I tested a biological agent in 96-well microplates, using 9 serial dilutions (1/2 to 1/1024) against three bacterial strains . It turns out that I didn’t get a dose-dependent response, that is, both the lowest dose and the highest showed antimicrobial activity, however, how can I explain this pattern: 1/2 reduced 60% of microorganisms, 1/4 (80%), 1/8 (70%), 1/16 (90%), 1/32 (70%), 1/64 (90%) and so on. There were no pattern. Does anyone know how to explain what dynamics this is? thanks
Hi Daniele,
I think that your vehicle (or solvent) may be toxic to your bacteria. But I can not be sure until you share your experimental conditions.
This kind of result can also be seen, if the bacteria number is not equal in all wells.
Another possibility can be pipetting error or contamination during the pipetting.
To solve your problem, we need to see your positive and negative control results and your sample numbers, in short all of your raw data.
It also may be a factor that your biological agent has no effective dose response, would be interesting to grow up the remaining % of cells and reapply the antimicrobial and see what happens too, could be that the susceptible portion of cells died? But also run a negative control well also, always important
Hello, Dr. Cigdem Ozen ! Thank you for the prompt reply. The inoculum were prepared according to the protocol M07-A9 (CLSI), where isolated colonies were selected, and a suspension was prepared at 0.5 Mcfarland density scale. The microbial suspension were prepared to 1x106 CFU / mL for bacteria). Sample were Serially diluted in sterile Milli-Q® ultrapure water (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512, 1/1024 ). One hundred microliters of each sample dilution and 100 μL of microbial inoculum were incubate in a 96-well microplate, in triplicate. Positive control included 100 μL of microbial inoculum with 100 μL of sterile Milli-Q® ultrapure water; the negative control included 100 μl of sample with 100 μl of medium. The microplates were incubated at 37°C and the results were read after 24 h.
Thanks so much
Hi, Dr. @ Phil Geis my sample was diluted in sterile Milli-Q® ultrapure water
thanks
Hello dr. Stephen Reynolds I did several experiments and in all of them there was this pattern of response. It is as if all doses reduce bacterial cells, however, without the traditional pharmacokinetic pattern where the higher the dose, the more effective the response is. In my case, I can't explain what happened. I did the experiment with 2 bacteria and 1 fungus, following CLSI protocols. After 24h incubation of the 96-well plates, we transferred, in triplicate, 50uL of content from each well to agar plates and incubated for 24 h to count the number of colony units, and the average of the three replicates gave these result models - for example:1/2 (60% de redução bacteriana comparada ao controle), 1/4 (83%), 1/8 (65%), 1/16 (85%), 1/32 (80%), 1/64 (85%), 1/128 (86%), 1/128 (82%), 1/256 (81%), 1/512 (85%), 1/1024 (86%). The other experiments followed the same pattern of responses. How can I explain this? Thanks
Daniele Lourinho Dallavecchia, super strange, what I am seeing is relatively consistent % reduction of around 80% with the two 60% ones compared to control. There is clearly an effect seeing as the control is unaffected but it may be that 24hrs was too long to leave the bacteria in contact with the antimicrobial and your seeing the plateau of the response due to that instead of the dose? Have you ever taken the remaining bacteria and re-cultured them and tested them against the antimicrobial again to see if there was some for of resistance properties in them or the selective pressure of the antimicrobial brought them out at a certain point. If this is a novel antimicrobial that might be it but if there is previous literature how have strains responded in the past?
I have used disk diffusion methods in the past for sensitivity testing, agar plate with a paper diffusion disk of x conc. might give you a little more clarity on how the drug works if you see some colonies still growing inside of the clearing in the diffusion zone.
Hope that helps somewhat
Dear Daniele Lourinho Dallavecchia ,
I want to repeat again, I need raw data to say something meaningful. I need to see all 3 value of every sample vs controls. Percentages do not help us to solve your problem. We need to see standart deviation and cv% data (=standart deviation/avarage*100). If the standart deviation is higher than 10% of your avarage value, you should take the pipetting errors to the consideration.
We did something similar with B. subtilis and E.coli. If you add your bacteria into each well with manually, you should vortex every time your bacteria culture, I mean before every well. Otherwise every time your bacteria number will be different in each well, due to gravity bacteria sink in to the bottom. This effect causes unexpected results.
I realized that you use pure water as solvent. It is biocompatible, however every time you dilute your medium with water and it can also cause your problem. If I were your, I woul try to solve my test compound in medium. As long as your compound solves in water, it will solve in medium too.
I hope this hepls
Dear Daniele,
Have you checked out the purity and sterility and solubility of your test compound, that would help in doing troubleshooting for your test
Cigdem Ozen Thank you so much for all this information. Yes, we pipet manually and always had to vortex before inserting the sample and inoculum into each well. It was exhausting! I will review my data and analyze if there was a pipetting error. One person told me that there may have been a paradoxical effect, as with echinocandins upon fungi, I will investigate further. Thank you!
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