I'm doing disc diffusion experiments with E. coli TOP10 carrying my plasmid. I noticed that there was no difference between the TOP10 carrying the empty vector (my control) and TOP10 carrying my plasmid. But what is very strange is that neither ampicillin nor carbenicillin (solutions made fresh on the day) are killing TOP10 - which does NOT have ampicillin resistance.
My plasmid and the empty vector have kanamycin resistance. Originally I was including kanamycin (50ug/mL) in my LB agar plates. Then I lowered the kanamycin to 10ug/mL. Then I removed it entirely. This was in case there is some generic antibiotic resistance mechanism. Still the same result regardless of kanamycin inclusion.
I also have arabinose in my plates for the induction of the gene in my plasmid. But I do 0%, 0.002%, 0.02% and 0.2% and again this doesn't effect the result (although I have noticed that the lower the arabinose% the better growth I get - I tend to get lawns with 0% and 0.002% and single colonies for 0.02% and 0.2%.)
Antibiotic disc preparation: I'm using Oxoid blank antimicrobial susceptibility discs. I prepare a solution of 100mg/mL carbenicillin/ampicillin then do a few 10-fold dilutions to reach 1mg/mL. I filter-sterilise it then from this prepare solutions of 5, 10, 15,20 ug/mL. I then soak the discs (by dropping several discs into the solution tubes) for 30 mins minimum. I then remove them with sterile tweezers and lay them on sterile petri dishes by the blue flame to dry. Everything sterile.
I use the bacterial culture around OD600 0.5-0.7. I dilute it 1:1 then spread 200uL on plates with a sterile plastic L shape spreader. Immediately I place the now-dry discs onto the quadrant of the plates. I also have discs soaked in sterile water as controls. I then incubate them at 37oC for 20-24hrs.
Is the reason for not having zones of inhibition with carb or amp because the concentration is too low - do I need to go >20 ug/mL? Since the strain should be susceptible I would've thought that any concentration of carb/amp would inhibit growth. Alternatively, could my commercial chemically competent One Shot TOP10 have acquired resistance to amp/carb?
I won't have the results until tomorrow, but today I have tried the non-recombinant commercial TOP10 strain with discs of 5, 10, 15, 20, 100, 1000 ug/mL carbenicillin discs. I guess if it still isn't killing the TOP10 then it has somehow acquired amp resistance....?
Sorry this is not very concise but I wanted to include all the details .
You will probably need a higher amount of ampicillin, standard amount is 100 mg/ml so you are well under that concentration. Antibiotics are only effective if they reach a threshold level.
See what results you get for your ongoing test with the higher amp amounts.
It is unlikely to have resistant bacteria as you grew initially grew the cells in the bacteria in the absence of ampicillin and cells tend to lose resistance plasmids in the absence of selection.
Good luck!
Amy Klocko Thanks for your answer.
100 ug/mL is the standard working concentration (100mg/mL is usually the stock concentration).
The result from my test with the non-recombinant commercial TOP10 strain is that 1mg/mL carbenicillin has a nice zone of inhibition. But 100ug/mL doesn't.
I'm now going to make agar plates containing 100ug/mL carbenicillin (rather than on discs) and if this kills the strain then I know the issue lies in my preparation of the solutions and discs.
A concentration does not really have meaning when it comes to a disk. So if your starting solution is at some concentration (say 100ug/ml) then you are adding some volume of that solution (say 10ul) to the disk, so the disk is loaded with only 1ug of ampicillin, which is pretty negligible. Then it is diffusing in the plate which is a much larger volume (many milliliters of practical volume). So I agree with Amy Klocko that you probably just have too little drug you are adding. I think you need at least 10ug amp on a disk to see a nice zone.
Michael J. Benedik The reason I didn't think of it like that is because I'm soaking the discs in my solutions, rather than pipetting a certain volume on. So, is it the case that discs can only hold a certain volume (even when being soaked) so in reality they're only really holding 10/20/30uL of solution? If this is the case then I will modify my disc prep method and calculate my concentrations based on adding 10 or 20uL to discs.
I don't actually know how much volume a disk would hold but it probably is less than you think, which is why I think you are only seeing effects at the highest concentration.
You could start with your highest concentration and then add different volumes to the disks and let them dry and then see if you observe a nice somewhat linear reduction in zone size.
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