Science Buddies: "Ask an Expert"

I was testing the effects of honey against e coli k12 using the well diffusion method but there was no range of inhibition at all! The bacteria actually grew over the samples, what did i do wrong! I started off by spreading the culture across the agar plate using a cotton bud. I used 6mm filter paper from a hole punch to test the different honey samples. i used a 50% solution of 6 different honeys, as i've read from over 15 pubmed sources that almost all honeys are really antibacterial at 50% dilution. I know there's something wrong as i used manuka honey 20+ which can kill superbugs like MRSA at 10% dilution!


Once i mixed the honey and the deionised water to make the 50% solution i used a pipette to transfer the solution to the 6mm hole punch paper, i then used tweezers to put the 6mm paper with the samples onto inoculated agar plate.

Hi,

I don't see anything that you did wrong. Did you check some lliterature to see if perhaps that bacteria is resistant? I found this abstract, so you may not be the only person to get that result.

http://www.ncbi.nlm.nih.gov/pubmed/22047137

Could you test again but include another inhibitor as a positive control?

I'm hoping anohter expert or two has other thoughts.

Tonya

Hi,

My thought is that your method may be too insensitive to see the effects of the honeys on the bacteria. E coli is pretty tough and honey is a nice source of sugar for them (glucose). I think maybe if you added the E coli to a liquid salts medium (there are recipes for this online) containing 50% honey and let them grow for a couple of hours then made appropriate dilutions and plated them on minimal salts agar, you could see a difference in colony counts. Alternatively, you could make minimal salts agar plates with or without 50% honey, plate several dilutions of an E coli culture on them, let them grow up overnight at about 37 degrees C and count the bacterial colonies. If the honey inhibits growth, there will be fewer colonies.

If you don't know how to do serial dilutions and bacterial plating, here's a video you can watch: http://www.youtube.com/watch?v=pmRUBYlPMBM

Let me know what you think about this and we can help you do more tests. Experiments frequently have to be modified until they work the way we need them to. Hopefully the colony counting method will give you more quantitative results, and you will be able to do statistical analysis on the averages you get to prove that any difference between honeys is significant.

Best wishes,

Sybee

I used iodine as a control this time as well as using full strength honey for each sample.
I suspected there was something wrong with the way i spread the colony over the agar plate, "https://www.sciencebuddies.org/science- ... plates.pdf" I was very careful to cover every area and i used a lot of starter culture.


Honey is such a difficult substance to test, the viscosity of all the honeys was different meaning that they'd diffuse differently right? That doesnt tell me much about their antibacterial properties.....

Plus the 6mm papers are so small it was almost impossible to put the same amount of honey on each of them, how can i make it more accurate? Usually the 6mm paper would be saturated with a liquid so it wouldnt be a problem......

Honey is literally the most annoying substance to test!

sunmoonstars wrote:Hi,

I don't see anything that you did wrong. Did you check some lliterature to see if perhaps that bacteria is resistant? I found this abstract, so you may not be the only person to get that result.

http://www.ncbi.nlm.nih.gov/pubmed/22047137

Could you test again but include another inhibitor as a positive control?

I'm hoping anohter expert or two has other thoughts.

Tonya

forgot to quote you on my other post! Please refer to my other post on this page

SciB wrote:Hi,

My thought is that your method may be too insensitive to see the effects of the honeys on the bacteria. E coli is pretty tough and honey is a nice source of sugar for them (glucose). I think maybe if you added the E coli to a liquid salts medium (there are recipes for this online) containing 50% honey and let them grow for a couple of hours then made appropriate dilutions and plated them on minimal salts agar, you could see a difference in colony counts. Alternatively, you could make minimal salts agar plates with or without 50% honey, plate several dilutions of an E coli culture on them, let them grow up overnight at about 37 degrees C and count the bacterial colonies. If the honey inhibits growth, there will be fewer colonies.

If you don't know how to do serial dilutions and bacterial plating, here's a video you can watch: http://www.youtube.com/watch?v=pmRUBYlPMBM

Let me know what you think about this and we can help you do more tests. Experiments frequently have to be modified until they work the way we need them to. Hopefully the colony counting method will give you more quantitative results, and you will be able to do statistical analysis on the averages you get to prove that any difference between honeys is significant.

Best wishes,

Sybee

This sounds good! But how can i accurately dilute honey using a high school lab? And what dilutions would you recommend in this case?

Hutchdan wrote:I used iodine as a control this time as well as using full strength honey for each sample.
I suspected there was something wrong with the way i spread the colony over the agar plate, "https://www.sciencebuddies.org/science- ... plates.pdf" I was very careful to cover every area and i used a lot of starter culture.


Honey is such a difficult substance to test, the viscosity of all the honeys was different meaning that they'd diffuse differently right? That doesnt tell me much about their antibacterial properties.....

Plus the 6mm papers are so small it was almost impossible to put the same amount of honey on each of them, how can i make it more accurate? Usually the 6mm paper would be saturated with a liquid so it wouldnt be a problem......

Honey is literally the most annoying substance to test!

Yes, the full strength honey would be difficult to test because it is very thick and hard to work with. If you dilute it with (sterile) water, it should thin nicely and you could even use a pipette or eyedropper as you would any other liquid. Also, it thins if you heat it slightly. If you need to put straight honey on the discs, you could try dabbing it with something small like a Qtip - again, if you heat it a little it might be easier to do this.

Trying another method as the other expert suggested is a good idea, too. You can do both.

Hi again,

You asked me: "...how can i accurately dilute honey using a high school lab? And what dilutions would you recommend in this case?"

I suggested testing the honeys at 50% first, which would mean one part honey to one part sterile water or medium. You could do the dilution in two ways--by weighing a volume of honey and then adding an equal weight of water or by taking a 50 ml (or whatever size your lab has) graduated cylinder, adding honey to about 20 ml, noting the actual volume after the honey has all drained down then adding an equal volume of sterile water or medium. Put some plastic film over the top of the cylinder and invert it several times until the honey has all dissolved.

The usual method for testing a substance for antibacterial effect is what you are doing with the paper discs, but there are other ways to do it. If your paper discs soaked in honey do not kill the E coli or inhibit their growth measurably, then you may want to try the colony counting method i suggested. Does your lab have the capability of sterilizing media and pouring agar plates? If so, then you can make the agar medium with various concentrations of honey starting with 50% and working down in concentration to 0%.

If you can find someone to help you who knows how to work with bacteria, have them show you how to make serial dilutions and spread E coli on a plate to get individual, countable colonies. I suggested using minimal salts agar because there is less chance of interference from substances in the usual nutrient agar. This is a very good lesson in basic bacteriology and well worth the time and effort. Plus the colony counts give you numbers that can be compared statistically, which helps greatly in proving that a particular honey or concentration of honey is more effective.

Let us know what you decide to do.

Best wishes,

Sybee