Thanks a lot, very usefull your information. We are on touch

I agree with P. Srinivasan comments. As LD50 means the dose needed to kill 50% of the fishes, it depends very much on the strain tested. I suggest you to get a model strain (maybe some ATCC or some other that has been already published) and validate such virulence and/or determine its LD50 (we use to inject 20 μL of bacterial suspensions at concentrations ranging from 10^6 to 10^10 CFU/mL). Then choose a suitable concentration and compare other strains. There are several papers you can consult anyway. Maybe this is also of your interest:

Kinkel, M. D., Eames, S. C., Philipson, L. H., and Prince, V. E. (2010). Intraperitoneal injection into adult zebrafish. J. Vis. Exp. JoVE. doi:10.3791/2126.

Cheers.

Thanks a lot Pol. In General all are agree in the range of dose, and, as you say, there exists some papers about that . My problem is: how to standarize the dose per fish?

I think the volume of inoculum must have a relationship with the weight of the inoculated fish, If you could express the volume to inoculate as a percentage of the fish weight , the experiment could be repeated in "same condition " with different sizes of fish.

I´ll read the paper that you recomend me

Regards

Yes, that could be a problem, but are you infecting zebrafish or some other sp? Of course every fish has its own particularities (weight, inmune system...), but if you replicate the experiment and take a big sample (several fishes) the likely error will be minimized. Otherwise the experiment could become really arduous.

Cheers

Alejandro, you are correct in your statement that "the volume of inoculum must have a relationship with the weight of the inoculated fish, If you could express the volume to inoculate as a percentage of the fish weight , the experiment could be repeated in "same condition " with different sizes of fish."

For Aeromonas hydrophila it is usual to inject the fish with 10 microliters of known bacterial CFU suspension per 1 gram of fish weight. (assuming you are working with small laboratory fish model species). You have to weigh each fish before injection.

You can check my recent publication on more methodology details. (link included)

And yes if you have the different strains of bacteria you want to test you are absolutely correct that the only way to compare them is by calculating LD50 after 96h post-exposure. However in order to do that you need to inject every fish with 10 microliter of suspension per gram of fish weight. Otherwise the results are not comparable. You need to use 5 concentrations for each bacterial strain you are testing and at least 30 fish per concentration to get accurate values. As far the concentrations are concerned as Mr Srinivasan correctly pointed out to you for Aeromonas good concentration to start with  is around 1.6x10^8 cfu/ml  but do only 50 % dilutions  dont do 10 fold dilutions as Aeromonas hydrophila have a very narrow toxicity curve in fishes, and if you do 10 fold serial dilutions you wont be able to accurately calculate LD50 and would have to repeat experiment with more narrow doses. I am not familiar with A. veronii but it may happen that for A. veronii you may need to select different concentrations. I would recommend for A. hydrophila that you start with 2x10^8 cfu/ml and do 50% dilutions(1, 0.5, 0.25, and 0.125) injected as 10 microliter per gram fish weight. As for LD50 calculation itself you can use the free EPA toxicity estimation software - http://www.epa.gov/nrmrl/std/qsar/qsar.html, or you can use other statistical software. MiniTab statistical software has a very nice LC50 build in tool with nice graphs. StatFi is another software option with good LD50 option, or alternatively you can calculate LD50 by hand if you know how to work with probit functions.

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Thank you very much Boris, thats the information that I was looking for. I just read your paper, very good work, congratulations.

Tomorrow I´ll start the first infection experiment.

Regards

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