like ethanol or quaternary ammonium disinfectants that directly disrupt the cell membrane. They do develop resistance to other antimicrobials such as antibiotics. What antimicrobial agents were you planning to use.

If you run out of time, you can reduce the number of cycles to 3 or 4 rather than 5; however, doing 5 cycles will be best.

Good luck!

Donna Hardy

[godelbor]

Thanks for the reply. I believe I finally understand the original instructions. It is set up for one disinfectant.
Our immediate problem is we do not seem to have near enough agar plates. We have acquired 3 nutrient 3.5" plates and one 5.5" nutrient plate. We also acquired 3 TSA 3.5" plates. We were hoping to avoid using the TSA plates since this is not what the project calls for plus to avoid adding another variable to our experiment.
I have uploaded a diagram showing what we are considering but request you verify if its valid. Using two side of each dish for either two different disinfectants or perhaps even to different dilutions would help. In the drawing I uploaded, it shows a different disinfectant on each half of each dish with arrows showing where the most resistant bacteria will be transferred two on the next round. Two disk on each half of each dish is what were are considering. Not sure if colonies will overlap or affect each other in this configuration. We had another drawing which should each dish divided into four parts, each part with one disinfectant but with a 50%, 25% 12.5% and a control dilution. Limited to only one uploaded file thus could not send. In this latter configuration, we were considering only placing one disk in each quarter dish. I know compared to the original instructions, in each dish, we are reducing the number of disks from 4 to 2 (as per drawing) or to 1 (as per drawing not sent). We know we can acquire possibly four more agar plates.
Please advise what you think of our approach Or what options we have with the number of plates we have on hand plus possibly acquiring 4 more.

On a related note, are there two expected results from this experiment even though the title indicates only one? Result one - weather different dilutions of disinfectants affect development of bacteria. Result two - Is bacteria affected differently with repeat exposure to same disinfectant.
The antimicrobial agents we want to use is 409 All Purpose Cleaner (Antibacterial) and Clorox Toilet Bowl Cleaner with Bleach (kills 99.9% of germs)
Is the use of more than one disinfectant just for the purpose of verifying results of the first disinfectant?
The due date has been changed to 11/24/09 so we have a bit more time. We decided to do this at home since my daughters schedule will not allow her to do it at the lab. We have already prepared our dilutions and will await your response.
Thank you for your support and attention.

[donnahardy2]

Hi,

Thanks for posting the diagram of the experimental set up. Your protocol is valid, and I think you will be able to do the experiment using one half of the plate since you will be doing just 2 dilutions. You should go ahead and plan to use the TSA plates, even though it won’t be a strictly controlled. Just have your daughter state her protocol with a note that she knew it would be better to use the same medium for all of the plates. Nutrient agar and TSA are both general all purpose growth media and E. coli should grow well on both types of plates. If you can acquire the additional 4 plates, you can add one more round of testing, and that would be a better project.

Both of the products you have selected can be described as disinfectants or sanitizers and contain chemicals that interact directly with bacteria to kill them quickly. Here is some information on disinfectants.

http://en.wikipedia.org/wiki/Disinfectant

You should have your daughter read the list of ingredients and do research on the mechanism of action on bacteria. You will find that bacteria don’t develop resistance to disinfectants like quaternary amines, alcohols, bases (high pH), and oxidizers (bleach). If you have not started the experiment, it would be better to switch one of the products to and antimicrobial agent that is associated with development of resistance, such as an antibiotic or triclosan, an antimicrobial agent found in a variety of products.

http://www.beyondpesticides.org/antibac ... oducts.htm

What is the question and hypothesis for this project?

I’m glad the deadline has been extended to the 24th; you have just enough time to complete the project. Please let me know if there are any other questions.

Donna Hardy

[godelbor]

Thanks donnahardy2 for the info.
A couple of additional questions. We have the plates in an incubator but there is alot of condensation on the lids. We initially stored them in the fride right side up but later we learned the should be stored upside down. So we stored them for about 5 days right side up and about two days upside down. We now are finding out that perhaps we were suppose to store them upside down after the culture in the incubator. Can you confirm this?
Also, we received live k12 in a tube and stored it in an incubator at 37 deg c for 5 days. We wonder if it was still alive when we cultured the plates or were we suppose to transfer it to an agar plate as we have read on some sites.
Please advise and sorry for asking so many questions.
Thanks

[godelbor]

donnahardy2,
I forgot to mention, the reason for the additional questions is that it has been 24 hrs since we incubated our plates and no sign of growth. We know it may take longer but read if we incubate, it should grow within 24 hours.
Thanks again.

[donnahardy2]

Hi,

Yes, you are supposed to store and incubate Petri dishes upside down to avoid condensation.

However, this did not cause the lack of growth of your E. coli. You should check the temperature of your incubator and make sure it is not too hot. If your culture were viable when you received it, you should have seen growth during the 5-day incubation and you definitely would have seen growth after 24 hours on the Petri dishes. It's a good possibility that your culture was not viable.

How old were the plates that you used? If they were several weeks old, it is possible that the surface of the agar was too dry to support growth.

Can you get another sample of the K12 E. coli and try this again? You need to find out why the E. coli did not grow and include this information in the write-up. Just think about how much you are learning!


Donna Hardy