Disc Diffusion (Kirby-Bauer) Antimicrobial Susceptibility Testing

In this video you’ll learn the principle of the Kirby-Bauer disc diffusion test and see how it is performed and interpreted.
Useful links:
CLSI veterinary interpretive criteria: clsivet.org/Get...
European Committee on Antimicrobial Susceptibility Testing (EUCAST): eucast.org
For more educational materials visit our Flickr page:
www.flickr.com...
To learn more the exciting research done in the Rubin lab, check out the website:
www.therubinlab.com

[@ABRIL6361]

Did this last week for my micro lab class .

[@alderamin1402]

I'll do it this week ✌️

[@therubinlab]

Hope this video was a helpful review after your class!

[@therubinlab]

@@alderamin1402 Hope your class went well and that this video was helpful preparation for you!

[@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?

[@therubinlab]

Hi Babu, I'm not sure I understand how you are performing your assays; in the disc diffusion test there are no wells in the agar. I am assuming that you are screening plant extracts for their antimicrobial activity? If this is the case it would seem generally reasonable to filter the extracts through a filter to ensure that you don't contaminate your experiment with the bacteria normally found on the plants. Generally speaking, when performing a susceptibility test you want to make sure that your reagents are free of contaminants and that the bacterial cultures you are assaying are pure isolates. I hope this helps!

[@babusheikh6652]

@@therubinlab Thanks a lot for your nice explanation.

[@Sfxxs]

Hi, I want to compare the effectiveness of commercial antibiotics and natural antibiotics. I am planning to use the Kirby Beuer method. However I am confused on which step I should use the antibiotic treatment. I really need an answer, thank you!

[@simondahdouh994]

Thanks this helped. I didn't think that there was a machine that could tell the McFarland of a tube. WOW. Since we are in practice, we just use a random amount. BUT, i guess, it is best to add more then less since you did take colonies twice from agar and into saline.

[@therubinlab]

I'm glad this helped! Making sure that you have a MacFarland 0.5 density of cells is CRITICAL - either too much or too little will negatively impact the test results. One thing that I could recommend if you don't have access to a densitometer would be to visually compare to a MacFarland 0.5 standard. To do this you can simply draw a dark, thick line on some white paper or card and hold it behind your suspension and and the standard at the same time; what you're looking for is an equivalent level of "blurring" of the line. I found this video which might be helpful (although they don't do the dark line trick) kzbin.info/www/bejne/r5vMfZ5vlL92Z6s Hope this helps!

[@Khaledf]

Thank you. Is there a specific medium used for this test? Can we use nutrient agar? 😊

[@therubinlab]

Good question! Yes, there is a specific medium - both the CLSI and EUCAST state that you need to use Mueller Hinton agar. Nutrient agar does NOT give zone diameters which you can interpret with recognized criteria for classifying an isolate as susceptible or resistant. It's also important to make sure that plates are 4mm thick so that the drug diffuses through the media in a predictable way. Stay tuned to the channel... I just filmed demonstrations for a new video and took photos for a new visual guid that I'm preparing - I'm anticipating that video should be out in the next 3-4 weeks :) In the mean time, if you have any questions please just leave another comment below!

[@Khaledf]

@@therubinlab Thank you! Much appreciated!

[@bioworld896]

Very nice explanation 🎉🎉

[@therubinlab]

Thanks for the comment! I just recently uploaded a new video, and step-by-step guide you can download, about disc diffusion testing - you can find it here: kzbin.info/www/bejne/iIOpnYZ_gLuGe9U Good luck with your studies!

[@marlenetorres2198]

Super thankful, thanks!

[@therubinlab]

I'm glad this helped!

[@emilygordon6625]

Awesome video!

[@therubinlab]

Glad you found it helpful!

[@jeffronylpausal5395]

hello! may i ask what kind of agar did you use for culturing in this video? we are going to perform disc diffusion method on p. acnes and we are suggested to culture it in fastidious anaerobe agar specifically blood agar. if so, would the new plate which we will transfer our p. acnes on during the disc diffusion still contain the blood agar?

[@therubinlab]

You've brought up an important point... for aerobic and non-fastidious organisms we always use Mueller-Hinton agar. I haven't worked with P. acnes before, but I would suggest taking a look at the CLSI standards for anaerobes: clsi.org/standards/products/microbiology/documents/m07/ If you have a clinical diagnostic lab at your institution they might have standards on hand that you could review. Testing anaerobes can be a bit of a challenge, and for many bacteria it is actually recommended to NOT test using dilution based methods (either discs or E-tests); you might be better off trying agar dilution or broth micro dilution. There are number of commercially available products for doing broth micro dilution on anaerobes (the MicroScan or Sensititre products are a couple that I'm somewhat familiar with). Best of luck with your project!

[@therubinlab]

I realized my second sentence wasn't very clear... for some fastidious bacteria we DO use blood containing media (some Streps etc.). But before doing this, it would be good to see if there is a CLSI standard; that way your results will be comparable with other studies in the literature.

[@RywaTaka]

great video

[@therubinlab]

Glad you found it helpful!

[@RywaTaka]

@@therubinlab Hai Rubin thanks for the video. you have broth dilution method also but e test is not available. can you post a video on e tests for antimicrobial sensitivity testing?

[@therubinlab]

@@RywaTaka I don't have one for E-tests because these aren't tests that we regularly use in our lab... BUT I would say that performing an E-test is technically very similar to doing a disc test (so this video essentially shows you how to do it). One small difference is that because E-tests use a gradient of drug concentrations, the strip can't be moved once it touches the plate; so unlike discs which can be adjusted immediately after being applied, E-tests can't. I am hoping to put together a video on agar dilution in the coming months which may be of use to you as well - this is an inexpensive method for determining MICs - so stay tuned for that one!

[@RywaTaka]

@@therubinlab okay thank you. 👍

[@Thinker-gd8ck]

Hi, May I ask you how to access CLSI on VET08 ED4? I could not find it.

[@therubinlab]

No problem, you can find the freely available veterinary and human guidelines no the CLSI website here: shop.clsi.org/standards/products/free-resources/access-our-free-resources/

[@Thinker-gd8ck]

@@therubinlab Thank you very much!

[@therubinlab]

You are most welcome, good luck with your research

[@nhatminhtran5629]

Hi I want to ask that can I test antibacterial activities of solid sample by this method, for example ZnO nanoparticle

[@therubinlab]

I'm not aware of a method (at least a clinical method) that could be used to assess the ability to test the inhibitory effects of a surface on the growth of an organism. If these nanoparticles can be solubilized, I think you would have better luck with a dilution based technique - either agar dilution or broth dilution; these methods will let you determine an MIC which will give you some quantitative data regarding exactly how much of the compound is required to inhibit bacterial growth. Here's a link to a video that I made describing the procedure for agar dilution - hope fully this will give you some methodological ideas: kzbin.info/www/bejne/Z6XChoSeqbdqpsk Best of luck with your research!

[@TheRedKnight101]

I am using blank disks and disks where only solvent is added as a negative controls, Yet I still get small zones of inhibition for both. Are these zones okay as long as they are a standard diameter? Or is there an issue with how I am plating the disks or time between making the bacterial lawn and adding disks?

[@therubinlab]

What solvent are you using? This could certainly be having an impact on bacterial growth; and I could imagine a situation where it is either enhancing or inhibiting antimicrobial activity as well... Are you testing regular (already approved antimicrobials) or novel chemicals? If you are testing regular antimicrobials, I would encourage you to take a look at the CLSI guidelines where you can find a list of recommended solvents and diluents for preparing antimicrobial containing media (for most drugs we just use sterile water). If you're testing other chemicals which are not soluble in water, then this could be a challenge and I would encourage you to think about trying a different method (agar dilution or broth dilution) which are better suited for these types of experiments as you'll get MICs vs. zone diameters. I hope this helps!

[@TheRedKnight101]

@@therubinlab I am using methanol as a solvent as I am working with plant extracts.

[@Jjannaa_.]

sir if the outcome was resistant does that mean the treatment was weak?

[@therubinlab]

Good question, and I think that's a common misconception. I don't think I would say that this means that the treatment is weak - resistance means that the drug is not predicted to work if used according to it's label indication (in the species intended, dosing regimen - dose, route of administration and duration, and for the type of infection it's labeled for). I've put together another video which you might also find helpful, there is also a link to a written/visual guide which you can download: kzbin.info/www/bejne/iIOpnYZ_gLuGe9U I hope you find this helpful!

[@tanigirl1]

I'm doing fungal research study these days. We couldn't find perfect method to test antifungal activity. Can't we do disk diffusion method on fungi

[@therubinlab]

Hi Tania, I think the issue you're coming up against is that until recently there haven't been standardized methods available for these tests. However, for the tests which are used, I believe dilution (MIC based) methods are the ones which are used. See this link to the EUCAST mould susceptibility testing webpage, they also have one for yeasts and dermatophytes: www.eucast.org/astoffungi/methodsinantifungalsusceptibilitytesting/ast_of_moulds/ Hope this helps!

[@abdullahfrehat1127]

please i want to ask you, it is normal to use paper disk that submerged with specific antibiotic ? to test the effectivness of other antibiotic ?

[@therubinlab]

I think there are 2 things I would keep in mind. 1. In order to interpret the test, the disc must contain a standardized amount of antimicrobial, the standard contents are listed in the EUCAST guidelines I linked in the description. 2. There are specially made filter paper discs for this type of testing, you can purchase commercially prepared ones that come with the correct antibiotic concentration or blank discs which can be prepared in-house. However, if in-house prepared discs are used then it is even more important to include quality control organisms to ensure that the assay is functioning within the standardized ranges. I hope that helps!

[@abdullahfrehat1127]

@@therubinlab yes it is help, thank you very much to your information. however, my study need blank paper disk from where can I purchased it ?

[@therubinlab]

@@abdullahfrehat1127 I think any major scientific supply company (maybe VWR or Fisher Scientific) should be able to sell you these. I believe the discs that I have purchased in the past are BD brand - they also make a lot of antibiotic containing discs.

[@Thinker-gd8ck]

Hi Dr.Rubin, it seems you replied my questions, but I couldn't see the reply.

[@therubinlab]

To make the McFarland standard there are 2 approaches that I am familiar with. One would be to spectrophotometrically measure the density of your culture suspension, this could be done with something like a densitometer. The other would be to visually compare your test suspension with a commercially prepared McFarland 0.5 standard visually. Both the CLSI and EUCAST publish details of how to perform this quality control step.

[@Thinker-gd8ck]

@@therubinlab Thanks for your reply sir!

[@jozefinachadzynska6825]

Hi where can I find interpretive criteria for Lactococcus?

[@therubinlab]

As far as I'm aware there aren't clinical interpretive criteria for this genus... BUT I took at look at EUCAST, and in their "expected phenotype" tables, they state that these species are intrinsically vancomycin and teicoplanin resistant (see link). I hope this helps! www.eucast.org/expert_rules_and_expected_phenotypes/expected_phenotypes

[@jozefinachadzynska6825]

@@therubinlab Tkank u so much!

[@malvikasingh6256]

I did swabbing but instead of uniform growth, there were only scattered bacterial colonies on the media.. Can anybody explain why it happened? What precautions should be taken while doing swabbing to get uniform growth?

[@therubinlab]

Hi Malvika, my guess is that the suspension you spread on the plate didn't contain enough bacteria. Make sure to inoculate your plates with a suspension of MacFarland 0.5 density (~1-2 X 10^8 CFU/ml). Hopefully that helps you troubleshoot!

[@salmaallah3703]

Do you incubate the plates upside down

[@therubinlab]

Yes! We always incubate plates upside down, otherwise you can get condensation on the lid which drips down onto the agar surface. When doing disc testing, it's important to gently push on the disc after it's places so that the discs don't fall off.

[@salmaallah3703]

@@therubinlab thanks for your prompt response. I think it’s really important to mention this step in your video so the method will be clear to the end.

[@therubinlab]

That's a really good point... I'm not sure if you saw, but I recently published a video which includes a link to a guide to performing disc diffusion testing kzbin.info/www/bejne/iIOpnYZ_gLuGe9U My co-authors and I tried to make this guide as clear as possible... have a look and let me know what you think. I hope this resource will be helpful as well!

[@rosieyves]

can we text oinment using this method

[@therubinlab]

Hi Livy - I don't think I would recommend using the disc method for testing ointments; disc diffusion results would be very difficult to interpret for chemicals which don't have CLSI or EUCAST breakpoints. I would recommend doing a broth micro dilution test or perhaps an agar dilution test for other compounds - You can check out this video I made about broth micro dilution here: kzbin.info/www/bejne/Zqm5kIBsYrV-Zs0

[@rosieyves]

@@therubinlab ok sure thank u so much i will check it out i prepared a beeswax based salve i wanted to check it is agar dilution method okay for beeswax cream?

[@therubinlab]

@@rosieyves It could work, I can imagine 2 challenges: 1. whether the product you want to test is water soluble and 2. how you will interpret the results - you will be able to determine an MIC but the question will be what does it mean exactly? Either way, sounds like an interesting project, good luck!

[@rosieyves]

@@therubinlab thank you so much im working on a green sythesis project for nanoparticles preparation. i prepared the beeswax cream with a very small quantity of the nanoparticles i wanted to prove it's an antimicrobial agent

[@therubinlab]

Hi Livy, I wanted to share a video that I recently posted about agar dilution which I think might be helpful for your study: kzbin.info/www/bejne/Z6XChoSeqbdqpsk

[@cook.king0844]

❤

[@jasonvoorhees2025]

TF IS THIS!!!!!