Kirby-Bauer Test

[kanmit]

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]

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

[kanmit]

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]

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.

[kanmit]

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]

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

[kanmit]

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.

[ncarter79]

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]

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

[kanmit]

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]

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.

[kanmit]

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]

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

[kanmit]

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]

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