Hello Syed, you need to experiment with different ratios and measuring the inhibition zones that appear. Furthermore, it is important to prepare disks with solvent only to check that itself has no antimicrobial activity. I also recommend doing MICs, because you would relate concentrations with inhibition zones and determine the criteria of sensitivity, resistance and intermediate resistance.

We normally initially use the plant water extract in the following dilutions 100%, 50%, 25% and 12.5% to give us a look see. Most of the plants we use are used in the community neat, so initially we try to mimic this. If the extract can be squeezed out of the plant without using water the better. The discs are then soaked in the extract, air dried and used on the plate.

Hellow Syed, As others suggested you may test with different concentrations. For me I suggest you prepare a stock solution of 500 mg/ml and then pipette 10 micro and load the disc (this will give 5 mg/disc . leave the solvent to evaporate if possible overnight. then test for activity. do not forget to include the solvent soaked disc as the negative control. I also suggest you find the MIC.

Hi Syed. As Denis said, you could prepare disks with defined content of the extracts. You should use a high purity volatile solvents which could dissolve whole extract. Don't use DMSO! Because it trap many of the probable antibacterial compounds and don't allow them being diffused in the aquas environment of Agar based plates. However I prefer determination of MIC by using broth micro-dilution assay recommended by CLSI with some modifications regarding the concentration range of mg/ml instead of microgram/ml (which is suitable for current antibiotics!).

Dear syed, disc diffussion have the problem of diffussion of several compounds in the agar nonpolar compounds can not diffuse in antimicrobial activity. I recommend you use a broth dilution method for evaluate this extract, you can use fluorescent dyes as alamar blue for avoid turbidity troubles. With best regards

Hello Mr.Syed, it is a good question and a great query in mind, when we used to start our work on this. Actually I am saying this as I have passed through the same situation. I have scrrened antimicrobial activity of some herbal drugs, I was wondering that how a 6 mm disc can soak to an amount of 5/ 25/50/ or 100 mg and more amount of drug.But now as I have now passed through that I think I can be of some help to you.

Ok Mr.Syed

1.The drug extract :if you are taking 25 mg of drug you must take 200 ml of the solvent, you can have the extract either by Cold percolation method (dissolve the drug in the said amount and left it unstirred for 24 hrs and than filter it or by Magnetic Stirrer i.e dissolve the drug and stirr it on the shaker for 6 consecutive hours and than filter it OR by hot percolation method (use Soxhlet apparatus or just reflux it) and filter the extract and than dry it and measure the extractive value.

2.For concentration of the herbal extract: As initially we are going to screen it for the activity, we never know that how much amount of our drug is going to be effective, so we should take atleast four or five different concentration like 5 microgm/ml than 10 25 50 100microgm/ml.

3.if you are using Kirby Bauer's disk diffusion method than take the sterile disk from himedia or what ever u like or u can just have the paper puncting machine and take out the disk from whatmann filter paper, autoclave it and than use it. Just take the required amount of drug and make it to absorb on d disk first let ot soakk the amount than again drop the next till it reaches saturation and go on.

4. I would suggest you that you should use Agar well diffusion method that is easy, you just dig the well in the prepared plate and than just fill the whole well by slight amount of molted agar to fill up the base and than pour the required amount of drug in the well.

5.So, in this way you can use all four or five well in the plate with same drug and different concentration and in the Centre you can put the Standard disk of the Positive Control of the antibiotic , but please keep in mind that you should always need to make a well for the solvent which you have used in dissolving the drug extract (in this i would suggest you that you should use DMSO (dimethyl sulphoxide) it dissolves easily most of the components and is neutral towards strain.

ANYWAY ALL THE BEST FOR YOUR STUDY.

(If you want any more description, you can mail me at [email protected])

GOOD LUCK

Hello again, considering that each extract is different, what you would do is a series of previous experiments: In principle there are no estandad concentrations to use and you can begin a series of dilutions with undiluted extract, but you always need to determine conditions and concentrations experimentally. Attention, because some very concentrated extracts not diffuse well in the agar. You also have to try several diluents. The obliged are water, ethanol, and DMSO, but then, according the composition of your extract you can include others, raise or lower the pH ..... The result may not be the same with all of them, but you always have to prove the diluent alone to see its influence on the result. Of course, ideally is water, or if you use other with the first dilution, then you can do the rest with water. Prepare the discs in the moment of its use, to prevent degradation of some compounds or some volatile compounds may disappear. Furthermore, with this type of extracts I would do the MICs with constant stirring to encourage contact with the hydrophobic compounds that may contain the extract. This that you want to do is not easy. Courage and patience to adjust conditions.

Please use disc diffusion method and determine the mic value. Don't forget to use standard antibiotic disc for gram positive/negative and fungi as control. Experimental procedures can be obtained from many published papers. I have few papers published using disc diffusion method. I think few listed in my research gate.

HI... if your extract is in the solution form and you are not making the disc, it is better to perform the antimicrobial assay through well diffusion method, rather than disc diffusion method. For the better comparison the it would be good if you can make the same concentration of extract and standard drug. But the problem is extract in general have very low potency than that of standard drug. So you can just make the different concentration of extract to identify the minimum inhibitory concentration at you can make the same concentration of standard drug. Sometime you can make the10:1 of extract and standard drug concentration as well (with reference).

I hope the following will be helpful (if not, please get in touch again): The disc diffusion method (Jorgensen et al., 1999; Joseph et al., 2006) was used to screen for antibacterial activities of the crude extracts against the five bacterial strains. Approximately 9 mL of Müller-Hinton agar (Oxoid, UK) was poured into petri dishes (9 cm in diameter) and inoculated with the respective test organisms. Wells (4 mm) were punched out of the solid agar using pipette tips, and 1 ml of 50 μg/mL of the test extracts or control antibiotic Ciprofloxacin (5 μg/ml) placed in different wells. The petri dishes were incubated at 30 oC for 20 h and the average diameter of the inhibition zones surrounding the wells computed from three sets of replicates.

I do not recommend to perform a direct test on the crude plant extract. I prefer to make first some processing as for example aqueous and organic solvent extract phase separation. Then, you can do a simple Petri plate assay, but to prevent diffusion problems of certain compounds in the agar from the disc you can use the following methods: (1) a drop test (5-10 microliters drops) onto the surface of an agar overlay of the microbial indicator, or (2) the liquid contact test in which you incubate in a test tube given amounts of the extract plus the indicator microorganism in water or buffer and then after a given time you take a sample and plate a drop (drop test)in suitable agar plates. In the later case, if you use low cell concentrations of the indicator microorganism you can plate it directly without serial dilution and this is very easy to do. This contact test gives you an indication of the microbiocidal capacity of the extract.

As it was said by others it is extremely important to test also your solvents for antimicrobial activity. Also, use always you can substrates and growth media with a low capacity to interact with your compounds/extracts. Very often phenolics can react with many media components, even with you antimicrobial components, then masking the effect.

All here are giving the suggestions for your study, have you applied any of them . I would like to hear if u r using disc method that how u r preparing disk from that ?

We screen hundreds and thousands plant extracts (and other extracts) in our High Throughput Screening plateform for antimicrobial activity. You should adapt your solvant to your microbial models and assays. For example, som do not support other solvants than water. Some can support DMSO, etc... Concerning the ratio, it also depend on the source (part of the plant) and antimicrobial assays. Always keep in mind that your process should be easily "scalable".

Thankx all for your suggestion. I have completed my investigation by disc diffusion method, and used cipro for std. First I prepared my extract solution of suitable amount, and dissolved in distilled water. First I cultured the organisms in broth medium and then to agar medium. I got good result. Now I want to know that how can I measure the antimicrobial potency of my extract. I mean what is the calculation method to compare with the std I used.

Hi, to know the antimicrobial activity MICs are need. You can do it in a solid medium as you diluted with water and you can get an homogeneus mixture. As I previously say, then you can perform a regression line with the MIC data and measures of the inhibition zones obtained for different concentrations by agar diffusion, using a reasonable number of bacteria in the calculation.

You can follow the reference below to determine the MIC accordingly the NCCLS procedures:

Barry AL, Thornsberry C 1991. Susceptibility tests: Diffusion Test Procedures. In: Balows A, Hauser WJ, Hermann KL, Isenberg HD, Shamody HJ 1991. Manual of clinical microbiology. 5.ed. Washington, DC: American Society for Microbiology, p. 1117-1125

Dear Moreno, the link you have given is in Portugal language I think. If you have any English version, please let me know.

Dear dr dewan, sorry it shouldn't be a link it was supposed to be a reference we used

in an article from our group, sorry. If you cannot access it send me a messsage I can try to make a copy for you

Yep, Dr. Moreno, I can't access, I tried. If you please manage me a copy, it'll be great pleasure to me.

My suggestion is to make an extract by taking phosphate buffer (pH 7.4) solution and that too by maceration process. Don't forget to see the anti-microbial activity of the solution (here it is Phosphate Buffer (pH 7.4) solution) you are using to extract your plant extract. Then for the rest you can go through any standard book to get the protocol for conducting the anti-microbial activity.

If you want your reults to be publishable in high impact journals, do not use agar diffusion methods. It is impossible to compare the activity of different plant extracts using agar diffusion. By far the most antimicrobial compounds have intermediate polarity or are non polar. This means that these compounds do not diffuse easily in the aqueous agar matrix. You can use agar dilution metods by making a dilution of your extract in different agar petri dishes and then testing the extract. Here the compound does not have to diffuse because it would be in close contact with the microbe. This is still much less sensitive than serial microdilution methods. I will send you the method that has been cited nearly 600 times. [email protected] as well as the best extractant cited nearly 400 times

There will be problem with the microdilution method if the solution of the extract is turbid and coloured

My plant solution is of light greenish color, and not crystal clear.... @Mr. Alli.

if your extract is colored and you decide to perform a broth microdilution method, you can inoculate agar plates with a small amount of culture with each concentration of extract. The MIC / MBC is that in which not grow anything. In where there are growth, you can count the colonies to determine growth reductions

IAM also doing antimicrobial activbity by using plant extract. spread pour plate method

You can also try Kirby-Bauer disk diffusion method for antimicrobial testing.

https://www.researchgate.net/publication/231590198_Anti-Helicobacter_pylori_and_antioxidant_properties_of_Emblica_officinalis_pulp_extract_A_potential_source_for_therapeutic_use_against_gastric_ulcer

Article Anti-Helicobacter pylori and antioxidant properties of Embli...

The Determination of minimum inhibitory concentrations (MICs) of antibacterial agents or other plant extracts by agar dilution, using European Committee for Antimicrobial Susceptibility Testing (EUCAST DEFINITIVE DOCUMENT E.DEF 3.1) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) is a good method to use. You may also use the MIC by broth dillutions, EUCAST, and if your plant extract is not crystal clear and difficult to read the inhibition you may spot a 5-10 uL on non-selective agar from each concentration, after 24 h incubation, in order to determine the bactericidal concentration of your extract.

Dear Sir,

You may use DMSO for preparing your extract for various concentration like 10, 20, 30 microgm/ml and son on......

Mr. Jacobus Eloff I would also like to know about the method which is more appropriate than agar dilution method. I am also working in the same field of screening herbal drug extract for their antibacterial activity.

Mr. Sumbul Rehman. Broth microdilution method is much more accurate and acceptable.

Dear Colleagues

If you use the tetrazolium violet method the colour of the formazan is so intense that you can clearly see the difference with all leaf extracts that we have used and colour of the extracts is o problem. (See ELOFF J N 1998 A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711-714). The problem with precipitates forming in your extracts at different dilutions makes it very difficult to use a microplate reader.

Acetone is less toxic to fungi and bacteria than DMSO and you can recover your sample easily. (See ELOFF JN, PICARD J, MASOKO P (2007) Resistance of animal fungal pathogens to solvents used in bioassays. South African Journal of Botany 73, 667-669.) I repeat if you want your work to be comparable to results of other authors and published in good scientific journals, you must use a serial dilution method. Resurin also works well as a growth indicator.

The agar dilution methods and disk methods work well for antibiotics and for single compounds, but not for plant extracts where there is a mixture of compounds with different polarities.

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. The absence of an inhibition zone in disk diffusion method in our experiment did not necessarily mean the compound was inactive, especially for less polar compounds, which diffuse more slowly into the culture medium.

For quantitative determination of antibacterial activity of plant extracts of phenolic components the broth microdilution method is a rapid and accurate way of testing antimicrobial efficiency for all the tested bacteria. The best evaluation for normal growing bacterial strains we found by colorimetric determination using INT - as suggested by prof. Ellof (with amazing ideas for quantifiaction already in 1998) and it is very much useful and also used in many recent publications.

However, tetrazolium salts are not appropriate for microaerophilic campylobacters since they indicate the respiratory activity. But for microaerophilic species like Campylobacter spp. we used ATP determination by bioluminescence measurement, which works very well (2009, In Vitro Antimicrobial and Antioxidant Activity of Commercial Rosemary Extract Formulations, Journal of Food Protection). This method may be an acceptable alternative for quantitative determination of bacterial susceptibility to plant extracts. When ATP was used as a viability indicator other non-Campylobacter strains also gave results comparable to TTC or INT.

As prof. Eloff mention that also other growth indicators are possible– I would just like to add, that several dyes are good indicators of bacterial growth, but are not all appropriate for plant extract assay - difficulties arising because of autofluorescence, salt reduction and the antioxidant properties of plant products, especially for XTT, TTC and Resazurin make them less suitable indicators for MIC assay (2010, Evaluation of diffusion and dilution methods to determine the antibacterial activity, Journal of Microbiological Methods). Especially the Resazurin is less used for viability determination when we used plant extracts, because of interaction between the extract and the reagents, which can not be compensated.

1. Extract can be taken out by Hot Percolation/ Cold Percolation/ Soxhlet Method.

2. After taking out extract you have to dry or lyophilized your drug extract, so as to take off the solvent used.

3. Now go on for dissolving the drug extract for your Antimicrobial Study. For that as you are comparing it with Standard drug , it should be closer approximate of your Standard. So keep your ratio like that of Standard.

4. If you are not sure of any antimicrobial potential of your test drug extract, you can take 4-5 or more different concentration of your test drug. e.g 5 microgram/disk , 10 microgram than 20 , 40 and so on .

By this you can trace out that is it minimum concentration which is showing good activity or the higher concentration, so in the next part of screening you can take accordingly.

5. Other than this the concentration of test drug which is actually arresting the growth of a microbe can be assessed by MIC and than MBC.

GOOD LUCK

For more query you can write to me on [email protected]

Please i need to know how you solve this problem thanks

Have a look on this article and proceed it

Article Phytochemical Screening and In Vitro Antimicrobial activity ...

Can anyone suggest me the procedure of preliminary screening of antimicrobial activity of crude plant extract on TLC gel

Just prepare few range of desired concentrated solution (Crude Extract + DMSO or any other appropriate solvent except methanol). Then pour the same in different well that you have prepared in Disc. Observe the zone of inhibition...

For more reference you can refer few books like: NK Jain etc...

Can check the manuscript

link.springer.com/content/pdf/10.1007/s11738-012-1120-x.pdf‎

www.ijprd.com/June_12_Issue.html

www.ijcrr.com/journals/Vol%203%20issue%206.pdf‎

There are two aspects to be considered the first is the quantitative screening of antimicrobial activity.  

Quantitative

Here determining the minimum inhibitory concentration is the only method that makes it possible to compare results of different laboratories.  Detecting turbidity works with large volumes, but is dangerous with small volumes because, some compounds may precipitate from the extract/growth medium mixture and confuse the determination of turbidity. To overcome problems with coloured plant extracts a compound has to be added that will change colour reproducibly  to indicate at what concentrations the microbial growth was inhibited.  Two compounds rezurin (or derivatives) or tetrazolium violet (NOT TETRAZOLIUM RED) can be used. My paper JN Eloff 1998 Planta Medica 64, 711-714 has been used widely  (cited 933 times).

Qualitative

To determine how many biologically active compounds are separated by TLC, biography works well.  You need volatile TLC solvents.  After development the solvents have to be removed  in a stream of air.  The chromatogram is then sprayed with a high density of actively growing bacteria or fungi.  After incubation (time depends on the growth rate of the microorganism) the plates are dried  for a short period and sprayed with tetrazolium violet.  It is then incubated for another period. The white band of inhibition provides information on how many compounds were separated and the Rf values gives information about the polarity and makes it easy to isolate the active compounds. The method works very well with bacteria and we have succeeded with fungi as well. See the open access journal (MASOKO P and ELOFF JN (2006) Bioautography indicates the multiplicity of antifungal compounds from twenty-four southern African Combretum species (Combretaceae). African Journal of Biotechnology 5, 1625-1647).

Hope this helps, Kobus Eloff

I do agree that this method is very efficient. Just keep in mind that you will miss potential synergies/antagonisms

Thank you for this point you are 100% correct.  

One of the frustrations in trying to find compounds that could possibly be used as a single compound antibiotic is that we expect the isolated compound to have say 1000 times higher activity than the crude extract (present in 0.1% of the mass in the crude extract) yet we hardly ever find isolated compounds with an activity higher than 5-10 times that of  the crude extract.  My explanation is that synergistic activities, possibly related to absorption, distribution, metabolism or excretion of the active compound may be influenced by other compounds that are present.  

We now focus on using extracts as therapeutic agents.  For quality control purposes it helps to know the identity of the active compound.  

In any case if you want to publish in good journals or want your PhD students to graduate, identifying active compounds are important.

Kindly. For MIC test of the crude extract using microplate , do you have proper method for this test? I found an article which they take 256 mcL from a 10,000 mg/L stock to prepare 128 mg/L first dilution . How come they reach these calculations?? 

Dear Shumoos

The method we use is  as follows:

We make up a 10 mg/ml solution of he plant extract in acetone.  This is an excellent extractant for plant metabolites and is not toxic to microorganisms  at a 25% concentration.  We place 0.1 ml water in all the wells of a 96 well microplate, add 0.1 ml of the extract.  This will dilute the concentration to 5 mg/ml in the first well.  Then 0.1 ml is taken from the first well and mixed with the water in the second well.  The process is repeated to give a series of concentration from 5.0 to 0.04 mg/ml.  The we add 0.1 ml of an actively growing culture to each well decreasing the concentration series the microorganisms are experiencing to  2,5 to 0,02 mg/ml before incubation overnight or longer depending on he microorganism.

Be careful of measuring growth by determining turbidity because compound in the plant extract may cause precipitates from the growth medium.  We determine growth by adding 0.04  INT (p-iodonitrotetrazolium violet) to each well.  Actively growing cells change the light yellow colour to purple.  The MIC (minimum inhibitory concentration) is the lowest concentration where the colour change was decreased.  This method is used widely has been cited nearly 1200 times.  The publication is 

 ELOFF J N 1998 A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711-714

Good luck with your research

Dear ELOFF J N,

I need a published paper that can show the superiority of broth dilution over agar dilution assay for testing the antimicrobial activity of natural compounds. please send me via email: [email protected]

If you extract is soluble in water, water would be fine for the bioassay.  In more than 95% of our experiments the active compounds were relatively non-polar.  If the active compound does not dissolve in water you will have very low activities.

Another problem with water is that you may extract sugars that serve as a good growth medium for fungi even if your samples are stored in a refrigerator,  

Finally if you want to recover your extract and store in a dry condition where it would be very stable acetone is excellent because it will evaporate very soon in a stream of cold air.

 jacobus can u plz share the link of plants extract method and to check their actifungal activity .thanks 

Thanks Prof. J. N. Eloff, i agreed with you in all the  recommendations. I find it funny when i read journal articles where authors claim that they find MIC of plant extracts using microbroth dilution. I hope those in doubt will take heedof your recommendations.

The paper  (ELOFF, J N 1998 Which extractant should be used for the screening and isolation of antimicrobial components from plants? Journal of Ethnopharmacology 60, 1-8) is available on ResearchGate with the link

https://www.researchgate.net/profile/Jacobus_Eloff/publication/51325813_Which_extractant_should_be_used_for_the_screening_of_antimicrobials_components_from_plants/links/09e41510c0b2e820c0000000.pdf

Article Which extractant should be used for the screening of antimic...

This is a link to the paper 

 ELOFF J N 1998  A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64, 711-714.

This method is used very widely and has been cited 1320 times already

here is a link to the paper

http://s3.amazonaws.com/academia.edu.documents/43994547/serial_dilution.pdf?AWSAccessKeyId=AKIAJ56TQJRTWSMTNPEA&Expires=1471466591&Signature=1LRqa4544MMNd2foQW3l4GiJD4Y%3D&response-content-disposition=inline%3B%20filename%3DA_Sensitive_and_Quick_Microplate_Method.pdf

Prof., i will like to suggest that after preparing the stock solution of the plant extract, the solution should be filtered through sterile micropore filter papers before dispensing into the microplate. Thanks.

Dear Abdulsalaam

Thank you for this very useful suggestion.

In general I think that this is a good idea especially if you use water extracts because these may contain carbohydrates that could encourage microbial growth even if the extract is stored in a refrigerator.

The micropore membranes are also relatively expensive.  .  To get around these problems we use acetone as extractant.  In our experience it is the best extractant to determine antimicrobial activities (See   KOTZE M and ELOFF J N 2002 Extraction of antibacterial compounds from Combretum microphyllum (Combretaceae). South African Journal of Botany 68, 62-67). At 100% acetone no microbes will grow on your extracts.  (At 25% acetone the highest concentration the microbes are subjected to  in our serial dilution method, there is no growth inhibition.)  Extracts can be safely stored in acetone and should have no microbial growth.  I was surprised that acetone can evaporate through different stoppers during storage.( ELOFF J N [2003] The container and stopper used may have important implications for the storage of plant extracts? South African Journal of Botany 69, 603-604).  You should use a stopper with an aluminium foil insert or alternatively mark the level of the acetone extract on the side of the container and fill up to the mark before the analysis.

Because I developed the serial microplate method when we had very little sophisticated apparatus I use a 50% microbial inoculation.  If a number of spores of another microbe landed in the extract after dilution, the effect would be minimal with an inoculum of in the order of 100 to a thousand million cells of the test microbe.  

I am also wary if there is some particulate material in the extract that would later dissolve during incubation, filtering through a micropore membrane may remove the particulate material.

I hope this would explain why we do not filter sterilize our extracts.

hi can u plz tell how much DMSO is require to dilute plants extracts,and how to make ppm solution ,in our lab we use disc diffusion method to check antifungal activity but in some cases fungus grow in filter paper and extracts show no antifungal activities. thankx :)

Start with 10% DMSO 

We investigated the concentration of different solvents that would inhibit the growth of five different fungal species. 

ELOFF JN, PICARD J, MASOKO P (2007) Resistance of animal fungal pathogens to solvents used in bioassays. South African Journal of Botany 73, 667-669

Based on these results fungi can withstand 45% DMSO, 51% acetone, 30% ethanol and 32% methanol.  Concentrations higher than these would inhibit fungal growth.  The bad thing about DMSO is that it is practically impossible to recover you sample after the bioassay becasue it is so difficult to remove the DMSO.  Acetone would evaporate quickly at room temperature under a stream of cold air.  This is why we prefer acetone.

Hope this helps.

I want to determine MIC for my crude actinobacterial secondary metabolite. Please suggest how do I make different concentrations. I have a total of 70 mg of crude..

Dear Alam,

You have to prepare dilutions. But first, you have to (Step1) prepare a solution (SM) for Ci = 1mg/ml or 5mg/mL according to the weight of your extract (Goal is to take the smallest possible volume from SM and keep in darkness at 4°C). Think to Water or DMSO for a totally solubility.

Step2/ calculate for a choicest high concentration for test (i.e. Cf=100µg/mL or 200µg/mL) by simple formula Ci.Vi = Cf.Vf (you search Vi / Ci for SM / Vf = Total of V(bacteria medium+indicator solution+Vi)

Example: Vi = [Cf(100µg/mL=0.1mg/mL)xVf(2mL)]/Ci(1mg/mL) = 0.2mL = 200µL

So, Vf = 0.8mL (O/N Incubated Liquid Bacteria Medium) + 0.2mL (Vi your extract solution) + 1mL (indicator) = 2mL

Step3/ and than you can prepare a serial dilution: 100µg/mL - 50µg/mL - 25µg/mL - 12.5µg/mL ...etc. For preparing that you can use this

Step3.1/ Prepare serial tube (1, 2, 3.... z) with 1mL of Liquid Bacteria Medium to facilitate dilutions (work aseptically !)

Step3.2/

- Tube1: calculating for Cf(SM)=200µg/mL in Vf= 2mL (only Incubated Liquid Bacteria Medium(ILBM)) / Attention: Vf = V(ILBM)+Vi(calculated) / And you will add 1mL of indicator (so it's another dillution to have 100µg/mL for that we calculate for 200µg/mL)

- Mix well and take 1mL from Tube1 to another tube (2) with 1mL of Liquid Bacteria Medium to have a dilution (100µg/mL) in tube2

- Mix well and take 1mL from Tube2 to another tube (3) with 1mL of Liquid Bacteria Medium to have a secon dilution (50µg/mL) in tube3... and repeat the same process to Tube(z)

- You will have 1mL in each test-tube (containing ILBM + Extract dilution). Than add 1mL of colored indicator in each tube (So all concentration will devided by 2 and you will have your dillutions).

Step4/ Incubate O/N at 37°C and learn the results = MIC (color change).

Good Luck !

Thanks for all the tips! This was extremely helpful

I am also to try evaluate plant extract as antimicrobial  in PDA media but very difficult to know because some microbe disperse  so very difficult to know the inhibition. Thank for Mr Yacobus for sharing and comment

Extract the dried plant powder with different solvents (1:10 w/v), filter, take the supernatant, dry by rotary evaporator, suspend each dried extract in DMSO in different concentration, and test the antibacterial effect on plates

Dear Ali

I am not sure what you mean by testing the antibacterial effect on plates. If you mean using an agar diffusion assay this will not be accepted by good journals.

Agar diffusion studies do not work well for plant extracts because the active compounds are not polar enough to diffuse easily in the aquaeous agar matrix.

The problem with DMSO is that you cannot get rid of it after the assay. Acetone is less toxic to fungi than DMSO and is very easy to work with. DMSO is also a teriible solvent if you want to do TLC or bioautography of your extrac becasue it is not nearly as volatile as acetone.

I have been invited by the Executive Editor of BMC Complemenary and Alternative Medicine to write a paper to help authors to do research on plant extracts that will be publishable in good journals.

This paper is freely available

ELOFF, JN 2019 Avoiding pitfalls in determining antimicrobial activity of plant extracts and publishing the results. BMC Complementary and Alternative Medicine (2019) 19:106 https://doi.org/10.1186/s12906-019-2519-3.

Thanks for all the tips! This was extremely helpful

Glad that it is useful. Good luck with your work.

Jacobus Nicolaas Eloff Thanks for the update. Can you please send me the protocol? [email protected] Thanks

Jacobus Nicolaas Eloff your guidance is very insightful. However, I have a few concerns. I am trying to compare the antimicrobial efficacy of a herbal formulation (extract) vs synthetic antimicrobials in the management of bovine mastitis in vitro. If I use agar diffusion for the antimicrobial efficacy (as I want to quantitatively analyse the data from the zones of inhibition) and then use the agar dilution for MIC determination, will my findings be publishable in a good journal? Is there any way to quantitatively analyse the agar dilution results. Could you also kindly send me the protocol? [email protected] Thanks