Dear Madam,

I feel that disc diffusion method is good. As Mr. Thavasi said, we can use Whatman filter papers for making discs. After sterilizing it, while working in LAFC in sterile condition, we can place the disc in a petriplate separately and add the extracts using micropipettes. Suppose if we have to add 10 microlitres of extracts to it, first take 10 microlitres of extracts in a micropipette and add a drop to the disc and wait for a few minutes it'll dry and then again take the remaining extracts and add a drop to to the disc again. Do this repeatedly. Finally the disc will absorb the phytocompounds present in the extract and it will be concentrated. And then place the disc in the medium.

Thanking You

With regards,

Dr. Tresina

It all depends on the diffusion of the drug molecules in agar or in discs. Some molecules won't diffuse in agar, in that case you have to use disc or 96 well liquid method. Solubility of the molecules play very important role. If the drug molecules you want to screen are soluble in the solute you are using then both method should work well.

Regarding quantification (how much is gone into the disc) you can try with whatman filter papers. Cut them with paper punching machine and autoclave the discs. You can check their absorption capacity by adding different vol. of the extract and fix the appropriate vol.

Regarding quantification in agar plates, it is very simple, since you have a well and it should hold 50 to 100 micro-litter.

Thanks Thavasi, But I want to know that as many researchers are using Disk method , and they mention different volume of drug absorbed in same size Disk . So this point I did not get into that a 6 mm sized Whatmann papaer Disk can absorb so much varying amount and how ??

The well method is best,

in disc method was need you know for Concentrations of plant extractions . And the need to sterilize the paper may be focusing less than the estimated loss occurs in concentrations

Dear Madam,

I feel that disc diffusion method is good. As Mr. Thavasi said, we can use Whatman filter papers for making discs. After sterilizing it, while working in LAFC in sterile condition, we can place the disc in a petriplate separately and add the extracts using micropipettes. Suppose if we have to add 10 microlitres of extracts to it, first take 10 microlitres of extracts in a micropipette and add a drop to the disc and wait for a few minutes it'll dry and then again take the remaining extracts and add a drop to to the disc again. Do this repeatedly. Finally the disc will absorb the phytocompounds present in the extract and it will be concentrated. And then place the disc in the medium.

Thanking You

With regards,

Dr. Tresina

according to your extract as follow:

if your extract oil the best method is disc diffusion

if your extract non oil the best method is well diffusion

agar well diffusion is better than disc diffusion method .see attachment

Article ECONOMIC IMPORTANCE OF CHEWING STICKS IN ORAL HEALTH CARE IN...

Agar diffusion well-variant method the bacterial inoculum is uniformly spread using sterile cotton swab on a sterile Petri dish MH agar. Nine serial dilutions of

yielded concentrations of 100, 80, 60, 40, 20, 10, 5, 2.5, and 1.25

mg/mL for extracts and fractions and four serial dilutions yielded

concentrations of 20, 15, 10 e 5 mg/mL for pure substances is taken.

50 µL of natural products is then added to each of 5 wells (7 mm

diameter holes are cut in the agar gel around 20 mm apart from one another).

The systems is incubated for 24 h at 36ºC ± 1ºC, under aerobic

conditions. After incubation, confluent bacterial growth is

observed. Inhibition of the bacterial growth is measured in

mm. Reference commercial discs used can be chloramphenicol

30 mg.

But in Agar diffusion disc-variant method the natural products are dissolved and diluted with solvents as mentioned previously. Same number of subsequent dilutions

is performed as described above. However, natural products

serial dilutions is performed out of initial concentrations 2.5

greater than the ones performed for well-variant method (i.e.

250 mg/mL for extracts and fractions and 50 mg/mL for pure

substances); 7 mm filter paper discs (Whatman, no. 3) are

impregnated with 20 mL of each of the different dilutions. The

discs are then allowed to remain at room temperature until complete

diluent evaporation and kept under refrigeration until ready to

be used. Discs loaded with natural products is placed onto

the surface of the agar. Commercial chloramphenicol discs (30

mg) and paper discs impregnated with 20 mL of diluents could be used to

dilute natural products used as control.

I have used discs with blended raw plant juices, or teas brewed using dried plant materials, (but never alcohol extracts) to evaluate and discover many plants active against MRSA, acne, and also found a few useful against DRSP and one that kills VRE, that you can see at http://www.ecoseeds.com/mrsa.html.  

I would not be concerned as much about the amount of material on the discs themselves, but just keep track of the measurements of the plant material used--was it undiluted expressed plant juices, or was it a tea made from a particular weight of plant material brewed in a particular volume of water, at what temperature, for how much time.  

The comparisons with the antibiotics should come as a second cut, after you find an active plant, extract the active ingredient, and then test a weighted amount on the disc against the chosen antibiotic disc---plus you may need to make your own weighed antibiotic discs also. 

I used the two methods and I suggest that the disc diffusion method is more suitable as explained above by Tresina. Else, it should be considered to determine the Minimal Inihibitrice Concentration (MIC) as it'll be very useful to know how volume you can poor on the paper disc. For the agar well method you should be careful for which kind of agar or media you will use as it can interfere with the extract;

Other than the person asking the question here and myself, are there any other viewers or followers of this question, actually assaying plants for antibiotics, and what pathogens are you looking to kill?  I have been working on MRSA, acne, DRSP and VRE. 

Disk method is the best method to use as the first cut, as you can see at http://www.ecoseeds.com/mrsa.html.  

By assaying plants you can find that there are crude plant juices that are much more potent in killing topical MRSA than any OTC preps. currently on the market, and faster-acting plant juices against other skin problems like acne.    For my crude plant juices, I expect to see no inflammation and healing beginning within 24 hours of application to a skin infection or acne.

How can we make a hall in media agar?

Dear Aseel T. Issa,

It is easy to make holes on agar plates with 1 mL micro pipette tips

Disc diffusion method is better in my point of view, disc paper you can buy it from Himedia ready made.

Regards

Article Antibacterial activity of annona squamosa l. And annona reti...

I love your article, and I agree 100% that the disk method is the easiest and best to screen potential herbal drugs, as you show in the photos of your article.

May I ask how many micro-liter  the well in the agar can hold? 100 , 200 300? thank you

Compare to DD method well diffusion is better. Because most of the people will calculate zone of inhibition around the 6mm paper disk,  what about the 6mm diameter which is covered by disk it self. In the care of Well diffusion method that 6mm is no media and organism. we can measure zone of inhibition clearly. 

For evaluating antibacterial activity of herbal drugs, disc diffusion method was most preferred one than agar well.

Dear Prof.,

The Disk Diffusion Method is the right choice in many ways.

1. While preparing the disks for studies use sterile blank disks available commercially for easiness.Those disks can hold uniform amount of substance. Dry the disks aseptically before use. Reported comparison of sterile commercially available antibiotic disks and disks prepared from Whatman Filterpapers for various antibiotics is available and showed comparable results.

2. Most of the drugs/antibiotics are in solid forms and if you want to mimic the actual environment of application the Disk Diffusion Method might be the best.

Regards,

Abhilash

depending on your extract but the well is best when you want to study the effect of different concentration

Agar well method

Disc diffusion method is better in my point of view,

Thank you for your response but I think Dr. Huda for disc method, you are not sure all quantity is absorb by a dsic.

Disk diffusion method for antibacterial screening of herbal drug extracts are better as per my opinion.

if your extract is hydrophobic then disc diffusion may not perform well better do broth dilution methods. However no standards are developed for extracts and mm of diffusion is resistant, intermediate and susceptible, broth dilution followed by CFU gives good reproducible data, there are limitations with disk diffusion , the type of media used, agar percentage, thickness of the media , solubility, etc... leads to lots of ambiguity.

QUESTION FOR Dr. Sumbul Rehman--

WITH THESE 28 ANSWERS over a 5 year period, which method did you find works best? For the simplicity of use, I am still voting for 6 mm disks, as you can see at http://www.ecoseeds.com/mrsa.html - but suggest using the same ATTC culture of the pathogen, so you have comparable results every time.

By using the disk you can prepare the herbal drug extract material and have it easily stored on the disk for future testing.

Prior to making an extract, all of my testing so far has involved using a blender and have the disk absorb the plant juices as an initial test. If the raw plant juices are very active, then you know that when you produce an extract or purify the active compound, you should have a very powerful material. It is also a quicker method of evaluating a lot of different plants and plant materials, by initially skipping the plant extract phase.

Well diffusion is better than disc in case of crude extraction, it is so difficult to add the crude extraction into paper disc(6mm),

Do we need to dry the disc in an oven at 40°C overnight? or need to soak 2-4 hour, or just can directly used the disc after we dropped the herbal drug extract?

If you use the disc method, you extract the juice and do not need to dry--I used a blender, then a garlic press, and expressed the juice right onto the disc, then put that disc on your petri dish after you have put on your pathogen culture with a loop, and incubate, like what you see at http://www.ecoseeds.com/mrsa.html

If you heat the disc with the juice on it, you might change the active compound.

Agar well diffusion is better than disc diffusion in case of crude extraction of drug,