Kirby-Bauer Test

deleted-297845 · Post by **deleted-297845** » Tue Sep 08, 2015 5:47 pm

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

deleted-297845 · Post by **deleted-297845** » Tue Sep 08, 2015 5:55 pm

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

deleted-297845 · Post by **deleted-297845** » Thu Sep 10, 2015 11:48 am

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 · Post by **deleted-284605** » Thu Sep 10, 2015 1:01 pm

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 · Post by **SciB** » Thu Sep 10, 2015 6:58 pm

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

deleted-297845 · Post by **deleted-297845** » Thu Sep 10, 2015 8:20 pm

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 · Post by **SciB** » Thu Sep 10, 2015 8:46 pm

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

deleted-297845 · Post by **deleted-297845** » Tue Sep 15, 2015 10:58 am

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 · Post by **SciB** » Tue Sep 15, 2015 4:48 pm

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

deleted-297845 · Post by **deleted-297845** » Thu Sep 17, 2015 3:26 pm

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 · Post by **SciB** » Thu Sep 17, 2015 5:01 pm

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

deleted-297845 · Post by **deleted-297845** » Tue Sep 22, 2015 4:06 pm

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 · Post by **SciB** » Wed Sep 23, 2015 11:53 am

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

deleted-297845 · Post by **deleted-297845** » Thu Sep 24, 2015 10:11 am

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 · Post by **SciB** » Thu Sep 24, 2015 12:40 pm

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

deleted-297845 · Post by **deleted-297845** » Sun Sep 27, 2015 1:44 pm

Hello,

Thanks for your help!

I have decided to just use pure oils without diluting them. I will be using different agar plates with each agar plate having different amounts of the same oil. Other agar plates will have different oils. I was just wondering how much liquid can a 6 mm filter disc can hold. Based on that, I will decide the different amounts of oil to use.

Thanks!

SciB · Post by **SciB** » Sun Sep 27, 2015 3:11 pm

If you are not going to dilute the oils just dip the discs in the oil and let the excess drain off before you place it on the agar. I don't know how much oil one disc can hold. That is why I suggested weighing the disc before and after loading it.

If you plan to test different amounts of the same oil you will need a fine dropper. Count the drops until the disc is saturated. Now decide the range of drops to use from 1 to whatever is the saturation number.

Oil does not dissolve in water so there won't be much diffusion of the oil from the disc into the agar. Hopefully there may be some water-soluble components of the essential oil that are able to diffuse enough for you to measure a clear zone where the oil inhibits bacterial growth. Here's a reference I found where they did use the Kirby-Bauer test with oil: https://books.google.com/books?id=ukmHB ... ar&f=false

Good luck,

Sybee

deleted-297845 · Post by **deleted-297845** » Mon Sep 28, 2015 11:26 am

Hello Sybee,
To simplify, I'm thinking of following setup.

One agar petri dish can be divided into 6 sections. One section is control disk - a filter disk with nothing added to it
Others will have 5 different "amounts" of 100% essential oil. For example, if i choose rosemary oil - 100% oil.
Then Ill add 0.25 ml on one filter disk,0.5 ml on another one, 0.75 ml on another one and so on.
I will not add any water additionally.

This way i can plot the zone of inhibition to the amount (in ml) of the essential oil used.

I do not know for sure how much liquid a filter disk can hold - thats an open issue right now.

Any advice on this setup?

thanks
kanmit

SciB · Post by **SciB** » Mon Sep 28, 2015 6:40 pm

Why don't you first test the capacity of the disc by pipetting increasing amounts of the oil onto the disc before you put it on the agar? A 6 mm paper disc is not going to be able to contain much oil. If you try to pipet 0.5 mL onto it, the oil is just going to run off onto the agar and coat the surface. Find the maximum volume the disc can hold and work back from there to the control.

deleted-297845 · Post by **deleted-297845** » Tue Sep 29, 2015 11:39 am

hi

1) What is the best type of pipette to buy for measuring small quantities. I looked up amazon for a micro pipette and not sure if thats the right one. Are there any materials' recommendations you can provide.

2) I have another question about initially doing the swab. Say I do an oral swab for one person, and swab it on agar in one direction. Typically i have to rotate the petri dish 6 times and keep swabbing ....but each time I rotate, should I use a fresh q tip and do another oral swab from the mouth. Also is it advisable to mix n match swab from multiple people?

Thanks
Kanmit

SciB · Post by **SciB** » Tue Sep 29, 2015 7:29 pm

Hi Kanmit,

Pipettors are available in several different volume ranges--1 to 50 microliters (uL), 10 to 100 uL, 20 to 200 uL, 20 to 1000 uL--so it depends on what volumes you think you will need. Adjustable pipettors are quite expensive--$300-500--so you might want to consider a less expensive alternative. I suggested using a dropper before but if you want to use a pipettor you can get fixed volume ones that don't cost that much: http://www.amazon.com/Kartell-MiniFIX-M ... B007A3NKGG

Use only ONE swab for each plate. To get the best lawn you need to spread the bacteria over the entire surface of the agar uniformly. This is difficult to do with a swab especially if the surface of the agar is dry. If the agar is dry put a drop or two of sterile distilled water on the surface, rub the swab in the water for a few seconds and then swab the entire plate. Take your time and be patient with this as it is critical to get bacteria spread over the entire plate. Hold the swab gently so that you don't dig into the agar surface. When you are done, drop the swab into a bit of rubbing alcohol to sterilize it before you throw it away.

Good luck!

Sybee

deleted-297845 · Post by **deleted-297845** » Wed Sep 30, 2015 3:54 pm

1) I am trying my first culture . Swab on agar seems easy to do, however, after Im done, and invert the petri dish, am i supposed to seal the dish using something ? Or just close it regular...because it seems easy to open.

2) I know you mentioned using dropper, but that would mean I can simply soak the filter disk in the oil ? Is that what you meant ?
I would not be able to measure out the exact quantity with a dropper e.g 5 microliter, 10 microliter etc. I did some research on youtube and found that each disk can hold maximum of 20 microliter only. So if I have to vary the amount, Ill have to do 5, 10, 15, 20 microliter perhaps?

3) I am doing a trial culture without any antibiotic right now. I got LB Agar plates. Is it better to use the generic Nutrient Agar for oral bacteria.

deleted-288920 · Post by **deleted-288920** » Wed Sep 30, 2015 4:45 pm

Hi there!

That is great you are trying your first culture! After streaking the culture, just invert it. You don't want to seal it up as the bacteria still need oxygen to grow. But yes the plates are easy to open.

I took a look at the pipettors SciB sent you the link for. Those are very similar to what we use in the lab. If you are looking to test multiple volumes, which it sounds like you are, the price can add up to buy 4 of them (assuming you are looking to test 5uL, 10uL, 15uL and 20uL). Depending on your budget you could purchase just the 5uL. Then you could test the various amount of oil, pipette once for the 5uL, twice for 10uL, three times for 15uL and 4 times for 20uL.

Hope this helps! Let us know if you have more questions.

Nikki

SciB · Post by **SciB** » Wed Sep 30, 2015 6:01 pm

Hi Kanmit,

When I said 'seal' the tops of the Petri dish to the bottoms i just meant to attach them with one piece of tape on each side so that they cannot come off accidentally. Since the bacteria on the plate may be human pathogens, DON'T take the lids off after the bacteria have grown up. You can measure the zones of inhibition through the lid or you can photograph the plate and measure the ZOIs on a computer screen after making the image exactly life size.

If you haven't already done so, read the Scibuddies standard procedure for measuring ZOI, https://www.sciencebuddies.org/science- ... #procedure

Nikki explained better what I meant by giving you the ad for the fixed pipettors--just buy one of a small volume like 5 uL and use it multiple times to get the volume you need. This is not as accurate as using an adjustable pipettor but it won't break the budget.

I would test the discs first with your oils to determine exactly how much oil they can hold. It may be more than 20 uL.

Are your plates moist enough to be able to swab all over easily? This is important, so if necessary add a drop of sterile water to make sure. Also, were you able to construct an incubator to keep the temperature of the plates between 90 and 95F? This is also important. Human bacteria won't grow as well at lower temperatures and you want your experiment to be as close to human conditions as possible.

Keep us posted on your project.

Good luck!

Sybee

deleted-297845 · Post by **deleted-297845** » Wed Sep 30, 2015 6:49 pm

Is there any advantage of using "NUTRIENT AGAR" plates over "LB AGAR" plates. I am getting LB AGAR plates from Amazon at a good price and the ones at Carolina are "Nutrient Agar" and are at least 40% more expensive.

Please advise.

Thanks,
Kanmit

SciB · Post by **SciB** » Thu Oct 01, 2015 5:10 am

LB agar plates are what we use. LB is the best general purpose agar since it will grow a wide range of bacteria. See here for more info on agar:

https://www.sciencebuddies.org/science- ... Agar.shtml

Just make sure that you buy them from a reputable supplier. You can also buy powdered LB agar and sterile disposable plastic Petri dishes and pour your own plates, but since you don't need that many it is probably cheaper to buy them already prepared.

deleted-297845 · Post by **deleted-297845** » Thu Oct 01, 2015 9:36 am

hi My incubator works well - Maintains temperature around 92 F.
I did a test culture with no antibacterial substance for now.
Just an oral swab .

1) how much after should i expect to see results ? What should i expect to see in a regular oral swab culture.
2) I saw some water condense on the inside of petri dish. Is that an issue
3) Problem is the sterile filter disks will reach me only next week. Is there a make shift I can do to test one of the oils.
Can i punch from coffee filter paper or something else? Just to make sure i do a test of the zone of inhibition.
4) Also incubator has a bulb so it stays dimly lit but warm inside. I read somewhere bacteria need warm DARK place to grow.
So am i supposed to cover the petri dish with something?

SciB · Post by **SciB** » Thu Oct 01, 2015 10:00 am

Yes, cover the dishes with aluminum foil to keep them in the dark.

You can cut discs from coffee filters, just wear clean rubber gloves to handle the paper. Don't touch it with your fingers as this may contaminate the paper with bacteria from your hands.

If there is a lot of water condensed on the inside of the dish you can shake it out into a sink and then replace the lid. When you put the dishes into your incubator after you swabbed them and put on the discs, remember to attach the lid to the bottom with a couple pieces of tape. Place the dishes upside down so any water that condenses runs into the lid and not onto the surface of the agar.

Did you rub the swab over your teeth as well as gums and cheeks? You want to get as good an amount of mouth bacteria as possible. Was the agar moist when you swabbed it? Did you try to rub the tip on every part of the surface? It will take 24-48 hours for the bacteria to grow into visible colonies on the agar. You may see different colored colonies--white, yellow or reddish depending on the bacterial species. Streptococci are some of the most common and they are milky colored.

Keep us posted on your progress an be sure to take photos of the plates.

Good luck!

Sybee

deleted-297845 · Post by **deleted-297845** » Thu Oct 01, 2015 3:48 pm

A basic question about observing the agar plates and taking photo.

1) Should i observe it from the lid side (the top) or from the opposite side...(which is technically the bottom of the petri dish)

2) If agar is suspected to be not moist, should i add sterile water and spread over Agar prior to streaking with the swab ?

3) For cleaning , I could not get 70% ethanol. Is 70% isopropyl alcohol ok (antiseptic) ?

SciB · Post by **SciB** » Thu Oct 01, 2015 7:07 pm

In answer to your specific questions:

1. Do your measurements of the ZOI and take photos from the top--WITH THE LID ON!

2. If the agar surface looks dry, put a drop of sterile water in the center and immediately dip the swab that you just used to swab your mouth into the water and carefully smear the liquid all over the surface. If the surface still seems too dry add another drop of water and swab that around. What you are aiming for is an even lawn of bacteria over the entire surface, not a spotty bunch of colonies here and there.

3. Yes, 70% isopropanol, rubbing alcohol, will kill bacteria. When you are all done with your plates, fill a bucket with 10% Clorox ( 8 0z of Clorox plus 72 oz of water) and immerse the plates in this overnight to kill all the bacteria. Then you can dispose of the plates in the regular trash.

Good luck!

Sybee

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