Out of 50 mg/ml stock solution of plant extract, 20 μl is taken which is equivalent to 1000 μg/ml and transfer to 5 ml of broth (first tube). Other tubes were filled with 4 ml of broth and 1 ml is taken from the first tube and transferred to the second tube. Likewise the dilutions were done for concentrations 500 μg/ml, 250 μg/ml , 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml and 8 μg/ml. Each tube after dilution is inoculated with 20 μl of bacteria with turbidity of 0.5 McFarland standard.
Finally the tubes were left for 24 hours in the incubator.
Hi! Mac Dz.
The procedures as you have stated, I doubt if you can achieve the desired working concentrations. This is because, the concentrations are apparently products of two-fold dilutions, and the serial dilutions as indicated are wrong. Adding 20μl of 50mg/ml into 5 ml broth, resuits in 0.199mg/ml (approximately 200μg/ml). Subsequent dilution of this concentration by addition of 1ml in 4ml broth, reduces the concentration by 5 (i.e approximately 40μg/ml). Thus the subsequent dilutions are 5-fold such as 40μg/ml, 8μg/ml, 1.6μg/ml, etc.
N.B: The final working concentrations also changes with the addition of the 0.5 Macfarland inoculum.
Incubation period also varies, depending on the organism(s).
The microdilution technique using a microplate photometer to determine the minimal inhibitory concentration of plant extracts.
Hi! Mac Dz.
The procedures as you have stated, I doubt if you can achieve the desired working concentrations. This is because, the concentrations are apparently products of two-fold dilutions, and the serial dilutions as indicated are wrong. Adding 20μl of 50mg/ml into 5 ml broth, resuits in 0.199mg/ml (approximately 200μg/ml). Subsequent dilution of this concentration by addition of 1ml in 4ml broth, reduces the concentration by 5 (i.e approximately 40μg/ml). Thus the subsequent dilutions are 5-fold such as 40μg/ml, 8μg/ml, 1.6μg/ml, etc.
N.B: The final working concentrations also changes with the addition of the 0.5 Macfarland inoculum.
Incubation period also varies, depending on the organism(s).
In addition to the above information, you need to have similar concentrations of your plant extract in broth without the test microbes (Positive control for extract); broth inoculated with the test microbes (Positive control for test microbes), and broth (negative control). These can be done in 1.5 ml Eppendorf tubes.
But it is better to use microplate photometer to determine the MIC of plant extracts, as suggested by Abdulridha Taha Sarhan .
@Mac Dz
Yes, I can help. Since your plant extract stock concentration is 50mg/ml which is equivalent to 50,000μg/ml. To dilute it to 1000 μg/ml, withdraw 20μl from the stock and transfer into sterile test tube, and make it up with 980μl of diluent i.e RPMI 1640 medium (or any of other broth medium for the test). Then dilute 1000μg/ml to 640μg/ml by withdrawing 640μl and make it up with 360μl of the diluent. Fill a set of sterile test tubes with 100μl of sterile broth medium each, and into the first tube, transfer 100μl of the 640μg/ml extract, take the same volume from the frst tube again into the next and continue serially. The concentrations from the first tube now become 320μg/ml, 160μg/ml, 80μg/ml, 40 μg/ml, etc.
Now, for broth macrodilution methods, the extract concentrations should be 10× the final concentration. The 0.5 Mc Farland standard inoculum will be further diluted first to 1:100, and then adjusted to 1: 20. Pipette 900μl of the
inoculum suspension into each of the test tubes containing the 100μl extract dilutions. This results in the final concentrations of 32μg/ml, 16μg/ml, 8μg/ml, 4μg/ml, etc.
This would appear fine.
However, I would plates out each dilution on to PCA or YEA, using the Miles and Misra (1938) technique:
https://en.wikipedia.org/wiki/Miles_and_Misra_method
This allows a much better visualisation of the number of organisms per given volume.
Paul Chidebelu
Thank you for your explanation.
Since 980μl is a very small amount, it is not enough to visualize the growth of bacteria as I have tried before in the macrobroth dilution test. However, 5 ml seems sufficient. Is there any way to make it up to 5 ml & apply the method you have suggested?
Well, 980μL is, near as dammit, 1mL, for a start.
Apart form tat, I don't know what you mean.
@Mac Dz
Yes, you can always scale up the volume of your working concentration. For instance, if you are targeting 5ml, multiply both proportions by 5 and maintain the scale to the last step. So, in the first step, withdraw at least 0.1ml and dilute with 4.9ml. Also, in the last step make sure the inoculum was also scaled up to 5 times the volume I stated in the earlier explanations (before this response), so that you have 4.5ml inoculum suspended into tubes containing 0.5ml diluted extracts, and the volume of the final solution now equals 5ml (without affecting the concentration because you maintained 5ml scale throughout the experiment from the first step to the last step).
Paul Chidebelu
Sounds like a good method. So I should do as follows?
1.Take 0.1 ml of extract from 10 mg/ml stock solution where the fraction is equivalent to 1000 μg/ml and mix it with 4.9 ml of broth (first tube).
2. Then take 0.5 ml from the first tube and dilute it with the second tube having 4.5 ml of broth to top-up to 5 ml.
3. Likewise continue till the last tube..
How about the bacterial inoculum? is that also 0.1 ml from 0.5 McFarland standard.?
Thanks.
“Out of 50 mg/ml stock solution of plant extract, 20 μl is taken which is equivalent to 1000 μg/ml and transfer to 5 ml of broth (first tube). ”It's "1000 μ g" instead of "1000 μ g / ml".
Yes it's very effective method for studying the susceptibility of a no pf microbes against different bacteria ...
I can suggest another better way which will cause reduction in loss of plant extract during such experiments.. which is spot on lawn prick test.
As Paul Chidebelu and others already stated, you can't obtain your desired concentration of 1000 μg/ml with the calculation you did. Next time that you want a fixed concentation in a fixed volume starting from a known stock solution, try this formula:
C1·V1 = C2·V2
Where: C1, C2 = concentrations in solutions 1 and 2; V1, V2 = volumes of solutions 1 and 2.
In your case, as you want a final concentration of 1000 μg/ml, or 1 mg/ml (C2) in a final volume of 5 mL (V2) and you start from a stock of 50 mg/ml (C1) you just have to get V1 to see which volume of the stock solution you have to take in order to achieve your desired concentration:
V1 = (C2·V2) / C1 = (1 mg/ml · 5 ml) / 50 mg/ml = 0.1 ml = 100 μl
You should pipette 100 μl of stock solution.
Mac Dz your stock has a concentration of 50 mg/ml, or which is the same, 50 μg/μl. It means that for each microliter of your solution, you have 50 micrograms of plant extract.
Now, take 100 μl of your stock solution. If you have 50 micrograms in each microliter, in 100 microliters you will have 100 times more, which is 5000 micrograms. But let me hihglight: 5000 micrograms in total, not 5000 μg/μl. When you take a single volume from a concentrated stock, the concentration is the same as long as you don't dilute it. You have taken 5000 μg in a volume of 100 μl, if you divide it means you still have a concentration of 50 μg/μl in your pipette tip.
Lastly, if you take that volume of 100 μl containing 5000 μg to a higher volume (5 mL, or 5000 μl), it will be diluted, and the concentration will decrease. Now you have your 5000 μg in 5000 μl, which is 1 mg/ml.
I hope it helps, don't hesitate to ask more questions if it's not clear enough.
Jesús Perales-Adán
I see! so that 5000 μg gets diluted in 5 ml (5000 μl) then it becomes 1000 μg/ml is that it? Excellent answer!
So after that I can further dilute it by taking 0.5 ml fractions from that as Paul Chidebelu suggested.
How about the bacterial inoculum is it also 100 μl from 0.5 McFarland?
Thanks! :)
Mac Dz
I've always done the microdilution assay to test antimicrobial susceptibility, so my answer may not fit your requirements. I resuspend bacterial cultures in PBS to 0.5 McFarland standard (1.5 x 10^8) and then further dilute them to 10^5 UFC/mL. In each well of the 96-w plate I put 100 μl of diluted bacteria and 100 μl of culture medium containing 2X antimicrobial compound (it is 2X because you're going to dilute it to half when you mix it with the bacteria, so if you want to test a 10 μg/ml concentration for instance, you have to put 20 μg/ml).
You can search CLSI (Clinical and Laboratory Standard Institute) guidelines for standardized protocols in antimicrobial susceptibility testing.
I agree absolutely with Dr. Paul, there is no need for this series of dilutions, you can use the well known dilution procedure.
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