Agar well diffusion assay

Sensitivity test by well diffusion method.
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[@TheYuenKim]

Hi Mr. Rejo. This is such a good and educational video. I just want to ask, what were the amount of melted agar and extract that you used?

[@rejojacobjoseph]

U can add 2-3 drops of melted MHA to the well (purpose is just to seal the lower part of the well. But if the extract is viscous no need to seal the well. Extract u can add up to 100μl.

[@TheYuenKim]

@@rejojacobjoseph Thank you for the reply Mr. Rejo. Have you ever worked with quercetin or other flanovoids as potential antimicrobial agent?

[@rejojacobjoseph]

@@TheYuenKim I worked mainly with leaf, plant,& seed extracts.

[@kabitaneupane3652]

The sample extract as well diffusion, and the positive control or standard as disc diffusion in the same plate. Can we differ like this sir?? Just curious to know 🤔

[@goodnessonukwube3068]

Sir, I want to know, when one is using a plant extract, unlike conventional antibiotics that have a standard, how can one determine the concentration of the plant extract that inhibits the bacteria when using MIC

[@ayasya.]

should the plate place inversed or not when incubated?

[@banupriyabanupriya8570]

Sir in comments ,you said that if the extract is viscous ,we dont need to add melted MH agar at bottom, in your experiment also the extract is viscous then why you added MH agar on bottom?

[@jessadesoloc1159]

Thank you for the video! May ask in what book can I find this step-by-step process? Just so I can put a reference book for an activity for school. Thank you!

[@Liki_Bu]

thanks for good video. does big size of well have the more powerful antimicrobial activity? or small size of well ?

[@siddhigosavi3683]

I dont think the size of the well matters in the case. Its what you add to the well that gives you the clearance zone. so the more powerful the antimicrobial activity of the solution/extract, the bigger the zone of clearance and vice versa.

[@babusheikh6652]

Dear Sir, would you please tell me about how to prepare the sample? In my case, when I filtered my sample through 0.2 micron filter inside a laminar flow cabinet, my sample's concentration as well as the volume also get decreased. So, I am now in confusion about how to prepare the sample correctly. Please help!!

[@rejojacobjoseph]

Different methods r there for the sample preparation. Best way is first follow the procedure from 2-3 good articles related to your study.

[@babusheikh6652]

@@rejojacobjoseph Thank you sir for your suggestion

[@sarangas8688]

Very help full

[@alexmares5322]

That you very much, very well explained.

[@KristelleMaeTardecilla-eh6ci]

Hi what is the diameter of the well using blue pipette tip

[@vikasashraf1672]

Dear sir How to measure the ZOI in agar well diffusion method. since incase of Disc Diffusion assay the ZOI is measured end to end which also includes the area under the disc. in this case there is a well and couldnt be counted as clearing zone

[@rejojacobjoseph]

Measure same like disc diffusion method if the zone is clear.

[@lilyrose1673]

Hello Sir, what media you sugest for antibacterial testing for staphylococcus aureus?

[@rejojacobjoseph]

MHA

[@anjua3771]

Sir, in the case of using sterile disc is it possible to add 100 microlitre on a single disc???

[@salettedepintosouza5455]

Hello my products are neem extract nutmeg extract combo of neem & nutmeg extract herbal gel & marketed preparation. My need extract is viscous with what solvent I should dilute with?? Tween 80?? Can't I add above products directly?????

[@rejojacobjoseph]

Plz check some article related to neem extract study. So that u will get an idea about it..

[@anjanaramesh6327]

Sir,Which is media is used for sauropus androgynus plant extract for antibacterial test?

[@rejojacobjoseph]

For antibacterial test better use MHA or MHA with blood

[@anjhere]

Thank you for the video sir. What control disc have you used in this video sir? I have a similar experiment with curcumin nanoparticles, but they are in paste form, not liquid form, do you have any suggestion in how I should load my sample inside the wells?

[@rejojacobjoseph]

When u do this procedure first u add full con: of ur extract in to the well and check the zone of inhibition and compare the zone with antibiotic zone (commonly used antibiotic used for the bacteria u inoculated). If the zone is big then u can perform the MIC/ dilution and again repeat the procedure to get a good result with less dilution.

[@anjhere]

@@rejojacobjoseph thank you for the response sir

[@tanishakumari2958]

Sir when I pour my extract into well then zone of inhibition is not clearly visible due to color of extract then what I do please suggest

[@rejojacobjoseph]

Before adding the extract, seal the underside of the well. When adding the extract, ensure not to exceed 100 microL. After incubation, if no clear zone is observed or if bacterial growth surrounds the well, it indicates that the extract concentration is insufficient........

[@tanishakumari2958]

@@rejojacobjoseph okay sir thank you for your suggestions

[@chemist291]

How to perform antibacterial activity of CuO nanoparticles

[@reetkaur4662]

Sir can we use this method for candida albicans...? And if yes then the media will be MH or SDA..?

[@rejojacobjoseph]

U can use MH for candida

[@ankitabarla8534]

hello sir, i am working on fungus but I am unable to culture it because of contamination, and how else can I try to culture and isolate fungus from seed cake surface.please help by suggesting something

[@rejojacobjoseph]

U can transfer the fungus to the media by loop. When fungus grows, u can see different colours (means different type of fungus). With a sterile loop Touch only on to the top (spores) of one type of fungus (touch only 1 color) and transfer to the media and incubate the plates in an inverted position.

[@ankitabarla8534]

@@rejojacobjoseph yes sir I've tried that. Any more techniques or ways to culture it?

[@ankitabarla8534]

And it's turning out to be milky in consistency

[@sheikhsaad5368]

Sir in agar well addition of melted agar drop is necessary

[@rejojacobjoseph]

For better results add 1-2 drops of melted agar to seal the lower border of the well..

[@ran2182]

Hello Mr can you provide the link to the protocol used in this video thank you

[@tribikramdahal5998]

Sir can you please make video on aloe gel extraction

[@halamohammed6961]

What is the composition of Muller Hinton Agar media?

[@happychemist]

what is the name of possiitive control? and what is it purpose?

[@sahiba5396]

Sir can we use PDA media for candidal species. Please reply sir.

[@rejojacobjoseph]

I didnt try PDA: but u can use it.

[@sahiba5396]

@@rejojacobjoseph thankyou so much sir.

[@akarshs8482]

Use SDA media for candida

[@sawsansawsan470]

hello, thank you for the video! . I want to ask, how do you choose the positive control?

[@rejojacobjoseph]

U can select one commonly used susceptible antibiotic for the inoculated bacteria. eg. For staph aureus u can use erythromycin as PC.

[@sawsansawsan470]

@@rejojacobjoseph thank you

[@bindumangat333]

Sir pls also upload video of poison food method

[@bindumangat333]

Pls do reply me sir

[@youthaventure2725]

Can we use plates with nutrient gelose medium instead of a MH one for bacteria's?

[@rejojacobjoseph]

U can use, but MH is more good becoz it contain starch, which can absorb toxins released from the bacteria.

[@youthaventure2725]

@@rejojacobjoseph okay thank you

[@aaliyafatiwala8633]

Can nutrient agar plate be used?

[@rejojacobjoseph]

Better use only MHA

[@drsamsonsoon8611]

Are the plates with mueller hinton medium?

[@rejojacobjoseph]

Yes

[@bhuvanachandrikamandapalli5853]

Hello sir I'm inter 2 year student can you please tell me this mothad: tube dilution and agar dilution please explain me sir

[@rejojacobjoseph]

www.slideshare.net/RejoJacobJoseph/antibiotic-sensitivity-test-by-dilution-method

[@CarTrackers]

Sir how to calculate MIC of this kindly tell

[@rejojacobjoseph]

Well diffusion technique helps to check whether ur extract is having good antimicrobial activity or not. When u get good zone of inhibition after this technique, U can perform broth dilution method with ur extract for MIC. www.slideshare.net/RejoJacobJoseph/antibiotic-sensitivity-test-by-dilution-method

[@ashoknutkut1]

nalada aairnu