Can anyone suggest me the best method for MIC antibacterial testing of silver nanoparticles. In my recent experiment of MIC testing using broth dilution method, I noticed that when Mueller-Hinton broth added to silver nanoparticles, the colloidal silver nanoparticles destabilizes immediately and nanoparticles settles down as black precipitate in 96 well. This precipitation causing the absorbence errors at 600nm. Hence I am not able to calculate accurate MIC values. I believe that to be effective antibacterial agents silver nanoparticles should stay in the stable colloidal solution form. Can someone provide me more information and help me with experiment.
The medium should be chosen based on the bacterial species being tested. There are standard MIC testing conditions at the CLSI web site.
Here's an old version of the guidance document:
http://www.asm.org/ccLibraryFiles/FILENAME/000000002484/Manual%20of%20Antimicrobial%20Susceptibility%20Testing.pdf
Thank You Dr Adam.
A very useful information I got from provided link. I am working with metal nanopartilces (Silver and Gold). As per my observations these particles (atleast my samples) have the tendency to react with media (broth), that we use in testing. Can you suggest me any specific method which can help me deal with metal nanoparticles.
Thank you Dr Amit.
My nanoparticles solution is stable brownish colloidal solution before testing. But during antibacterial susceptibility testing,when I add MH broth to it the color immediately changes to black and nanoparticles settling down. That means they loosing their identity during testing. During the incubation for 24 hrs, the absorbance contributed by nanopartilces (at 600nm) is changing i.e decreasing may be due to their degradation, which is causing problem to me in calculating MIC values. Is their any specific method or formula to negate all these background absorbance problems created by nanoparticles
The media in which you are adding nanoparticles to cause coagulation. This may be due to the high salt concentration or pH. Try using a highly viscous media, it would weaken the coagulation.
Dear Dr Gannimani,
I really do not think so that your highest problem is the color changes of your reaction mixture of silver nanoparticles and the MH. Definitely, I would not recommend turbidimetric method for antimicrobial assay of suspensions of metal nanoparticles because usually you will have the deposition of part of these particles in the bottom of the well plates and it will interfere in your spectrophotometric readings. I advise you, in the end of your experiment, to do bacterial counting for the different concentrations of the suspensions of the silver nanoparticles tested. I think it will be the best way to determine the most satisfactory antibacterial concentration of your silver nanoparitcle suspensions.
Cheers.
MH broth or the IB broth are general purpose media for MIC studies. These are not suitable for MIC studies of nano particles. This is because nanoparticles show different chemical and physical properties from their parent elements. I feel you should try different techniques like exposure of the microbial culture to the particles before growing them on a medium, etc.
If something in the growth medium is causing the destruction of your nanoparticles, you are going to have trouble moving forward with the project. You will have to identify a different medium that does not cause this problem, but one in which the various bacterial species will grow well. The media that are used for MICs were presumably chosen because they favor the growth of the bacteria. Other media may not work very well. I suggest you consult a professional microbiologist for help with this problem.
Broth media are complex mixtures. Perhaps it would be possible to use a synthetic medium in which you can omit whatever ingredient is causing the damage to the nanoparticles, while still containing the right nutrients for the bacteria. However, it will be complicated to identify which ingredient(s) is (are) causing the problem. See if you can find an analysis of the composition of the media you have been using. It may suggest possibilities for the troublesome substance(s). It could be salts, metal ions, or reducing agents, for example.
Perhaps you could analyze the composition of the precipitated nanoparticles to see what has changed.
Dear Dr Gannimani,
I have a similar problem. It seems to me that chloride ions react with AgNPs. I showed that Mueller-Hinton Media contains chloride ions, because it created cloudiness after the addition of silver nitrate. It is a characteristic reaction of the formation of AgCl. I suggest diluting the medium 100-200 times and conducting the experiment.
What method did you use to get out of the problem?
Generally MHA media is selective for MIC but you can also try LB broth
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