For other susceptibility testing techniques I found good videos, but for agar dilution, this video is perfect, specially showing how replicator works 🎉
Hello sir! We are currently conducting a research about antibacterial activity of plant extract and we are about to perform MIC using agar method (MHA) for p. aeruginosa and k. pneumoniae. Can you suggest an easy explanation of how we can perform it keeping in mind that we also prepared different concentrations of the plant extract? Do we still need to determine each MIC for each concentration of extract? Thank you!
Hi Jamie - sounds like an interesting project. I might be misinterpreting your question, but think there might be a bit of confusion with the definition of MIC - this is the minimum inhibitory concentration; in order to determine the MIC you have to test multiple concentrations. If for instance you test 1, 2, 4, 8, and 16 micrograms/ml and you see growth of an organism at 1, 2 and 4 micrograms/ml but not 8 or 16, then the MIC would be 8 because this is the lowest concentration which inhibits growth. I hope that makes sense and helps! If I've misinterpreted your question please just write back.
Hello Omo, I'm glad you found the video helpful - hopefully your student is able to utilize this method! I know the CLSI does provide example calculations which can be used for preparing antimicrobial dilutions; although they suggest a slightly different approach; I believe using the most concentrated solution to make several lower concentrations and then diluting from those vs. a simple series of 2-fold dilutions like I present here. I think the most important thing is to work out all of the calculations on paper first and then having another person check them over to make sure there are no errors before preparing the media! I would suggest that your student could just clearly describe how he/she makes the dilutions (that way others could check the math) as opposed to needing a reference. Honestly, I've published data using agar dilution in the past and have never been asked by reviewers for a specific reference for how the dilutions were made. I hope that helps!
@@omorugged1 Here's one in PLoS One - it's for a bit of an unusual bacteria, so this isn't the best example for non-fastidious organisms, but we describe in detail how we did agar dilution: journals.plos.org/plosone/article?id=10.1371/journal.pone.0286594 Just make sure to use the conditions that are correct for your organisms (probably way simpler than in this study!). Here's another where we used agar dilution for colistin susceptibility testing: ami-journals.onlinelibrary.wiley.com/doi/10.1111/jam.14658
I think the most important consideration is, what is the resistance breakpoint? You definitely want to make sure that concentrations surrounding that breakpoint are incorporated within the dilution series you test. Beyond that, I think you can decide whether you are interested primarily in identifying resistance (maybe then you only need several concentrations immediately around the breakpoint), or generating a detailed susceptibility profile - perhaps for surveillance purposes (then you might want to test a wide range of MICs - maybe 10 concentrations far above and below the breakpoints). Hope that helps!
I was actually lucky enough to inherit this piece of equipment from a colleague who purchased it decades ago and has since retired - I am not sure if they are still available for purchase? This device is called a "Cathra Replicator". An alternative that you could try, would be to inoculate plates with a multi-channel pipette - the only thing to be aware of is depending on the volume that your pipette can dispense, you will have to dilute your McFarland 0.5 suspension so that your final inoculum is ~5 X 10^4/spot. I hope that helps, best of luck with your research!
Yes, thanks so much for the quick response! I think a multichannel pipet might be the way to go. And thanks for the video, it was super helpful!@@therubinlab
Oh you are most welcome! When we last performed this test in my lab I thought it was a great opportunity to capture the process on video... good training material for my students so why not share more broadly :)@@peterfowler6753
That's a really good suggestion, I have some time-kill experiments planned sometime in the next year and I'll try to remember to take some video so I can post an explainer video here! The basis of the technique is to grow your organism in broths containing various concentrations of your antimicrobial of interest and at various timepoints (ex. 0, 2 hours, 8 hours, 24 hours) do a quantitative culture of the broth (spread plates) to measure changes in the bacterial population. The shape of these curves, can help to determine whether a drug-organism interaction can be classified as static or cidal - I believe a 3-log reduction in bacterial concentration after 24 hours is generally considered the benchmark for bactericidal activity.
Hi sir! We are in the process of conducting our research study in college, I would like to ask if agar dilution is still possible if we are going to use a dry plant extract as an antifungal? And can you also explain what the mcfarland standard is for? We are testing if this particular plant has antifungal properties or not. Your reply will be much appreciated.
Good question. I'll try and answer in point form for clarity: 1. Yes, you could use agar dilution for the plant extract. You just have to be able to dissolve it into a liquid form prior to incorporating it into agar. Depending on how much of the extract you have, it might be more practical to use a broth-micro dilution method (since plate volumes are 20ml you will likely need to use a lot of the extract). 2. The McFarland standard is to make sure you're testing a consistent, standardized concentration of bacteria (although with fungi there may be a different method). I'm not familiar with anti-fungal susceptibility testing, I would suggest looking into the CLSI guidelines to see what the standard procedure is. 3. For this study, I think it will be really important to also include a positive control (no extract plate) to ensure that your fungus will grow on the media you use. Again, here I'm not sure what's recommended for fungi, Mueller Hinton is the media used for non-fastidious bacteria. The M27 and M38 documents describe broth dilution methods of anti-fungal susceptibility testing (so broth might be the way to go rather than agar!) I hope that helps, good luck with your experiments!
@@therubinlab Thanks sir for replying! We’re not really sure if broth-micro dilution will be feasible in our part since our university still lacks some of the equipment needed. Do you have any other methods that you can recommend for testing anti-fungal activity specifically by using a plant extract? I’ve been trying to research about it but the results are so minimal.
@@kelseymallow8428 I think the best approaches would all be based on determining an MIC, and I think either broth or agar dilution would be the way to go. For the broth method, you could also do "macro-dilution"; here instead of using commercially prepared 96 well plates, you prepare broths with doubling dilutions of your compound in larger tubes (maybe 2-3ml volumes). This should eliminate the need for any specialized equipment (just tubes, pipettes, incubators etc.). If you can get your hands on the M27 document (clsi.org/standards/products/microbiology/documents/m27/) you should find some detailed instructions on how to perform this test.
@@therubinlab Is it possible to use agar well diffusion method? It’s the most feasible method in our part. How is the MIC usually measured if this is the method used? In terms of making the dry plant extract into a liquid, can we use distilled water for dissolving the dry extract? Thank you much, your replies are really a big help in our research.
@@kelseymallow8428 I'll try and answer in point form for clarity: 1. I'm really not sure about the appropriateness of agar dilution vs. broth dilution for anti-fungal testing, unfortunately this just isn't an area that I have expertise in! I'm not familiar with the agar well diffusion method; is this putting extract in a well in the plate and looking for a zone of inhibition caused by diffusing anti fungal compounds? If so, I think this sounds like a reasonable approach but would be very qualitative (I think the only question you could reliably answer is - does extract X have any impact on the growth of the fungus we tested). Further studies would be required to quantify the inhibitory effects. 2. In agar dilution, the MIC is defined as the lowest concentration which inhibits the growth of the organism - so if you have growth on the plate with 2ug/ml, but not 4ug/ml then then MIC would be 4. 3. Distilled water may be an appropriate solvent for the extract, but it really depends on the chemical properties of the substance present. When preparing antibiotic suspensions for agar dilution we do use water for most drugs, but there are some where we first need to solubilize the powder with ethanol, DMSO or PBS and then dilute the solubilized compound with water. I hope this is helpful; I appreciate that there are a lot of complications!
Sir We are going to do MIC taking five doubling dilutions starting from 1ug/ml so our highest dilution we will take is 16ug/ml in 10ml media We need to do this experiment of 6 diff microorganisms So we will have 5×6 of each dilution in nutrient medis I need to make a stock solution that do all the work Please help
So, if the highest final concentration that you will be preparing is 16ug/ml, then I would suggest that you should make a stock solution of 160ug/ml - this will allow you to do a final dilution of 1:10 (1ml stock + 9ml sterile broth) to achieve your final concentration. I would suggest figuring out how much of this highest concentration of stock solution you'll require and then prepare 3 times that volume (so if you need 10ml, prepare 30). This will allow you to prepare your highest concentration stock solution and then make 1:2 dilutions to get your other stocks while still having a little extra just in case you make a mistake or spill some. Ex. If you prepare 30ml of 160ug/ml, take 15ml of this solution and dilute it with the appropriate diluent (probably sterile water) to get 30ml of your 80ug/ml stock - repeat this until you have all of your stocks prepared. One other tip is that depending on the accuracy of the analytical balance you're using it might be more accurate to mass out more of the antibiotic, and prepare a higher concentration initial stock solution (perhaps 640ug/ml). Of course if your antibiotic powder is very expensive this might be hard to justify; perhaps consider testing higher concentrations - why stop at 16ug/ml, maybe go all the way to 128! The best piece of advice I can give you is to write down all of the calculations for all of the media you want to prepare - this way you can have a colleague check your math and ensure that it's correct. I hope that helps!
Hey man, for my school biology project, I'm planning on conducting an investigation which involves testing antimicrobial properties using Kirby-Bauer method. Plz Plz Plz reply to me I have a few questions.
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Fantastic video and very helpful in preparing the research student I support.
Really helpful video great mini lecture
For other susceptibility testing techniques I found good videos, but for agar dilution, this video is perfect, specially showing how replicator works 🎉
Oh thank you so much! I was hoping to fill that gap in content, so I'm glad you found it helpful :)
Are there different bacterial strains in each holes of well plate?
Yes, definitely! One of the benefits of this method is that you are able to test multiple strains at the same time.
Hello sir! We are currently conducting a research about antibacterial activity of plant extract and we are about to perform MIC using agar method (MHA) for p. aeruginosa and k. pneumoniae. Can you suggest an easy explanation of how we can perform it keeping in mind that we also prepared different concentrations of the plant extract? Do we still need to determine each MIC for each concentration of extract? Thank you!
Hi Jamie - sounds like an interesting project. I might be misinterpreting your question, but think there might be a bit of confusion with the definition of MIC - this is the minimum inhibitory concentration; in order to determine the MIC you have to test multiple concentrations. If for instance you test 1, 2, 4, 8, and 16 micrograms/ml and you see growth of an organism at 1, 2 and 4 micrograms/ml but not 8 or 16, then the MIC would be 8 because this is the lowest concentration which inhibits growth. I hope that makes sense and helps! If I've misinterpreted your question please just write back.
Great video. Please could you provide reference for the formulas you used. A student urgently need it.
Hello Omo, I'm glad you found the video helpful - hopefully your student is able to utilize this method! I know the CLSI does provide example calculations which can be used for preparing antimicrobial dilutions; although they suggest a slightly different approach; I believe using the most concentrated solution to make several lower concentrations and then diluting from those vs. a simple series of 2-fold dilutions like I present here. I think the most important thing is to work out all of the calculations on paper first and then having another person check them over to make sure there are no errors before preparing the media!
I would suggest that your student could just clearly describe how he/she makes the dilutions (that way others could check the math) as opposed to needing a reference. Honestly, I've published data using agar dilution in the past and have never been asked by reviewers for a specific reference for how the dilutions were made. I hope that helps!
@@therubinlab helpful. Please give me link to some of your papers on this.
@@omorugged1 Here's one in PLoS One - it's for a bit of an unusual bacteria, so this isn't the best example for non-fastidious organisms, but we describe in detail how we did agar dilution: journals.plos.org/plosone/article?id=10.1371/journal.pone.0286594
Just make sure to use the conditions that are correct for your organisms (probably way simpler than in this study!).
Here's another where we used agar dilution for colistin susceptibility testing:
ami-journals.onlinelibrary.wiley.com/doi/10.1111/jam.14658
Please I want to ask concerning the highest concentration of the antibiotic to use. How can I determine it?
I think the most important consideration is, what is the resistance breakpoint? You definitely want to make sure that concentrations surrounding that breakpoint are incorporated within the dilution series you test. Beyond that, I think you can decide whether you are interested primarily in identifying resistance (maybe then you only need several concentrations immediately around the breakpoint), or generating a detailed susceptibility profile - perhaps for surveillance purposes (then you might want to test a wide range of MICs - maybe 10 concentrations far above and below the breakpoints). Hope that helps!
@@therubinlab It did thank you very much sir
You are most welcome! Best of luck with your project.
I was curious where you purchased the replicator you're using. We're having trouble finding one that is similar.
I was actually lucky enough to inherit this piece of equipment from a colleague who purchased it decades ago and has since retired - I am not sure if they are still available for purchase? This device is called a "Cathra Replicator". An alternative that you could try, would be to inoculate plates with a multi-channel pipette - the only thing to be aware of is depending on the volume that your pipette can dispense, you will have to dilute your McFarland 0.5 suspension so that your final inoculum is ~5 X 10^4/spot. I hope that helps, best of luck with your research!
Yes, thanks so much for the quick response! I think a multichannel pipet might be the way to go. And thanks for the video, it was super helpful!@@therubinlab
Oh you are most welcome! When we last performed this test in my lab I thought it was a great opportunity to capture the process on video... good training material for my students so why not share more broadly :)@@peterfowler6753
Could you make one video about Time-kill assay? It is hard to find one good about this subject.
That's a really good suggestion, I have some time-kill experiments planned sometime in the next year and I'll try to remember to take some video so I can post an explainer video here! The basis of the technique is to grow your organism in broths containing various concentrations of your antimicrobial of interest and at various timepoints (ex. 0, 2 hours, 8 hours, 24 hours) do a quantitative culture of the broth (spread plates) to measure changes in the bacterial population. The shape of these curves, can help to determine whether a drug-organism interaction can be classified as static or cidal - I believe a 3-log reduction in bacterial concentration after 24 hours is generally considered the benchmark for bactericidal activity.
@@therubinlab Next year will be later for me because I need it now. But, I wish all the best for the ones who will get this tutorial you will produce.
Hi sir! We are in the process of conducting our research study in college, I would like to ask if agar dilution is still possible if we are going to use a dry plant extract as an antifungal? And can you also explain what the mcfarland standard is for? We are testing if this particular plant has antifungal properties or not. Your reply will be much appreciated.
Good question. I'll try and answer in point form for clarity:
1. Yes, you could use agar dilution for the plant extract. You just have to be able to dissolve it into a liquid form prior to incorporating it into agar. Depending on how much of the extract you have, it might be more practical to use a broth-micro dilution method (since plate volumes are 20ml you will likely need to use a lot of the extract).
2. The McFarland standard is to make sure you're testing a consistent, standardized concentration of bacteria (although with fungi there may be a different method). I'm not familiar with anti-fungal susceptibility testing, I would suggest looking into the CLSI guidelines to see what the standard procedure is.
3. For this study, I think it will be really important to also include a positive control (no extract plate) to ensure that your fungus will grow on the media you use. Again, here I'm not sure what's recommended for fungi, Mueller Hinton is the media used for non-fastidious bacteria. The M27 and M38 documents describe broth dilution methods of anti-fungal susceptibility testing (so broth might be the way to go rather than agar!)
I hope that helps, good luck with your experiments!
@@therubinlab Thanks sir for replying! We’re not really sure if broth-micro dilution will be feasible in our part since our university still lacks some of the equipment needed. Do you have any other methods that you can recommend for testing anti-fungal activity specifically by using a plant extract? I’ve been trying to research about it but the results are so minimal.
@@kelseymallow8428 I think the best approaches would all be based on determining an MIC, and I think either broth or agar dilution would be the way to go. For the broth method, you could also do "macro-dilution"; here instead of using commercially prepared 96 well plates, you prepare broths with doubling dilutions of your compound in larger tubes (maybe 2-3ml volumes). This should eliminate the need for any specialized equipment (just tubes, pipettes, incubators etc.). If you can get your hands on the M27 document (clsi.org/standards/products/microbiology/documents/m27/) you should find some detailed instructions on how to perform this test.
@@therubinlab Is it possible to use agar well diffusion method? It’s the most feasible method in our part. How is the MIC usually measured if this is the method used? In terms of making the dry plant extract into a liquid, can we use distilled water for dissolving the dry extract? Thank you much, your replies are really a big help in our research.
@@kelseymallow8428 I'll try and answer in point form for clarity:
1. I'm really not sure about the appropriateness of agar dilution vs. broth dilution for anti-fungal testing, unfortunately this just isn't an area that I have expertise in! I'm not familiar with the agar well diffusion method; is this putting extract in a well in the plate and looking for a zone of inhibition caused by diffusing anti fungal compounds? If so, I think this sounds like a reasonable approach but would be very qualitative (I think the only question you could reliably answer is - does extract X have any impact on the growth of the fungus we tested). Further studies would be required to quantify the inhibitory effects.
2. In agar dilution, the MIC is defined as the lowest concentration which inhibits the growth of the organism - so if you have growth on the plate with 2ug/ml, but not 4ug/ml then then MIC would be 4.
3. Distilled water may be an appropriate solvent for the extract, but it really depends on the chemical properties of the substance present. When preparing antibiotic suspensions for agar dilution we do use water for most drugs, but there are some where we first need to solubilize the powder with ethanol, DMSO or PBS and then dilute the solubilized compound with water.
I hope this is helpful; I appreciate that there are a lot of complications!
Sir
We are going to do MIC taking five doubling dilutions starting from 1ug/ml so our highest dilution we will take is 16ug/ml in 10ml media
We need to do this experiment of 6 diff microorganisms
So we will have 5×6 of each dilution in nutrient medis
I need to make a stock solution that do all the work
Please help
So, if the highest final concentration that you will be preparing is 16ug/ml, then I would suggest that you should make a stock solution of 160ug/ml - this will allow you to do a final dilution of 1:10 (1ml stock + 9ml sterile broth) to achieve your final concentration.
I would suggest figuring out how much of this highest concentration of stock solution you'll require and then prepare 3 times that volume (so if you need 10ml, prepare 30). This will allow you to prepare your highest concentration stock solution and then make 1:2 dilutions to get your other stocks while still having a little extra just in case you make a mistake or spill some. Ex. If you prepare 30ml of 160ug/ml, take 15ml of this solution and dilute it with the appropriate diluent (probably sterile water) to get 30ml of your 80ug/ml stock - repeat this until you have all of your stocks prepared.
One other tip is that depending on the accuracy of the analytical balance you're using it might be more accurate to mass out more of the antibiotic, and prepare a higher concentration initial stock solution (perhaps 640ug/ml). Of course if your antibiotic powder is very expensive this might be hard to justify; perhaps consider testing higher concentrations - why stop at 16ug/ml, maybe go all the way to 128!
The best piece of advice I can give you is to write down all of the calculations for all of the media you want to prepare - this way you can have a colleague check your math and ensure that it's correct.
I hope that helps!
I should visit your lab, LOL😊
Hey man, for my school biology project, I'm planning on conducting an investigation which involves testing antimicrobial properties using Kirby-Bauer method. Plz Plz Plz reply to me I have a few questions.
Sure, what's your question? Also, did you see my video on Kirby-Bauer testing: th-cam.com/video/M-szotkpT00/w-d-xo.html