Kirby-Bauer Test

[kanmit]

hi
If i want to test antibacterial properties of a substance, whats the best test to conduct which can easily be done "safely" at home .
I do not have access to a lab at all.....
Also concerned about being able to culture bacteria, since it gets quite cold during Oct/Nov.

thanks

[kanmit]

1) I need a way to test antibacterial properties of a substance

2) Also compare #1 with antibacterial properties of a well known antibiotic (medicine).

how can i do that

[kanmit]

hi
I am considering doing Kirby Bauer disk diffusion test

- can these be safely conducted at home setting

- also for comparing, i want the medicated disk for a well known antibiotic. Where can i buy such disks?

[deleted-284605]

Hi Kanmit,

Great idea! Check out this link to a similar project: https://www.sciencebuddies.org/science- ... ml#summary There's tons of information under all the different tabs about how to conduct the experiment, what materials you'll need, and where to get them. I don't think it mentions antibiotic disks, but I found this pretty cheap set by searching around online: http://www.hometrainingtools.com/antibiotic-disc-set

I think you should be able to culture bacteria safely inside your home. Bacteria live all over everything, we just can't see them. That means you don't have to look far to find your material! You can use a cotton swab to swipe different places around the house, your own skin, your pet, soil, anywhere! Just wipe the swab onto an agar plate, and let it grow. Since you're selecting bacteria that grow inside your house and it's typically fairly warm inside, they should grow well.

Please be sure to read the safety information in the project I linked to above. It should be simple to do your experiment safely, but you do have to be careful! It's also important to ask a teacher or someone else in charge to make sure your science fair allows the use of biological agents such as bacteria, since sometimes they have rules about this.

Hope this helps you get started!
Megan

[SciB]

Hi,

Looking for antibacterial properties among things that may not have been tested before is a very interesting project because you don't know what will happen. I would suggest trying several different substance that you suspect may have antibacterial activity such as garlic, turmeric, salt, sugar, mushroom extract, coffee, green tea, etc. Use your imagination to think of things that no one else may have tried before.

The Kirby-Bauer diffusion method is a very nice way to demonstrate bactericidal properties. In order to use the disks effectively, however, you must grow bacteria in an even layer, called a 'lawn', on the agar in a Petri dish. The Scibuddies projects explain how to do this.

Let us know when you have more questions.

Good luck

[kanmit]

Hello,

I had a quick question. How can I carry out the Kirby-Bauer disk diffusion test at home?
I do not have access to a lab, though.

[SciB]

Hi,

One quick suggestion--PLEASE keep all your posts in ONE thread. Otherwise the experts may miss something that has already been discussed--like this.

You can grow bacteria at home and do the disk test without special equipment. I would suggest using the bacterium called Escherichia coli K12 because it is a standard strain for research and it is safe to use. You can buy cultures of E coli from Carolina Biologicals:

http://www.carolina.com/browse/product- ... ubmit=true

CBio also sells the agar, Petri dishes, spreaders, pipets and anything else you might need for doing the experiments.

Are you familiar with the procedure for the Kirby-Bauer antibacterial test? If not I can send you some links to videos showing how it is done.

Making water solutions of the various substances to put onto the disks to test them will require some thinking and planning. I would do some reading online about antibacterial extracts and how to make them. You can't use alcohol to make an extract because the alcohol itself would kill the bacteria, so you will be using distilled water. What i don't know is how much of a particular substance to add to a certain volume of water, what temperature the water has to be, how long you have to let it steep, etc. These are all things that you will have to work out as part of the project.

I think this is a really neat project, so keep posting so we can help you get started.

Good luck!

Sybee

[kanmit]

i wanted to test some bacteria of mouth - streptococcus....ideally want to get a pre done culture so faster to grow on Agar.
I tried looking at Carolina store, but cannot find it.

Anyone can help where i can order some steptococcus from?

[SciB]

Hi,

I understand that you want to work on a bacterium that is found in the mouth, but Streptococcus mutans, which is the one commonly found growing in the human mouth is a human pathogen and you cannot purchase disease-causing bacteria to use at home.

A more realistic way of testing oral antiseptics, I think, would be to use bacteria from the mouth itself. All you need is some nutrient agar and Petri dishes that you can buy from Carolina Bio (http://www.carolina.com/browse/product- ... ubmit=true).

The first thing you should do if you haven't already is read the microorganism safety guide that tells you the proper way to handle and dispose of bacteria safely: https://www.sciencebuddies.org/science- ... fety.shtml

To do the experiment you will need some sterile filter paper discs and these can be obtained from here: http://www.sigmaaldrich.com/catalog/pro ... &region=US

The procedure is given on this website, https://prezi.com/xqbhllixq_ry/mouthwas ... xperiment/, but basically what you do is use sterile tweezers to dip the discs into each of the solutions you want to test and let them dry. Get some clean Q-tips, swab the inside of your mouth (do this BEFORE brushing your teeth or using mouthwash!) and your front teeth along the gum line, then rub the Q-tip all over the agar in a zig-zag motion trying to cover it completely but being careful not to dig into the surface.

Label the discs (use sterile tweezers--don't touch the discs with your fingers) in pencil so you know which solution is on which disc then place them on the surface of the agar so that they are spaced about 4 cm apart. Remember to include one blank disc as a negative control and one disc that contains an antiseptic mouthwash as a positive control. In order to get a more representative sample, you should ask all members of your family to do a swab.

Seal the lid of the Petri dish to the bottom with tape so it cannot come off accidentally and put the dishes inside a box in a warm dark place until the bacteria form visible colonies. Bacteria from the human mouth grow best at 30-37C so try to keep the plates in that temperature range. The cooler the temperature, the slower the bacteria will grow and this might affect their sensitivity to the test substance.

When the bacteria have grown up you should see whitish or possibly colored areas on the agar surface with clear areas around the discs that have antibacterial substances on them. The blank disc should show no clear zone at all. Take photos of all the plates, but LEAVE THE LIDS ON. You don't want to expose yourself to potentially dangerous bacteria. Measure the clear zones in millimeters from the edge of the disc to the end of the clear area.

Fill a pail with 10% Clorox (1 cup of Clorox plus 9 cups of water) and place the Petri dishes with the lids still on so they are submerged in the bleach solution for at least two hours--overnight is better. After this treatment, the dishes and agar can be disposed of in the regular trash.

If you have more questions, let us know.

Good luck,

Sybee

[kanmit]

Followup question on culturing oral bacteria
I can see 2 ways based on what i have read
1) swab the agar, let the plate culture for few days, and grow a lawn of bacteria.
Then reopen the petri and introduce the antibiotic disk, and then let it stay overnight, and observe the zone of inhibition

2) swab the agar, and at same time introduce the antibiotic disk , seal the plate and let it culture...

are there any issues with following 2) v.s 1) reg using this Kirby Bauer test effectively to measure antibiotic effectiveness
i see 2) may be a better aseptic and safer technique since i dont have to reopen the cultured bacteria

please advise

[SciB]

Hi,

Method #1 won't work. The antibiotic test substances have to be present during bacterial growth not after.

What is your hypothesis for this project? If you test something like mouthwash that contains a bacteriocide or bacteriostat then you already know the result, so what's the point? You can include this as a positive control just to show that your method is working.

I suggested that you test substances that are not known for antibiotic properties. Is that what you plan to do? Pick a concentration of your test substance that is neither too concentrated nor too dilute and be sure to label the disc and the Petri dish so you are sure of which substance is on which disc.

In order to do statistical tests on your results you need to have at least three readings for each test. Then you can get the average and standard deviation and do a statistical comparison. This is really the only way to prove or disprove your hypothesis.

Are you planning to use nutrient agar? That would be the best choice for this experiment, I think. Do you have a place to incubate the plates that is as close to 37C as you can get? This is important. Mouth bacteria do not grow well at cool temperatures and you should try to duplicate the conditions in the mouth to make your test valid. You may have to use an old incandescent light bulb in a lamp to provide heat and put your plates inside a box near it. Just be sure it doesn't get too warm.

Let us know more about your procedure so we can help you with the details. One error in the method can ruin your results. Be sure to take lots of photos as you go along to record the work and the results.

Good luck!

Sybee

[kanmit]

Hello!

Thanks for your help. I had a question.

How can I create an incubator at home? In an earlier post, I was told that I can use an old incandasent bulb. Specifically, how can I do that?

Also, how can I measure the concentrations of the herbal or spice extracts?

[SciB]

Hi,

Happy to help!

You can make a simple incubator using a large cardboard or styrofoam box and a lamp with a 15W or 20W incandescent bulb as the heat source. Compact fluorescent bulbs do not give off enough heat. Here's some instructions for making the incubator: http://everydaylife.globalpost.com/make ... 39092.html

Try to keep the temperature between 90 and 95 F. Wrap the plates in aluminum foil to keep them out of the light. You should be able to see bacterial growth within 24 hours. Just let them incubate long enough to allow you to measure the clear areas accurately.

How you measure the concentration depends on the form of the compound that you are using. If you are testing a supplement that is a powder inside a capsule, you would cut open the capsule and pour the contents into a specific volume of warm distilled water. The label of the supplement tells you how much of the active ingredient is in one capsule and you can use that as your starting amount. If your test substance is a tablet like vitamin C (don't use chewables) you can crush it and add warm water to dissolve it. For spices that come in bulk you would need to weigh a certain amount and put it into a measured amount of warm water. Let it steep until the particles settle out and use the clear liquid.

To calculate your concentration, take the known amount per tablet or capsule--let's say 500 mg--and divide that by the number of milliliters of water you dissolved it in--say 10 mL. That gives you a concentration of 50 mg/mL. I'm assuming that you have an accurate digital scale that can weigh accurately to 1 mg. For example: http://www.amazon.com/Portable-Milligra ... B009334WPE
Put a piece of waxed paper and one filter paper disc on the scale and tare it to zero. Dip the disc in the test solution, let it drain and place it on the waxed paper. Record the weight. This is the weight of the liquid in mg that is on the disc. The number of mg of water is equal to its volume in microliters. Lets say the weight is 300 mg. That means the volume of your test solution that is on the disc is 300 uL (microliters). One uL is 1/1000 of a mL, so 300 uL = 0.3 mL.

If you have a micropipettor you could simply pipet an exact volume of your test liquid onto a disc. You can do it that way if you want. I just wanted to show you how to do it if you didn't have a pipettor.

Now, using the example I gave before of the 500 mg tablet dissolved in 10 mL of water to give a 50 mg/mL solution, you can calculate how much of the substance is in 0.3 mL. One tenth of a mL would contain 5 mg, so 0.3 mL would contain 15 mg. That's how you can determine the amount of test substance on each disc.

Once you find a substance that has bactericidal or bacteriostatic activity, you could try to do what is called a dose response. You would use say 5 discs. The first would have no test compound just distilled water. The next disc might have 1 mg, then subsequent discs would have 2, 4, 8 or 10 mg. To do this you would need to have a pipettor because you have to use a different volume for each dose.

I hope this answers your questions. If not, just send us another post.

Good luck!

Sybee

[kanmit]

hi
One of the ideas i had was use essential oil e.g Clove oil ..there are many essential oils.
Many brands are marketed as 100% pure cold pressed so we can assume its 100% the ingredient.
I was thinking of making simple ratio based dilutions e.g 5 ml of oil mixed in 5 ml of sterile water.
So this would be 1:2 dilution - similarly we can make 1:3, 1:4, 1:5 dilutions to vary the concentrations.

Then use a specific amount of this dilution say 1 ml on the filter disk since filter disks hold only specific amount.

This way i can simplify the weighing process....and no weighing is needed. I am doing this at home, so want to simplify within the constraints of the scientific method.

Then i could plot the zone of inhibition (if any) against the dilution strength...
Do you think that can work?

Few open questions
1) I will mix the essential oil with water (sterile) so it can diffuse on disk
2) Temp of water is a concern. I was thinking just keep it same as human body so 98.6F , so heat water until it reaches 98.6F ? Any suggestions on that.

What other handling precautions should I take.

[SciB]

There's one difficulty with using essential OILs and that is that oil does not dissolve in water. You can mix the two but they will remain separate. I did a search for 'clove oil bacteria' and found a paper that talks about testing the antibacterial activity of plant oils and extracts: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074903/

The authors of the paper used dimethyl sulfoxide in water to dissolve the oils and you could do that if you can get some DMSO. It is not harmful to humans and you may be able to find a small bottle of it at a pharmacy or online. You would have to test some different concentrations of DMSO to find out how much is needed to dissolve all the oil.Your control would be DMSO in water with no oil.

You may have to test a range of dilutions of the oils until you find one that has measurable antibacterial activity.

You really need to know what concentration you are putting on each filter so you either have to weigh it or measure the volume accurately. You can buy an inexpensive digital scale and then you will have it to use for other projects.

Here's another paper that tested clove oil (83% eugenol) on E coli: http://www.scielo.br/scielo.php?script= ... 2000400003
The authors used a different method from the Kirby-Bauer. They set up the test solutions and added the bacteria to them and incubated the tubes for up to 10 minutes. The bacteria were then plated on agar and the plates incubated overnight at 37C. The number of bacterial colonies on each plate were compared to show the activity of the clove oil.

I hope this helps to get your project started. Let us know as soon as you have more questions.

Good luck!

Sybee