Science Buddies: "Ask an Expert"

I'm testing how effective different types of honey is against e coli k12, probloem is i need a way of measuring the antibacterial properties of this honey that doesn't involve measuring the range of inhibition. Thats because i'm afraid the results will be too similar.

You see i'll be using the most antibiotic honeys in the world against a really weak strain :/

It would be ideal if i could allow the bacteria to grow until they reach their strongest point, and then see how quickly and effectively the bacteria are killed when i use the different types of honeys. Is this possible using a simple high school lab?

Thanks for your help!

Hi Hutchdan,

As I understand it, your hypothesis is that some types of honey have greater antibacterial properties than other types. You suspect that there is not a great difference in the effects, however. I think your question is how can you accurately measure small differences with your K12 system, right? What are you planning to measure?

Compounds such as honey may have two antibacterial effects on E coli. They can slow down the growth rate--bacteriostatic--or than can kill them outright--bacteriocidal. I don't know which effect predominates with honey and that is something you would need to determine. I think the most accurate way to measure the antibacterial effects would be to compare growth rates in the presence and absence of the various honeys. To do this you would need to count the number of bacteria that you start with, at time-zero, and again after a suitable time for exposure--maybe 4 hours. In order to obtain statistical verification, you would need to do a minimum of three cultures for each sample of honey and average the result. I would also test at least three concentrations of each honey to make your results more meaningful.

OK. The question now is how do you count the bacteria. First off you need to learn the correct techniques for working with bacteria safely and Scibuddies can help you with this Microorganism Safety Guide: https://www.sciencebuddies.org/science- ... fety.shtml

There are several methods for counting cells and I don't know what instruments and supplies are available to you. This website has an overview on bacterial counting methods and you can find more information on the individual methods online: http://www.disknet.com/indiana_biolab/b038.htm

When you decide on your experiments and methods, send another post and we can help you with the details.

Best wishes,

Sybee

Hi Hutchdan,

I think SciB gave you some really, really good suggestions. I would like to offer some of my ideas as well, and hopefully they will be helpful to you.

I agree that the easiest way to measure antibacterial effects would be to compare growth rates between the E. coli when they are grown in the presence or in the absence of honey. Here are some basic things that you should probably think about as you are planning your experiments:

1) How are you going to culture your bacteria?
It is important to determine the type of media in which you culture your bacteria (whether it is just for passaging them and keeping them alive, or for your actual experiments) because depending on the nutrients present, it can affect how your bacteria grow, and whether they would even grow at all. You will also need to pay attention to the optimal temperature at which your bacteria grow and whether it requires any shaking.

2) How do your bacteria grow overtime?
I think it will be important to first generate a growth curve for your bacteria (without the honey) where you will monitor their numbers at different time points starting at Time 0 over the course of growth. This will allow you to understand how long it takes for your bacteria to double and how long it takes for them to reach certain “phases” in the growth curve. Bacterial growth in liquid cultures can be divided into four different phases: lag phase, log or exponential phase, stationary phase, and death phase. Lag phase occurs in the beginning when you first start your cultures, where the bacteria are still adapting themselves to the growth conditions and are not dividing, log phase is when the bacteria are dividing, stationary phase is essentially when the bacterial numbers stop increasing usually due to a depletion of nutrients, and death phase is when the bacteria start dying. The lengths of these different phases will differ depending on how concentrated your initial culture is. Furthermore, it will be important to know when these phases occur because you don’t want to add the honey when the bacteria are in lag phase and are still adapting to their growth conditions, or in stationary or death phase when the bacteria basically stop growing or die. This is because you will not be able to tell the difference between bacteria dying due to the antimicrobial effects of the honey, or bacteria dying just because the liquid media they’re in can no longer sustain growth. You can generate a growth curve by measuring the optical density of your culture overtime and by plating the bacteria to get counts.

3) How will you resuspend the honey?
What is the solvent in which you will resuspend and dilute the honey? You have to make sure that the diluted honey solution will be soluble in your bacterial growth media. You don’t want your honey solution to be clumpy because that means that it will not spread uniformly throughout your bacterial culture, and all the bacteria in your culture would not be equally exposed to the antimicrobial effects of the honey.

These small details may seem trivial but they actually make a HUGE difference in your experimental results! Let us know when you start planning your experiments and need some guidance!

Best of luck,

Connie