I am doing MIC experiments for Recombinant BCG and R.mycobacterium Smegmtis.
How to dilute 20ug/ml of Rifampicin stock solution into 6.26ug/ml ,3.12ug/ml,1.6ug/ml,0.8ug/ml,0.4ug/ml,0.2ug/ml,0.1ug/ml,0.05ug/ml ?
Dear Nadeem Ullah
Supposing you want to make 5ml of 6.26ug/ml from 20ug/ml, you can use the dilution formula:
C1 = 20 ug/ml
V1 = ?
C2 = 6.26 ug/ml
V2 = 5 ml
C1 x V1 = C2 x V2
Substituting gives, V1 = 1.57 ml
This means, you should take 1.57 ml of the stock 20 ug/ml and make up to 5 ml with solvent which gives you 6.26 ug/ml. You may wish to apply same procedure for all.
Regards,
Egbuna, C.
Please read the methodology section of research URL mentioned below:
https://www.researchgate.net/publication/323303107_In-vitro_antimicrobial_activity_of_different_toothpastes
Hope it helps.
How to Serial dilute 20mg/ml of Rifampicin stock solution into 6.26ug/ml ,3.12ug/ml,1.6ug/ml,0.8ug/ml,0.4ug/ml,0.2ug/ml,0.1ug/ml,0.05ug/ml in 96 well plate Chukwuebuka Egbuna
Dear Nadeem Ullah please see the actual concentrations you will get from serial dilution: 6.26ug/ml, 3.13ug/ml, 1.57ug/ml, 0.79ug/ml, 0.40ug/ml, 0.20ug/ml, 0.10ug/ml, 0.05ug/ml
Step 1. As described above.
Supposing you want to make 5ml of 6.26ug/ml from 20ug/ml, you can use the dilution formula:
C1 = 20 ug/ml
V1 = ?
C2 = 6.26 ug/ml
V2 = 5 ml
C1 x V1 = C2 x V2
Substituting gives, V1 = 1.57 ml
This means, you should take 1.57 ml of the stock 20 ug/ml and make up to 5 ml with solvent which gives you 6.26 ug/ml.
Step 2. Serial dilution. starting now with the prepared concentration of 6.26ug/ml
1. For example, take any amount e.g. 5 ml of 6.26ug/ml sol and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 3.13ug/ml).
2. From it (3.13ug/ml sol), take 5 ml and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 1.57ug/ml.
3. From it (1.57ug/ml sol), take 5 ml and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 0.79ug/ml.
4. From it (0.79ug/ml sol), take 5 ml and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 0.40ug/ml.
5.From it (0.40ug/ml sol), take 5 ml and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 0.20ug/ml.
6. From it (0.20ug/ml sol), take 5 ml and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 0.10ug/ml.
7. From it (0.10ug/ml sol), take 5 ml and dilute with equal vol of solvent (5ml). This gives you a solution with the concentration of 0.05ug/ml.
Hope this helps? Our forthcoming book on phytochemistry, provided some guide on reagent preparation and methods of analysis.
https://www.researchgate.net/project/Collaborative-Book-Project-on-Phytochemistry
http://www.appleacademicpress.com/phytochemistry-3-volume-set-volume-1-fundamentals-modern-techniques-and-applicationsbrvolume-2-pharmacognosy-nanomedicine-and-contemporary-issuesbrvolume-3-marine-sources-industrial-applications-and-recent-advances/9781771887625
Regards,
C.E.
The explanation that @Chukwuebuka Egbuna just gave you also apply on 96 well plate.
Suppose you have already obtain your first concentration of 6.26ug/ml as well describe by @ Chukwuebuka Egbuna, the second step is introduce an equal volum of your medium which may be Müller hilton broth in your 96 well plate. Now take the may be 100 microliter of your 6.26ug/ml and introduce that in the first, when doing that the concentration will move from 6.26ug/ml to 3.13ug/ml.
Explanation: 100 microliter of MHB in the well + 100microliter of rifampicin at concentration of 6.26ug/ml will bring the concentration to 3.13ug/ml.
So your first well will have a concentration of 3.13ug/ml. Next step, take 100microliter in the first well and introduce to the second well just under containing 100microliter of your medium MHB and this second well concentration will move from 3.13ug/ml to 1.57ug/ml.
To summarize this:
1) first well 100microliter of medium (Eg. MHB) + 100microliter of rifampicin at concentration 6.26 = 3.13ug/ml in first well.
2) second well MHB + 100 microliter from the first well at concentration of 3.13ug/ml= 1.57ug/ml.
3) third well MHB + 100 microliter of the second well at concentration 1.57ug/ ml= 0.79ug/ml
You will do the dame dilution till you obtain what you want. Note that eachtime you will take 100microliter from a well thé volume will move from 200microliter to 100microliter un each well.
I hope that my explanation will help you.
Dear Nadeem, I'll try to help you.
The problem 1: actually you need the stock solution of 25 µg/ml for the testing of desired concentrations using a standard 1:1 microdilution MIC procedure in the 96-wells microtiter plates. In this case, if you fill each wells with 50 µl of growth medium suitable for Mycobacterium smegmatis and then add each 50 µl of 25 µg/ml stock solution in the row A, the concentration there will be diluted 1:1 to the 12.5 µg/ml. The following inoculation with 50 µl of bacterial suspension led to the next 1:1 dilution, which is resulted in the row A in the end concentration of 6.25 µg/ml. Thus the stock solution of 25 µg/ml is diluted in the row A to 4 times totally.
But if you have the stock solution of 20 µg/ml, you should dilute this concentration in the row A only to 3.2 times totally. For this aim instead of the 50 µl of 25 µl/ml stock solution you should add 62.5 µl of 20 µg/ml stock solution in wells of the row A, which is at the beginning should be filled with 37.5 µl of the growth medium. As a result you get 100 µl of Rifampicin with the concentration of 12.5 µl/ml (dilution 1.6 time). All next test rows should be filled with each 50 µl of the standard growth medium, because you then can obtain desired antibiotic concentrations in the next rows B to H, using a standard 1:1 microdilution procedure (take off 50 µl from the row A and transfer to the row B, mix, take off 50 µl… et cetera). After inoculation of all test wells from rows A to H with 50 µl of bacterial suspension you will have desired end concentrations of your antibiotic.
The problem 2: Rifampicin is mostly dissolved in methanol or in DMSO. The mixture of the 62.5 µl of the stock solution with the 37.5 µl of growth medium decreases the concentration of all medium components more then 2 times in the row A. To obtain the uniform medium concentration after the addition of your Rifampicin stock solution in organic solvent, the 2.7 times concentrated growth medium should be used for the row A. Standard concentrated growth medium should be used for the following rows B to H.
Please, see: Pharmaceuticals 2016, 9, 56; doi:10.3390/ph9030056
Dear Sir Nadeem Ullah you welcome. But please do remember to dilute 20mg/ml to 20ug/ml. To do this, see the following calculations:
1 ug/ml = 0.001 mg/ml
20ug/ml = 0.02mg/ml
Using dilution formula for the preparation of 100 ml of 20ug/ml (same as 0.02 mg/ml),
C1 = 20mg/ml (stock)
V1 = ?
C2 = 0.02 mg/ml
V2 = 100 ml
This resolves to V1 = 0.1 ml
This means, take 0.1 ml from the stock of 20 mg/ml sol into a 100 ml volumetric flask and make it up to 100 ml mark with solvent to give you the working concentration of 20 ug/ml which is same as 0.02 mg/ml.
For more information, please send me an e-mail to [email protected]
With kind regards,
Egbuna, C.
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