please some one verify my calculations i need to prepare 300mg/L augmentin , i want to prepare 100ml stock so i need to add 0.03g in 100ml dH2o , so how much amount i m suppose to use in 25ml plate, how to find out this so my required conc remains same i.e 300mg/L?
300mg/L is equal to 300ug/ml. what you should do is prepare a concentrated stock of your antibiotic so 100mg/ml then filter sterilize it (this depends on your antibiotic solubility). Then add it to the agar after it cools down but before it solidifies. since your working concentration is 300ug/ml you will need to add 3ul of your 100mg/ml stock per ml of media. Do not make the stock according to your calculation because then you will have a stock that is the same concentration as the working concentration. To make the 100mg/ml stock simply weigh 1g and dilute in 10ml of water then filter sterilize and aliquot to 1ml per aliquot then freeze at -20C and then thaw an aliquot and use it when you need to.
Add 7.5 mg of beta lactum antibiotic to 25 ml of MS. Thank you and good luck.
@ Hanna Alalam: generally you should make an antibiotic stock (eg in water at 1000x final concentration needed) and filter sterilize it, save aliquots by freezing. I understand you want to make agar plates, so you make the agar + medium sterile (maybe with a magnetic stirrer) by autoclaving, cool to temperature (~50C) that you can feel by touch, add the sterile antibiotic and mix with stirrer or by swirling with hand and pour around 0.5-0.6 cm deep media into petri plate or other dish, and allow to cool. This is best done in a laminar floor hood, or near a flame. The plates should be then inverted and stored.
1. Prepare 250 mg.l-1 stock solution by dissolving 250 mg beta lactum antibiotic in distilled water to volume at 100 ml by volumetric flask.
2. Then take 3 ml from stock solution at 250 mg.l-1.
3. Add 3 ml that take from 250 mg.l-1 beta lactum antibiotic to MS medium then complete volume to 25 ml by distill water.
It looks like that β-lactam antibiotics are used to kill a group of bacteria. I am interesting to know why are you adding this chemical to your plant tissue culture? Do your tissue-cultured plants got contaminated??
Yuan-Yeu Yau exactly you are right, beta lactum antibiotics are bactericidal, after co cultivation of plants tissue with agrobacterium the gene of interest is transferred to plants after that we need to kill the agrobacteria as it is of no use after transformation of gene of intrest into plants tissue so for that purpose we use beta lactum antibiotics (carbencillin, timentin, cefotaxime and augmentin etc)
Naseeha, most people or published papers would just use the word 'carbenicillin' or 'cefotaxime' for the agent used to eliminate Agrobacterium.
Try to calculate it using the following equation V1xM1 = V2xM2; V is volume, M is concentration.
(1) If your stock solution (V1) is 300 mg/lt, and you want to make 25 ml medium (V2) having an concentration of 300 mg/lt, then by solving the equation you have to pipet out your antibiotic as much as 25 ml (which is not possible, except you use the 25 ml liquid antibiotic to directly dissolve the solid medium and filter sterilize the liquid medium supplemented with antibiotik). You should not autoclave the mixture.
(2) The better alternative will be making your antibiotic stock more concentrated, i.e. M1 = 300 mg/100 ml, filter steriliized it, and aliquot it in 5 ml stock,. If you want to prepare only 25 ml media, pipet out 2.5 ml added into 25 ml media. Autoclave the media and wiat it until cool as suggested and add your antibiotic into it. Good luck.
Thank you all for your answers and valuable suggestions, i have prepared a stock of 2ml augmentin in which i dissolved o.6g augmentin powder form (0.6g/2ml)/L So the stock concentration is 300mg/L. NOw i use ul from this stock according to ml of media e.g in 25ml agar plate i use 25ul of this stock, and in 100ml media i use 100ul of this stock, so please let me know if there is somthing wrong in this whole calculation as in my concepts if i need to add 300mg/ml into one litre of media so its mean i need to add 100ul /100ml of this stock into MS media. can someone verify if thr is something wrong with this whole calculations ???
Dear Nasheera,
In my opinion - Your stock solution calculation is correct, and your use of 25 ul of stock to make 25 ml medium is also correct (as is 100 ul of stock for 100 ml medium).
However, 25 ul is too litle when you want to dissolve it in 25 ml of warm medium. I am affraid there may be a possibility it will not dissolve completely and you end up having a few plate with more concentrated antibiotic and some with less.
I suggest you take at least 500 ul of your stock solution. However you need to ajust your stock solution into less concentrated one.
S. Sudarsono i agree you are absolutely right, but what i do in daily routine in my lab work is i prepare stock solution in required amount and then aliqoute it for storage at -20, then i use single vial for use in my media in daily routine in lab work, so some times i need to prepare only one plate to check certain parameter ,so accordingly i use my antibiotic solution
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