Testing an Antibiotic Using a Disk Diffusion Assay - Kirby Bauer Method

[@daniellahogan2076]

this guy is the cutest thing I have ever seen. Super helpful, love the animations! Keep up the great work!

[@Goldbio]

+Daniella McLean Thanks Daniella, we appreciate it!

[@nathanzeligman103]

It's great that you are able to explain the science behind testing antibiotics. However, honestly it's cheaper just to buy new antibiotics in the first place. But it's a great way of testing your own homemade antibiotics

[@Goldbio]

@Alyssa Molina - Hi Alyssa. The quantity of antibiotic per disc is based on the minimum inhibitory concentration specific for that antibiotic, and is typically the same as the products found for medical testing. Best of luck with your project!

[@jatnarivas8741]

Hi! Thanks a lot for the video! What do you recommend when you are trying to find out the MIC of a novel substance?

[@Goldbio]

@ Deborah Frederick - Thanks for your question about the media. For the experiment shown, I was using LB agar, but depending on your standard bacterial species you can use whichever media is most appropriate for the organism. For this test, almost any media is fair game as long as you have good controls. Thanks for watching!

[@amnamohamed6145]

1) how did you prepare the antibiotic stock solution? 2) how did you know the desired concentration per disk Also, if i want to prepare a 10 ml stock solution with concentration of 100 mg/ml should i just do that and then dilute it a working dilution of 1/100?

[@DoctorCFlare]

I am so happy that you look happy! Most videos like this portray a monotone scientist, but you show the enthusiasm that I love!

[@Goldbio]

@ Khadijah Rafiq - Our apologies for the delay. I have stocks of my sensitive and resistant strains that I streak out on a fresh agar plate, grow them up and pick a fresh colony into an overnight culture. Unfortunately a fume hood won’t be good for drying the discs because it’s pulling in room air instead of filtered air. I’d just let them dry covered on the bench for best results. Thank you for viewing and checking in with us!

[@Goldbio]

***** Hi Joe - antibiotic effectiveness depends mostly on the organism being treated, and if they have any innate resistance to a specific class of antibiotic. While all antibiotics are grouped into a main category (chemical molecules that kill bacterial cells or prevent them from dividing), there are many different sub-classes that all work in different ways to achieve a similar goal.Some classes are just better at killing certain types of bacteria than others, based on their mechanism of action. When resistance is involved, things get even more complicated, which is why Antibiotics are a big research field for both doctors and microbiologists. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif;}

[@alexisv175]

This video is a life saver, thank you so much!!!

[@tomboy786]

Thank you so much for this video. the way my microbio textbook explained this assay was so confusing!

[@Priya_Dutta]

this was very helpful for me! needed this for my project work! thank you!

[@hassansuliman8886]

It is wonderful, you show something not available in the book, the technique. i will recommend this page to my friends. Thanks lot.

[@SinatSumAgri-tech]

how can I know about the a mount of bacteria which are killed by antibiotic? (0.1 mm of hollow zone). could you explain me?

[@yagnamehta6432]

56 dislikes are from bacterias.

[@hafizqaisar8693]

😂

[@yeongmingyue5604]

Hi! My research supervisor has advised me to place the disc on the NA plate that has been incubated overnight with well-spread bacteria culture, instead of placing the disc on the plate right after spreading the bacteria culture. She said the reason for doing this way is to observe the antibiotic killing the well-grown bacteria on the NA plate. The bacteria I am going to test against antibiotic is E.coli. However, I have not started my experiment. May I know how do you think of this suggestion? Can I have your advice? Thank you

[@amgadmohammed6825]

good lecture. i ask about the assay used to detect antibiotic efficacy intracellular Vs intracellular bacteria

[@adrianaquartey6428]

amgad mohammed sure

[@unknownperson-eg8mv]

Thanks very much. We have a research and my teammates are off to an official business and im on my own and this helped a lot

[@Goldbio]

Hi Laurice - That's great, we're happy to help!

[@eldhand]

I have done a similiar project in school, but I used household ingredients like honey and mustard. Can I look at the zone of inhibiation to see which ingredient is best at killing bacteria? I'm doubting it myself because they have different viscosity, so coca cola for example should have a larger zone than honey even if they are equals when it comes to killing bacteria, right?

[@Goldbio]

eldhand I would think that to get the mustard (or any thick substance) to absorb into the disk, you would need to dilute it in either vinegar or water to get it thin enough to compare directly to the cola. But it might be an interesting experiment to try!

[@ThePunkadagirl]

+Gold Biotechnology, Inc. But then if you use vinegar you could not tell for sure if the activity is coming from the extract you are testing or from the vinegar itself...as the vinegar would alter the pH of the compound and most bacteria would not survive on such an acidic environment.

[@djohnson4465]

my question though is how does one extract the bacteria? like if i was curious about a bacterial infection on a wound...

[@babusheikh6652]

Hi there, please help! I am conducting the antibacterial assay of my plant sample following the well diffusion method. But I have faced with a problem. Should I filter my plant sample with the help of 0.2 micron filter inside laminar flow cabinet before adding the samples into the wells. I found that when I filtered my sample, the concentration as well as the volume of my prepared sample also get decreased. I have also gone through severe literatures of my field but found no paper mentioning the use of 0.2 micron filter. So is it necessary to do so or I can skip this step?

[@yanebangos9489]

What kind of antibiotic should we used? Syrup or the capsule one? If capsule how can we apply it in using Kirby Bauer Method

[@electronicsNmore]

Great video!

[@gururajendiran5575]

Hello, this video was a great help. We have done the disc diffusion method using normal blotting paper as discs. We have used (clorox, Phenol, Probiotic solution and Dettol) as reagents. We did not see any halos around the discs. We have used milk bacteria for this experiment. Please help us find out the answer to the problem. What kind of paper have you used as discs? Thank You!

[@deborahfrederick3194]

Great video! Really easy to understand and great tips. I was curious, though. You didn't specifically say what type of agar you are using. Are you using Mueller-Hinton agar, or can some other kind be used if you are just testing to see if an antibiotic is still good?

[@nasirkhan-cx6uo]

While incubating the plates having disc agar side should be up or down?

[@gabrieladiaz983]

What type of filter paper did you use? I'm doing a bacterial growth inhibition project using filter paper to make the paper discs, but I don't know what type of filter paper is best to use. Also if I buy the discs would the 6mm size ones be the good to fit 5 of those in a 100 times 15 mm petri dish? Also, if you place the discs on the agar, put the lid back on, and then incubate them upside down, will the discs fall?

[@shellyannegnapragasan7211]

Very useful, Thanks. The video looks cool especially the sound effects when showing the materials of the experiment ;-)

[@srijanathapa2322]

Thank you so much for such a useful and informative video. Actually, I want to check the antibiotic activity of Photorhabdus luminescens bacteria against gram positive bacteria. Can you give me some suggestions?

[@yamisoaegyeo]

Can we do kirby bauer test with a bacterial colony from an agar plate instead of getting it from a liquid culture?

[@milky85ful]

fantastic demonstration!

[@sarashamseldin9864]

Can I use the disc immediately after adding the antibiotic without drying ??And thanks alot :-)

[@krafiqjkj786]

How would you grow the colony overnight? And also is a fume hood sufficient for drying the filter paper discs? Thank you

[@Drantisaad]

thank u you are hero you save me in my exam

[@khjw-yp4eu]

كان اختبارك ميكرو عملي..؟😂

[@MSAMM5]

this is a good video, but you are taking about antibiotic. what if i use disinfectant, what the optimum amount of disinfectant should i put on the disk, is it constant for all disinfectants or each one has its own parameters to perform its effect??? and thank you

[@nehatambe7383]

What gauge is the filter paper used? How did you prepare disk for drug?

[@SanguinaryStrife]

Why not use the Mueller Hinton agar? I thought that's what you're suppose to do *confused*

[@CoolChris-vn8hz]

You just secured my pass. Thanks man.

[@abdelraheemhussein9013]

Thanks for the video. How could you assure 0.5 log of bacterial concentration?

[@melissaxu2]

Could you do something like for testing whether chlorine based pool water is better at killing bacteria than copper based pool water

[@SapphireRavenmoon]

I have to do a project of my own and I've chosen to see if certain essential oils have an antibacterial effect, and I was wondering if I could use this method in the same way to test my oils? And would I have to dilute them or do you think can I just use the pure oil?

[@Goldbio]

Hi Moonsong - that sounds like a very interesting experiment. I would think the oils would absorb into the disk’s okay, but you might need a control (plain mineral oil for example) to compare the effects of the essential oils to. I’d also recommend a known antibiotic as a positive control. You could try doing dilutions, but you couldn’t use just plain water, so it would be a little more tricky. Let us know how it turns out!

[@SapphireRavenmoon]

For the dilutions I plan to use a carrier oil :) Thanks for the tips :D

[@chardonnayjones8188]

Instead of using the loop can you use Q-tips

[@Goldbio]

Hi Chardonnay - you can use q-tips as long as they’ve been sterilized in an autoclave first. Thanks for viewing!

[@niconics]

how do you compare the growth by recording the data? do we count the number of cultures per disk?

[@victorsjostedt4393]

Could anyone answer a quick question? In comparison to the E test, what are the relative advantages of the Kirby Bauer method?

[@tsvikaronen1516]

Patrick you're awesome!

[@shirsendumondal9197]

what is the pore size of the filter paper? do we have to prepare bacterial lawn before hand? yes/no

[@jankihingu1348]

Can you please make a video about penicillin chemical assay by titrimetric method??

[@angelicaroque8517]

I have a question, im doing this quiz and the question is if my unknown is Gram negative; and my partner's unknown is Gram positive. Which would you expect to have the larger zone of inhibition around the penicillin antibiotic disk? Please help thanks

[@isbykdi985]

THANK YOU FOR THE EXPLANATION

[@XxfuzzydragonxX]

@ Gold Biotechnology, Inc. I'm doing a similar Experiment for my senior EEI and was wondering if i could change this experiment slightly by replacing the antibiotics with household cleaning products and still have some success?

[@Goldbio]

Jk Fuzzydragon Sure! I would think the household cleaners (as long as they’re liquids that could absorb into the paper disk) would work just fine in an experiment like this. Gels or foams may not be as easy, but might still be possible. Let us know if you try it out!

[@Jenna-zw6zm]

can this be used to test the growth of bacteria against a specific substance?

[@ericy1]

What should the concentration be for the solution/broth with the antibiotic?

[@dayanacheong901]

hello. do you know what is the name of the antimicrobial disc with the code W 2.5 ?

[@Mona-vj5nm]

Hello! Is it possible to adapt this method to see the effect of visible light (blue, red, ..) on bacterial growth/inhibition? If not, which other method can be utilised? Thankyou!

[@takoyuuki4270]

Thank you so much for this video! It really cleared up the whole process for me! I’m doing an related experiment for my science fair project and I really needed to learn how to do this.

[@donneyongevans7698]

excellent demonstration,well explained.thanks

[@tristandyvalerian6632]

if you dont have filter paper disc...... would cotton do ??

[@nicolerosales9026]

how many dips in the fungi culture do i need to do, is there a maximum?

[@rellaferrer8835]

Can i ask where to get a sample of a bacteria

[@JaeJay0]

In my microbiology class we had to do the Kirby Bauer method today. I happened to stumble upon your video when trying to find out more information about the method. For class, we also have to do a lab report based on our findings. Do you have any suggestions or tips on how to do the report?

[@prettykat4567]

You try to give the video more brightness it will be great if you do

[@jeniantony2952]

What is the size of you petri dish? How many antibiotic discs i can place in 90 mm petri dish?

[@vanplatt8253]

Is there any way to quantitatively determine the effects using this method?

[@sujaltimsina8731]

at what temp should we incubate at 16-20 hours?

[@Farhana1910]

Can we use any other antibiotic other than Ampicillin?

[@nurulaliana5956]

how do you sterilize the filter paper disc?

[@fantastic1368]

What is use of antibiotic control ??

[@SanjaiKumar-sx9rr]

How to prepare antibiotics disc?

[@barcebarce8904]

is this method limited only to mueller hinton agar?

[@filipem.dossantos1190]

Thanks for you trial, very Helpful

[@maqdadullah8217]

Sir i want to know that how much mg of medicinal plants extract is to disslove in how much ml of dmso for well diffusion methode. Secondly sir that can we use water instead of dmso?

[@Sapphiresash]

shouldnt the tweezer be flamed for a longer time than that?

[@ThePunkadagirl]

+Sashene Brown No, as long as the tweezer is not on fire its ok. As soon as the alcohol evaporate the tweezer is not on fire anymore. The alcohol can also interfere on your experiment, so make sure that the tweezer is not on fire.

[@Sapphiresash]

Oh okay thanks. it just seemed like he moved through the flame for less than a split second. :D

[@prashanthgoud281]

please explain about cylenders cup method

[@maretafadi]

What happens if a non sterile swab was used ?

[@mohamedaden8637]

Thank you really so much sir , this clip is very helpfull. God bless you sir

[@alyssamolina3351]

Hi. Im sort of new to microbiology (like zero experience) but we need to use this method for this project we have. Im wondering how do you determine the concentration/dilution per disk?

[@innomaiden5009]

I just know your stem 😭😭

[@randischmidling4483]

Loved your video! Thanks a million!!

[@Goldbio]

Glad you liked it Randi. We are working hard on some new ones so stay tuned.

[@sarahrazak3421]

cool! how about antifungal and toxicity test? mind to demonstrate?

[@samiralkhatib7192]

THANKS A LOT SO SIMPLE AND INFORMATIVE .

[@maxlanzi8686]

I dont understand what control i would use

[@reeth9876]

Does anyone have a list of the equipment that he used in the video?

[@Goldbio]

Hi Ethan - we've added this list to the video description: -6 mm filter paper discs -sterile petri dishes -agar plates with no antibiotic -sensitive and resistant bacterial cultures -antibiotic to test and antibiotic for control Other items out on the bench is a Bunsen burner, lighter, tube racks, thermometer, pipette, pipette tips, antibiotics solution, forceps, ethanol, flow hood (optional) and a marker to label discs We also created a supplemental blog post about Kirby Bauer here: www.goldbio.com/blog/120/old-antibiotics-wont-spoil-research-with-this-test Thanks!

[@reeth9876]

Thank you! :D

[@nidaashraf4860]

This video is very useful.

[@lalitmohanjoshi4623]

Can u provide me clsi guidelines for abst .

[@quizonzuella2519]

I love it! Thank you so much sir!

[@drmahmoudkhalil7108]

very thankful for you thanx alot

[@polardog2396]

are the antibiotics liquid?

[@kimbondad7766]

can you pleaaaase send me the procedure? we need it for our thesis. thankssss!

[@Goldbio]

Kim Bondad Hi Kim - late last year, we created a blog post to supplement this video. It's not the procedure step-by-step, but it gives additional tips and information that may not have been covered. Thanks for getting in touch, we hope this can be of use! - bit.ly/1yXKCnv

[@suyashkanse508]

@@Goldbio i

[@lisztomaniacenid]

Thanks! Had been very useful. :)

[@priyankanilangekar3161]

How to sterile that filter paper discs

[@anbuk97]

In well diffusion method how to make a sample to 20 micro litre as test sample in gold nano particles

[@Goldbio]

anbu k Sorry Anbu, we don’t have any experience working with gold nanoparticles (despite the fact that we’re called Gold Biotechnology). Let us know if you give the experiment a try!

[@MariaGonzalez-ie7dw]

Excuse me what is OD?

[@mashaalfarraj9543]

Thank you for the video! :)

[@quirma6471]

hi can yall reply to me the materials that were said in the video? tenksss

[@Goldbio]

Good morning, here is the link to the protocol: www.goldbio.com/documents/1329/GoldBio%20Disk%20Diffusion%20Assay%20Protocol.pdf Materials needed are:  Antibiotic to be tested  Control Antibiotic (either previously tested product or commercially available disk)  1 overnight culture of resistant strain  1 overnight culture of susceptible strain  6 mm Whatman filter paper disks  Sterile petri dishes  2 Agar plates (with no antibiotic)  Tweezers

[@Goldbio]

Additionally, from the video description, here are some of the specific materials: -6 mm filter paper discs -sterile petri dishes -agar plates with no antibiotic -sensitive and resistant bacterial cultures -antibiotic to test and antibiotic for control

[@geoffreyhenstock4773]

this was so good thank you

[@alefkashikar8868]

thank you very much

[@sudesanprameela4343]

I'm a microbiology student😘😘😘😘it's my passion

[@ACHIEVEWN]

Thank you!

[@ricasaluria5145]

so intelligent of you.

[@monalizamarine3978]

you are the best

[@tarout82]

Thank you

[@sukhmandeepkaur6876]

this is really helping :)

[@Goldbio]

Sukhmandeep Kaur Thanks - we're glad to hear!

[@seedsofhope8643]

perfect thank you so so much :)