I found the attached paper that tested the influence of different diluents and their concentrations on the results obtained from the API 20E kit for marine and estuarine bacteria. Apparently, results will vary greatly depending on the kind of salt solution you use. They reported that adding marine salts in the concentration of 20ppm gave the best results and not so great results for NaCl. Higher salt concentrations seemed to cause interaction between the salt and the test reagents, while lower ones could provide false-negatives.

I have never performed those biochemical tests on my marine Bacillus (isolated in Zobell Marine Agar), but from what I see, I would try one of these options:

1) You could supplement the media with marine salts (the kind you buy on aquarium shops), but be sure to add a "blank" tube (media+salt, no bacteria) to see if the concentration you used will result in false-positives. So growth wouldn't be an issue and in theory the bacteria would have all sorts of ions necessary for the enzymatic activities.  

Edit: (Just a word of advice, I have tried Tropic Marin salts and they precipitated hardly upon autoclaving, making the media very "grainy". So, I would suggest to filter sterilize the salt solution, autoclave separately the nutrient solution  and combine both later when the temperature is around 40°C or so)

2) Test whether high salt concentration is really required for growth of your isolates. If it is not the case, maybe you could perform the biochemical tests without adding the salt. The isolates would grow, but maybe you would need some sort of positive control to exclude the false-negatives...

Please, let me know how it goes! =)

I suppose I would go for the option 2. I will try to grow them on 0 or low concentration of NaCl and see if there would be growth.

Thank you, Liany. Your suggestions were highly appreciated. I'll let you know soon :)

I did the same test to my Bacillus. I ran out of Zobell media and needed to subculture my isolates, so I tried to see if they would grow in nutrient agar, which has 5 g/L of NaCl. They did, and we were all very happy about it! :D

Dan, Marine heterotrophic bacteria requires salt for their growth, the concentration should be from lower level to higher because some isolates doesn't require salt and some needed the growth. I suggest 0.5 to 2% can add to the following biochemical media for your better understanding of tolerance results.

One more question, when I isolated my isolates from Seawater Agar (SWA; NA + Yeast Extract), I used the filtered seawater which I got from the sampling site as the diluent. Is it possible to use the seawater as diluent for my biochemical tests instead of adding marine salts to distilled water? 

Moreover, I already inoculated my urea agar (0.5% NaCl) with my isolates, so far no positive results yet. But I still have to observe them for 6 more days :)

I think the "problem" with using filtered natural seawater would be the amount of dissolved nutrients, which means you could be adding something else to the media besides the desired salts. I don't know to which extent this would somehow influence the results of those tests... You would have to know exactly what was in it to know where to check if anything went wrong with your blanks/controls. Plus, depending on few things like season, if it's open sea or coast line, or if there is some sort of extra input of nutrient close to your sampling area (like estuaries or sewer systems), the quality of the water would change greatly, so it would be a kind of unstable variable.

On Zobell's original publication, the author mentioned a kind of aging process. If I am not mistaken, it would be something like sampling the water and letting it rest for some time so that the microorganisms would consume everything. Then you would filter sterelize it prior to use in your media. Back on my isolation days I considered using seawater, but since my study area is a kind of an urban beach, we do have some estuaries, and on top of that I wasn't quite sure on how to perform that aging protocol properly, I chose to use the commercial marine salts. At least I would be able to "standardize" the media composition. BUT still, you could try it. It won't hurt. =)

Did you manage to get any marine strains to use as positive controls? It would be nice to have something to compare to your isolates... =/

I see. The "aging' of water makes sense and the composition could vary over time. I'll run again another round of tests after I get the reulsts of the other biochemical tests (no NaCl supplemented) and try adding marine salts.

No,  I haven't. But i'll try to ask the marine department if they have stock cultures of Vibrio sp., which I could use as positive controls :)

Hi Liany,  I already have results for my biochemical tests (TSI, SIM, Citrate, and MR-VP) no marine salts or NaCl added), and they did grow! It seems they can grow on medium without much salt.

I am really grateful for your suggestions/advice and for your time! God Bless you :)

Oh, that's great news!

I'm glad I could help. God bless you too. =)