Kirby-Bauer Test

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kanmit
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Re: Kirby-Bauer Test

Post by kanmit »

hi
1) I had started 2 cultures. After 48 hrs of my culture, i only see spotty growth. Not sure whats going wrong.
How can i send you a picture. Its spotty yellow dots over ...., but not like a fully white / yellow bacterial lawn

2) Is my LB AGar at fault. I bought the EZ research pre poured LB AGar plate
http://www.amazon.com/EZ-BioResearch-Ba ... B004MKHNJK
10x Pre-poured sterile LB-agar plates (100 x 15 mm Petri Dish)

3) Should i instead put oral swab in some "BROTH" ? I have read it different places some kind of broth can be used. Any suggestions. Like put oral swab in some kind of nutrient broth and spread that ?

The next part of my experiment which is the main part is testing antimicrobial effect, but if the lawn does not grow properly I have an issue.

4) Assuming i move to next step, when i actually put filter disks on it, how many filter disks should i put in one petri dish. Should i put 1 in each, or multiple...what is recommended given the size of the petri dish

Please advise
SciB
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Re: Kirby-Bauer Test

Post by SciB »

Well, what that means is that the person's mouth that you swabbed is quite clean. There's nothing wrong with your agar. It's just that there aren't enough bacteria on your swab to make a lawn. Maybe if the person avoided brushing their teeth or using mouth wash for half a day or so, there would be enough bacteria. You could use four swabs per plate and mix the water drop really well before you spread it.

What you said about the broth is absolutely correct. That is how a lawn is usually produced--by putting a drop of bacterial broth culture onto the agar and spreading it around completely.

Check with your teacher and ask them if you would be allowed to work with a broth culture of mouth bacteria. This may not be permitted under your school's rules.

Assuming you do get a decent lawn, the usual number of discs per 100 mm plate is 4. So, using a black Sharpie mark the bottom of the dish into 4 quadrants and label them 1-4 or a-d. Make a note of this in your lab book with the treatment that corresponds to each disc. Add the oils and incubate the plate 48 hours.

Overcoming problems with experiments is an everyday job for scientists. That is part of the art of being a successful researcher. Keep us posted on your progress.

Good luck!

Sybee
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

thanks.
I started a culture this morning again taking swab from all different places, cheek, gums, roof of mouth etc. Lets see what happens.

In the meantime, I want to research a backup option, if we can't product a lawn this way. With isolated colonies being produced only I see 2 backup options

1) We do not base antibacterial test on Kirby Bauer. Can we do some kind of counting of bacteria colonies ? Is there any test of that kind which might be acceptable.

2) I use a mouth culture bacteria. I researched oral bacteria, and unfortunately, I do not see any living culture of the bacteria on Carolina Biologicals website. The most common is Streptococcus Mutans, but I can't find that. Can you suggest what might be my options to look for closest bacteria to simulate mouth bacteria and how I can buy them?


Thanks
Kanmit
SciB
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Re: Kirby-Bauer Test

Post by SciB »

I told you to ask your teacher about the broth cultures. That is your best option. Grow your own liquid culture from a swab and spread a drop of that on the plates to make the lawn. Check your school's rules about working with potentially pathogenic bacteria,

It is unlikely that you can make a lawn from a swab culture unless the person you swab has an unusually high bacterial count in their mouth. And yes, there are ways to count bacteria, but they all require handling potential human pathogens. That is why I asked you to check with your teacher. Please do that and find out what you can and can't do!

If it turns out that you can't grow liquid cultures of mouth bacteria then you could purchase a liquid culture of a safe bacterium like E coli K12 from Carolina Biologicals, but then you would be looking at the gut and not the mouth. That's ok. You would just change your hypothesis to the effect of essential oils on gut bacteria.
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

Hello,

Would it be okay to buy lactobacillus instead of E. coli? (Research has shown that lactobacillus plays a small part in dental caries)

Thanks!
SciB
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Re: Kirby-Bauer Test

Post by SciB »

I've never grown Lactobacillus except in milk to make yogurt but I suppose it will grow on nutrient agar. What species of Lactobacillus is found in the mouth?
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

Lactobacillus Casei group is the one which is linked to caries
(It's not the main player though -Streptococcus Mutans is)
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

hi
Good news, my last culture from oral bacteria..the bacteria grew at least all over (not totally uniform) but much much better...

2 changes i had done
1) the swab was from a diff person - in general with healthy mouth, but did not brush for a good 12-18 hrs.
2) we had taken 2 drops of sterile water and spread on agar before swabbing with saliva.
SciB
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Joined: Fri Feb 01, 2013 7:00 am
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Re: Kirby-Bauer Test

Post by SciB »

Hi,

That is great news! Glad you finally got some growth. Next time why don't you have the person do TWO swabs so you get twice as much bacteria.

Make sure the surface of the agar is wet so you can smear the culture all over it. I think that is the secret to getting a lawn.

Are you taking photos? You could make a video of your procedure as you do it so you will have a great record showing exactly how you did it.

Let us know what happens when you test the discs with the oil.

Good luck!

Sybee
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

my school teacher is discouraging me to use oral bacteria...too many regulations in science fairs and may be considered pathogenic.

Hence i will now do experimentation with E-Coli K-12.
I bought the living culture from Carolina.

- any tips on how to do the lawn . Do i use single swab or multiple swabs?
- should i now use sterile water at all.
- any tips on how to make bacteria liquid culture last longer ..since my trials may take 1-2 weeks
- i have been storing LG Agar plates in the fridge (not freezer). Do i have to take them out and let them come to room temperature before swabbing them?
SciB
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Re: Kirby-Bauer Test

Post by SciB »

Don't use a swab for making a lawn. There is a better way to spread bacteria evenly. Scibuddies has a lot of good information on every kind of science project and if you search for 'spreading bacteria on agar' you will find out how to make a spreader from a paper clip and use it to make a lawn on agar:

https://www.sciencebuddies.org/science- ... #procedure

Follow the steps in the procedure for spreading the E coli. You don't need to add water to the plate. Just put a drop of your E coli culture on each plate and spread it evenly with the spreader.

Your E coli culture will stay alive for several weeks in the fridge.

It is best to take the plates out of the fridge before you need them so they can warm up. If there is a lot of water condensed on the plate, take off the lid and shake it out into the sink. Replace the lid quickly so that no bacteria or fungi from the air get on the agar.
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

1) is there problem in using a cotton swab for spreading bacteria ..?
If i use the paper clip option, how will I get baccteria out of the test tube.? I was hoping to just dip the sterile cotton swab
and spread it evenly on lawn ?
I am planning to start final cultures today

I have disposable pippettes. Should i transfer a set quantity from bacteria test tube to the agar plate and then spread uniformly using the improvised spreader.

2) The carolina website says, store bacteria at room temp, im assuming for a few days its ok and then i refrigerate?


3) is 70% isopropanol same as 70% isopropyl alcohol ? I have some in my medicine cabinet, but its over a year old. Can i use that. Is it same as rubbing alcohol ....70% ethanol has high shipping costs from amazon.

pl advise
SciB
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Re: Kirby-Bauer Test

Post by SciB »

If you read the instructions to the project in the link I sent you it explains exactly how to make a bacterial lawn. A swab has a cotton tip that absorbs almost all the culture liquid when you dip it in so that you will not get a good lawn. The only reason you used a swab before was so you could obtain bacteria from inside the mouth, but you saw yourself that this does not make a good lawn. The scientific way to do this is to use your pipet and put a drop or two of the E coli culture in the center of the plate and then use the spreader to spread it evenly all over the surface.

Watch this youtube video. It will show you the correct way to make a lawn: https://www.youtube.com/watch?v=nRRVWJQe6F8

Where you store the bacteria depends on how the culture was made. Stab cultures in agar can be stored at RT, but I and all the scientists I know always keep liquid cultures of E coli in the fridge at 4C [39F]. If you leave a liquid culture at RT it will continue to grow and divide until it uses up all the nutrients in the broth and then it will start to die. A couple of days at RT would be ok, but longer than that I would put it in the fridge.

Yes, isopropanol is isopropyl alcohol. In the USA, rubbing alcohol is usually 70% isopropyl alcohol. If your bottle of rubbing alcohol has been capped securely it should be fine to use for sterilizing the spreader.

Sybee
kanmit
Posts: 28
Joined: Fri Sep 04, 2015 10:14 am
Occupation: Student: 9th grade
Project Question: Boosting Cancer-Fighting Efforts of Immunotherapic CAR T-cells Against Solid Tumors Using Genetic Manipulation
Project Due Date: Nov 1 2017
Project Status: I am conducting my research

Re: Kirby-Bauer Test

Post by kanmit »

I will try a spreader.

this is the liquid culture i bought from carolina : http://www.carolina.com/bacteria/escher ... ?question=

So i will store in the fridge as per yr advice ,,my fridge is at 38F. I didnt store in fridge so far thinking that low temperature will deactivate the bacteria. This is the description of the culture from carolina website

This culture is the K–12 strain of Escherichia coli. It is Gram−, motile rods, and cultured in nutrient broth at 37° C in a tube. Each culture contains enough material for a class of 30 students. Culture is guaranteed as to species and purity.


thanks
kanmit
SciB
Expert
Posts: 2066
Joined: Fri Feb 01, 2013 7:00 am
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Project Due Date: n/a
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Re: Kirby-Bauer Test

Post by SciB »

Hi Kanmit,

The spreader is easy to use. Watch the video. They use a glass spreader, I think, which is easier to hold onto, but you can use the paper clip one if you are careful with it. Practice a bit first to get the feel of how to hold it so that you don't dig into the agar.

A temp of 38F will not 'deactivate' your E coli. It just slows their metabolism way down so that they stop dividing. They remain alive and as soon as you put the plate with the spread culture in your incubator they will start dividing happily and make a lawn. Take the plates out of the fridge an hour or so before you spread them so they are not cold.

The information from CBio does not tell you one important fact about the culture--how many bacteria per milliliter [mL]. If there is enough bacteria for 30 students, however, then you should have more than enough for spreading several plates. The amount of culture to spread is usually 0.1 to 0.2 mL or about one to two drops from a transfer pipet. Some transfer pipets are graduated in tenths of a mL so if yours are then you can use the markings to measure how much you put on each plate. If the agar is really dry you may have to add more culture in order to be able to spread it evenly over the whole surface.

Good luck and do let us know how your first experiment turns out.

Sybee
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