What is wrong with my Kirby Bauer experiment?

[Jannie1]

Hi, I'm in the 6th grade and doing a science project on whether green cleaners are as effective as chemical cleaners on bacteria. I am using the Kirby Bauer method and followed the procedures exactly. The experiment is not going well after a day and a half. I see bacteria growing, but there are no zones of inhibition around my filter paper (every plate has 3 discs soaked in the same cleaner and 1 control.) I am seeing some kind of results because the plates with the chemical cleaners are showing a lot less growth than the plates with green cleaners. Can anyone tell me what I am doing wrong? I couldn’t find chemistry filter paper so hole punched ¼” discs of coffee filter paper because someone had mentioned here that coffee filters could be used. Is my filter paper the problem? I am planning to repeat my experiment because I am not sure my teacher will accept the results I got. Also, I want to know what went wrong. How can I fix the problem so I see zones of inhibition? Thank you for your help.

[LilGreenFrog]

Hi TJR1, sounds like a cool project!
Your project is a great alteration of the Kirby Bauer test, but the test is designed for use with antibiotics, so don't be surprised that your protocol will require some tweaking. Also, even when you follow the steps of an experiment exactly, realize that this is only one version of the protocol, and you will likely find many more versions that other scientists changed to fit their needs; so you're just going to have to make your own version.

First, can you tell me your exact procedure? Size of plates, amount/type of bacteria, how long you allowed them to grow, incubation temperature for the plates, and how frequently you check your plates after adding the disks?

Thought 1: antibiotics have different abilities to kill bacteria, but also diffuse into the agar at different rates. You are using cleaning products which I'm guessing will diffuse at a much higher rate. It could be that they are diffusing throughout the media very quickly, which would keep you from getting a zone of exclusion, and just give you less bacterial growth in general. In that case, your 'fewer colonies on the chemical plate' is probably a legitimate result, but I agree with your choice to repeat the experiment.

Thought 2: Are you making your own agar? If so you could try making it more thick, to slow diffusion. You may also decrease how saturated your filter papers are, or decrease the number of them per plate.

Also: where are you keeping your plates to allow the bacteria to grow? If your conditions aren't ideal you might get patchy growth, which would make the zone around a disk harder to see.

Another thought: You could try plating your bacteria more densely (add more to the plate up front), or you could let some colonies appear before placing down your disks. The extreme option of that is to let a 'lawn' of bacteria grow, so that the bacteria covers the agar, and then put down the cleaning product disks. That would give you not a 'zone of inhibition' so much as a 'zone of death' - how big of a circle of bacteria die when exposed to the chemical. I think this is a totally valid alteration of the experiment. You will want to check them pretty frequently, as in every half hour, to catch the killing in action.

This is a lot of ideas, I know. And I actually just realized that your products likely contain alcohol, which antibiotics do not, so your products may be evaporating rather than spreading too quickly.

Because we don't know if you have too much or too little product, I would start with the extreme option: let bacterial lawns grow before adding the disks, and look for differences in killing (either sizes of 'death rings' or speed of death ring expansion). And even if your products are evaporating quickly, they should at least kill the bacteria directly beneath the disks.

If you get clear results with that, you can go back and try to trouble shoot the 'zone of exclusion' version of the experiment. Either way, the bacterial lawn experiment will be a good addition to your data.

Please let me know your progress!

[probiotics]

Hi!

This seems like a fun project, and I think that we can easily trouble shoot this problem.Using coffee filters is totally fine, and is probably not the cause of your problem. How did you coat the agar plate? You need enough bacteria to make sure that the zones of inhibitions are visible. Also, could you attach pictures of the agar plates? If the bacteria was not plated correctly or abundantly, there may not be enough growth. Also, make sure that you are keeping it at the right temperature for the bacteria to grow. How long had it been since you plated it? Often bacteria growth takes upwards of a week to show up. Let me know if you have any further questions!

- probiotics

[Jannie1]

Thank you so much for answering my post! I didn't realize Kirby Bauer was mostly for antibiotics so that is good to know. I didn't pour my own plates because it seemed complicated, but my mom bought me some already poured from Amazon. I used raw chicken for my bacteria. I swabbed the plates side to side, turned the plates a little, and swabbed side to side again. I tried to make sure that my swab touched every bit of the agar. I dipped my filters into the cleaner, put the filters on the agar, and made sure they lay flat. I put the plates in a box upside down, and put the box in a bathroom with my turtle’s old heat lamp pointed at the top. The temperature inside the box has been around 85 degrees. I checked my plates 2 times a day. It’s been about 4 days and there is a lot of bacteria growing on some of the plates, but still no zones. The plates for Lysol and Pinesol only have around 15-20 little spots growing, but other plates have more than I can count, even right next to the filters! My new plates are coming today. I had one extra plate left over so yesterday I decided to try to find better bacteria and see if I could make a lawn like you suggested. I swabbed 4 different things and even overnight it looks like 2 of the samples (a dirty place in my guinea pig cage and an old kitchen sponge) will grow enough for me to try the zone of death experiment. Weird, because I thought for sure chicken juice would have the most bacteria! Thank you for the ideas on why my first experiment didn’t work the way I thought. It makes me not sure whether I should use my new plates to do the zone of death experiment or repeat my first one with the better growing bacteria. I worry that if I repeat my first experiment, that it still might not work. Thanks again for everyone’s help!