Diffusion methods are attractive because of their simplicity and low cost, but they are, like all agar-based methods, labour and timeintensive. On the basis of the higher MIC values obtained when the disk diffusion method was used in our experiments, it appears that this diffusion method could not always be a reliable method for screening the antimicrobial activity of plant extracts. The absence of an inhibition zone did not necessarily mean the compound was inactive, especially for less polar compounds, which diffuse more slowly into the culture medium. The diffusion assay is not suited to natural antimicrobial compounds that are scarcely soluble or insoluble in water and thus their hydrophobic nature prevents uniform diffusion through the agar media. For quantitative determination of antibacterial activity the agar dilution method is more appropriate, where antibacterial activity of plant extracts was shown at lower concentrations compared to the disk diffusion method. The agar dilution and broth microdilution methods produced comparable results and a good level of agreement only for gram-positive bacteria. Our evaluation included colorimetric determination using TTC or INT, and ATP determination by bioluminescence measurement. Our results showed that the broth microdilution method with ATP measurement is a rapid and accurate way of testing antimicrobial efficiency for all the tested bacteria, including campylobacters. As the microdilution method by TTC or INT produced comparable results and cost may restrict the use of ATP indicator, we suggest using INT for quantitative antimicrobial determination for normal growing bacterial strains and ATP for microaerophilic species like Campylobacter spp. This method may be an acceptable alternative for quantitative determination of bacterial susceptibility to plant extracts.

For more detail information please read the manuscript: Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts (Journal of Microbiological Methods 81 (2010) 121–126) or contact me directly.

Anja Klančnik

For my was always teh higest concentrations normally starting at 100-500ppm

Disk diffusion assay is dependent on both the potential antimicrobial potency and the diffusing ability of the components of the extract. If the extract is rich of antimicrobials which do not diffuse well in the polar agar media, then it might read as a negative results or poor activity due to the low diameter of inhibition this is why micro or macro dilution assay is important. Thus it is not surprising to get non-correlating results. The attached pdf might shed some more light on the subject. where You've stated that you got activity with the disk diffusion assay at only three different conc, I wonder if these conc were higher than the MIC recorded or lower.

Dear Ouma, the concentration that shows positive results for disk diffusion is higher or same as in mic. means that if i have 6 concentration of plant extract, (0, 50, 100, 150, 200, 250, 500).. if only 200, 250 and 500 ppm of plant extract shows zone of inhibition, but for MIC, a lower concentration of plant extracts had also shows inhibition activity .

Plant extracts are poly -compounds in nature (with many active inactive phyto-compounds like gum resins etc. When you compare MIC values with diluted extracts and disc (with certain concentrations absorbed on discs) the diffusability of active compounds from the discs are minimized than pure extracts used as such hence variability in MIC values. It is always better to try discs only after certain confirmed concentration of extract are finalised (may be if you use pure or semi pure extracts rather crude extracts for disc experiments)

With plant extract the problem of diffusibility through the agar media leads to difference in the antibacterial activity. When we do mic and disc diffusion the difference in antimicrobial activity will be common.

Hello Shazzy, What are the extracts exactly?

Disk diffusion could be easily used to screen the antibacterial activity of plant extracts, however results are scarcely comparable with microdilution assay, because of problems related to antimicrobial compounds absorption on disks and to difficult diffusion through the agar. In microdilution tests, microbial cells get more easily in contact with active compounds and therefore results are usually better than in disk diffusion assays.

Depends on the methodology that you are using from one to another.

What methodology do you use? When I do, both give the same result. When this happens I make the triplicate again. Because the methodology for the disk diffusion can go wrong time to time.

the extracts are crude extracts of plant callus. Dear Patricia, the method means? i use the disk diffusion (kirby bauer) and the microdilution for MIC

Dilution method for antibacterial testing to calculate MIC (de Paiva et al, 2003)

1. Label the Mueller –Hinton broth tubes with name, date of inoculation, experiment number, and

name of bacterium.

2. The microdilution was performed in 96-well microtitre plates with U-shaped wells, label with

name, date of inoculation, experiment number, and name of bacterium.

3. Add 0.1 ml (100 μL) of sterile H20 in each well in rows A, C, E, and G.

4. Add 0.1 ml (100 μL) of sterile H20 in 2 wells in row H for control (Total wells=50).

5. A two-fold dilution reduces the concentration of a solution by a factor of two that reduces the

original concentration by one half. A series of two-fold dilutions is described as two-fold serial

dilutions. Use the micropipette to dispense 100 μL of test material (cinnamon, thyme, bacitracin)

to the first well and mix. This is the first two-fold dilution. Use the micropipette with the same

tip to carry out a second two-fold dilution. Continue the series of two-fold dilutions until the last

well of the microwell plate. Discard the quantity in the micropipette from this well.

6. Add 100 μL of cinnamon in row A in each of 12 wells by two fold serial dilution. Add 100 μL of

thyme extract in row C in each of 12 wells by two fold serial dilution as explained as above in #5.

7. Add 100 μL of Bacitracin n each of 12 wells in E row by two fold serial dilution as explained as

above in # 5.

8. Add 100 μL of tetracycline in each of 12 wells in G row, by two fold serial dilution as explained

as above in # 5.

9. Add 100 μL of 0.5 McFarland bacterial suspension in respective wells and also in control wells.

The bacterial suspension was prepared in T-soy broth .

10. The plates were sealed, placed in plastic bags and incubated at 370C for 24 h.

11. Measure the MIC which is defined as the lowest concentration of extract that exhibited no growth

by visual reading. It is expressed in mg or μg/ml.

12. Discard the plates by appropriate procedure

Disc diffusion method for antibacterial activity (Mukherjee et al., 1995a, b)

This method was used to assay the plant extracts for antimicrobial activity. The procedure, as

explained in detail in Appendix B was followed. In brief, the test quantity of specific extract as shown in

table 3 was dissolved in either distilled water or tween-80, depending upon the solubility of the extract.

In order to detect potential antimicrobial activity in the plant extracts, paper discs (diameter 12 mm) were

soaked in an extract solution containing different concentration as mentioned in table 3. The plant species

and type of extract tested are shown in Table 1, while the bacteria are listed in Table 2. Entire surface of

agar plate was inoculated with the culture of bacteria. The paper discs soaked in each of the test solutions

containing different extract solutions at varying concentrations, as well as the standard drug solution (an

antibiotic which is used as a feed additive) and the control-blank (sterile water discs or sterile tween 80

discs) were placed separately in each quarter of the plate under aseptic conditions. Multiple plates were

(four replications) done for each of the extract was done. The plates were then maintained at room

temperature for 2 h allowing for diffusion of the solution. All plates were then incubated at 370C for 24 h

and the zones of inhibition were subsequently measured in mm (Mukherjee et al., 1995a, b).

Diffusion methods are attractive because of their simplicity and low cost, but they are, like all agar-based methods, labour and timeintensive. On the basis of the higher MIC values obtained when the disk diffusion method was used in our experiments, it appears that this diffusion method could not always be a reliable method for screening the antimicrobial activity of plant extracts. The absence of an inhibition zone did not necessarily mean the compound was inactive, especially for less polar compounds, which diffuse more slowly into the culture medium. The diffusion assay is not suited to natural antimicrobial compounds that are scarcely soluble or insoluble in water and thus their hydrophobic nature prevents uniform diffusion through the agar media. For quantitative determination of antibacterial activity the agar dilution method is more appropriate, where antibacterial activity of plant extracts was shown at lower concentrations compared to the disk diffusion method. The agar dilution and broth microdilution methods produced comparable results and a good level of agreement only for gram-positive bacteria. Our evaluation included colorimetric determination using TTC or INT, and ATP determination by bioluminescence measurement. Our results showed that the broth microdilution method with ATP measurement is a rapid and accurate way of testing antimicrobial efficiency for all the tested bacteria, including campylobacters. As the microdilution method by TTC or INT produced comparable results and cost may restrict the use of ATP indicator, we suggest using INT for quantitative antimicrobial determination for normal growing bacterial strains and ATP for microaerophilic species like Campylobacter spp. This method may be an acceptable alternative for quantitative determination of bacterial susceptibility to plant extracts.

For more detail information please read the manuscript: Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts (Journal of Microbiological Methods 81 (2010) 121–126) or contact me directly.

Anja Klančnik

Do ensure you have the appropriate controls. Thickness of Gel, constant incubator temperature. Having an idea of the constituent of your plant extract will help determine how easily the active compound diffuses or how volatile and for that matter how much of these ingredients you lose whilst incubating. Other factors such as disc preparation and type should be considered. In the end for comparability majority of factors must be standardised. And finally, you could compare your extracts with that of commercial antibiotics in both ways i.e. MIC and disc diffusion. The results should go a long way in helping answer some of these disparities

These methods are no doubt differ in their sensitivity but not in their specificity, thus in my view you can adopt your own methods depending upon available infrastructure in your lab.

About different results obtained due to the use of different tipes of assays or methodologies, please consult Wayne PA. 2000. NCCLS (National Comitee for Clinical Laboratory Standards): Methods for antimicrobial susceptibility tests for bacteria that grow aerobically. Aproved Guideline (M7-A5), vol. 20 (2). or CushnieT and Lamb A. Antimicrobial activity of flavonoids. International J. of Antimicrobial Agents 26 (2005) 343-356. Good Luck! Maria

Isothermal microcalorimetry (IMC) provides a unique means for capturing the dynamic activity of any anti-microbial agent--including plant extracts. A specific paper on IMC assessment of antimicrobial activity of plant extracts is attached. For a general introduction to IMC,

see https://en.wikipedia.org/wiki/Isothermal_microcalorimetry

Thanks alot everyone

We did some work on plant essential oil and found that increasing the solubility of the essential oil can improve the antimicrobial activity. For your reference, please refer to:

http://onlinelibrary.wiley.com/doi/10.1111/lam.12082/abstract

Initial MIC/MLC investigation:

http://www.ijaaonline.com/article/S0924-8579(10)00476-0/abstract

Dear imran. Can u tell me the protocol. Thank you

hi Shazzy instated of disc diffusion method u can try hole–plate diffusion method. Where as in disc diffusion method solvent also plays major role.