You seemed doing every thing in wright methods so maybe the final concentration of your your extract in the wells not effective so you need to increase the concentration or it is from the beginning your extract is weak antimicrobial, also u can try agar dilution method add different concentration from ur extract into molten agar and then streak he strain

good luck

Sorry, is not so easy to understand the bacterial growth by a photograph. But it seems that in Petri dishes 1 and 2 (alchol extract and ether extract) you have no halos of inhibition. MRSA strain grew everywhere. In chloroform extract dish the MRSA din't grow or the growth was "difficult". Anyway, the coloured halos are probably to ascribed to curcuma extract. I suggest you to centrifuge the extarctions at 10000 rpm and to control the MRSA strain growth.

I think, the dilution you have is weakly so you have to do a strong one to make it possible, at the moment I am working on that.

if is no possible you cam employ a MIC concentration

Maybe your extracts are not effective? 50 mg/ml seems pretty fair concentration. Your wells seems a little bit overloaded, maybe paper disc (like from HiMed, Mumbai, 6 mm in diameter) will give you clearer picture? You could try as well some solvent that is not so volatile as ethanol, DMSO for example. Best of luck!

Firstly, use standard method for your extraction. Secondly, make sure the dilution tested is not too low.

I suspect the curcuma extract is not diffusing through the agar effectively. I would try using paper discs with different concentrations of the extract. Start with a higher concentration than 50mg/ml and see if you get a dose response.

Thank you every one for your valuable inputs. i will try and getback.

I'm not sure if i understood correctly. You used a borer to create a well in the MHB agar plates? I would suggest you to apply the extracts directly into the paper discs once they will assure a homogeneous diffusion to the agar plate. Please note that, if your compound has e.g. high molecular weight that may limit diffusion.

You might need to consider these points

(a) stage of growth and number of microorganisms (CFU/ml) you are seeding into the agar

(b) as your test antimicrobial is water insoluble its better to add emulsifiers like DMSO or T-80.

(c) Carry out agar-well and disc assay (6 mm) simultaneously

(d) While making wells using borer take care that there is no gap created between bottom of agar and plate. In that case you can seal that with soft agar.

(c) take care to run appropriate controls.

(d) while testing your study extract do not forget to consider the sensitivity of test organisms toward solvent.

You can refer

http://www.sciencedirect.com/science/article/pii/S1049964409000656

Good luck.

Because of the easy growing of your testing strains, I suggest that you perform MICs with broth-dilution method after concentrating your curcuma longa extract.

First of all, you must be sure that your extracts are not contaminated (you can sterilize them by filtration using 0.22 micrometer filters); also, you must be sure that your strains are pure (not contaminated with other bacteria), so a good idea is to prepare the inoculum using a single colony culture. Another cause of your results could be the low volume of extract per disc or a hetero-resistance phenomenon. Better, check the colonies grown inside the inhibition area in order to know if they belong to the same species you tested.

In this cade determination of MIC is more relaiable method than well or even disk diffusion method. You can determine MIC using broth or agar dilution method as recommended by CLSI, published elsewhere (Murray et al, Manual of Clinical Microbiology). Application of DMSO as solvent in well or disk diffusion method also is not recommended because this solvent could trap differebt substances of the samle and so inhibit the diffusion process onto aquos part of the agar.

Yes, it's a good idea to use EUCAST E Def. 3.1. method (agar dilution method).

First of all a very good work: did you mean the zone was irregular or you meant that there war double zone with in: first on e you may see when the well is not cut/or antibiotic disc is not properly prepared: second one is when there is a contamination with a resistant bacteria

You may try the extract without further dilution, or you can test the extract against other microorganisms beside the group you already have in the list. good luck.

In addition to what has been said by all our colleges, some plants (active components) are heat sensitive. You have used 60 °C to concentrate your extracts. usually, 40 °C is recommend. Try to consider this also

Good Luck

HI, My opinions:-

1) Concentration under 60oC seem too high for me. I ussually used 42oC. As we know some compound degrade under high temperature. so i suspect that your compound might have degraded. To test back just use the extract without concentrated and test directly the extract on your test microorganisms. If no activity means it is not active to your microorganisms.

2) 50mg/ml might be to low for some compound. So you might need to increase the concentration a bit. But if you have done the counter check procedure that i mention on (1) and you obtained the inhibition zone then something is wrong with your extraction procedure.

Hope my comments help you.

Agar diffusion tests are poor tests to test extracts as the large molecules of extracts don't usually diffuse well through the agar. Best is to use a broth or agar dilution test. When performing a broth dilution test use tetrazolium violet, added 20 minutes before reading the test, as your growth indicator as many extracts are coloured, making it difficult to see bacterial growth. Another way is to make dilutions of the extract in molten agar cooled to just before it solidifies and then pour plates. I have also used 48 or 96 flat-bottomed wells plates. All you then do is drop 10 microlitre of the bacterial suspension on the solidified agar and culture. The lowest concentration of extract that you see growth is the MIC.

Thank you everyone for your valuable comments. I faced a similar problem with DMSO as mentioned by Atousa aliahmadi. Thanks Mihai Maris for the document.

Dear Rajesh, it is all depends on the concentration (dose) of material used, antimicrobial properties of the of the compound used and also the type of the organism tested. I suggest you to perform the screening test with different concentrations of the material

http://with different ppm i.e lower to higher concentration (eg. 10 ppm-1000ppm), and find out minimum inhibitory concentration (MIC) of the material, I hope you will get an inhibition zone at some concentraion

Jackie picard very correctly said that agar well diffusion is an inappropriate method. U should actually try agar dilution & broth macro or micro dilution method. Also regarding ur pics, it seems that u haven't filter sterilised ur extract as in some cases there is growth of bacillus around the well, so it is must to filter sterilised ur plant extract before putting them into the wells.

In case u have mixed the culture with the hot agar & then poured the plates, then culture may die off & not grow at all & hence no zone of inhibition. Hence, surface spreading the culture on agar surface is safer option.

Boring uniform & proper well ( with no breaks) is also crucial, as it lead to improper diffusion which can be seen in ur pics.

On more important parameter in such type of study is - prediffusion, after filling wells with plant extract, keep the plates in refrigerator (4 degree) for 15- 20 mins & then transfer them in to incubator for incubation as this give some time for the extract to diffuse from the well & act on the organism.

You can refer book on industrial microbiology by Casida for details on these techniques.

If the test has been done properly, your pictures indicate that there is some inhibitory effect. Once you have sorted out the technical issues, it will be interesting to see how you go with the bioautography.

Your mehtod seems to have a problem. Ultimately the solvent needs to be removed and the extract dried onto a disk. The solvent present will affect the growth of the test organism.

Dear Mr. Rajesh,

Hope you are looking for curcumin and its ABS against some G+ve & G-ve. I have an idea for you, please try to do probe sonicator (pulse field) in ethanol with your fine powdered sample then dehydrate it gradually. Rotary evaporator will be help you to get partially purified dry extract. Better to avoid the temp. 45 degree & above to your extract. In respect to Bacterial culture, please check about purity of the culture. There may be chances for associative lab contamination. For that you can simply identify them by gram's staining. If you want any further help & doubts please feel free to ask. thanks.

Hi think you must concentrate the extract and must use this concentrate in different % dose

Properly cut wells is very significant and the concentration of agar also determines the diffusion of antimicrobial extract equally through the medium

I had some similar problems with agar diffusion in the past. Although I must admit that the method has some drawbacks, I have found it quite useful and rapid and, although it is true that large molecules do not pass easily, it is quite useful if you have substances that are not really soluble to water suspension so as to perform an MIC. Still in some cases a picture like the one that Rajesh has posted has emerged and the problem was solved by just one or two really simple steps.

First of all the petri dishes should be really dry (but not stale) when making the holes. After adding the inocullum in the surface you should put the dishes in an incubator so as to really dry the surface of the agar for approximately 15 - 30 min. For making the wells I have personally used the back of a glass Pasteur pipette and not a corkboarer because the last produced quite harsh edges in the well.

Finally it is almost certain that you have some kind of contamination - the last petri dish shows a quite distinct colony which is not close to a well. But I do not think that this could be attributed to your initial solvents (I find acetone, ethanol etc quite strong disinfectants by themselves), but to a later stage of extract preparation. Perhaps the final solvent that you use for the dissolution of the dried extract or even the glassware that you use for this. I would recommend the revision of every step so as to see what is going wrong.

Finally you have also the possibility to produce some paper discs impregnated with your extract so as to check the production of the zones.THe only drawback is that a 6mm paper disc could hold up to 10 μl of suspension. But if you dry your extracts then this is not a problem.

Why you have taken the supernatant of the extract after centrifuge? Is their any particular reason

as far as I think you have got the extract by Cold percolation already when you have soaked the drug in solvent overnight and on shaker.

You have proceeded with centrifugation of the filtrate and dried it and continued with its antibacterial screening ?

I would like to know the reason, please

Rajesh

Please let us know what worked best for you.

Please check your extract concentration do basic turbidity test and do spec analysis with and without extract. Increase the concentration of extract by rotovac and then do it.

thanks everybody for your valuable suggetions. I will get back to you with the results after sometime

Check the strain purity and sterilize the extracts before use. You can perform it by using a Millipore filter with 0.22 micrometer pores.

Hai Mr. Rajesh

my suggestion is during evaporation you used 60 °C, this temperature may be denature the components (curcumin). because they are protenacious component, similar kind of problem i faced during bacteriocin prodction form lactobacillus at that time i have used normal temperature for centrifugation i cant get the result but when i use cooling centrifuge (4°C) i got a good results..

You may try soft agar bioassay, also you need to do control for the solvent you use in dissolving dried extract

I think you must increase the conc. of extract.

curcuma longa is not soluble in water ..so it is not diffusing into the media

You can try organic solvents Such as

Methanol (lower boiling point) or ethanol (somewhat higher boiling point), pure acetone or acetone/water mixtures.

There are some extracts can not diffuse through the agar. Therefore this test is not reliable

This depends on the type of solvent used to prepare the plant extract and its polarity and its ability to withdraw the active compounds as well as the concentrations used in the inhibition of bacteria and the type of microorganism

Use antibiotic sensitive bacteria and try to test your extract using broth dilution method because the agar tests are affected by many factors.

use bilayer agar method. I tried it before with antifungal activity of lactic acid bacteria

May be the extract concentration needs to be increased

Even though curcuma longa is considered to have antibiotic properties it is not an antimicrobial and therefore has little or no effect on a bacterial culture growing in a nutrient agar, which means that a zone of inhibition test will not work. Did you try a serial dilution with a contaminated broth and then plating.