use R2A to isolate Bacillus, the water sample was boiling 10 minutes

Thanks. I'm trying to avoid spore formers as well!! Will give your ideas a shot and report back

Dear Colin,

The filter paper method can be a suitable alternative if you want to avoid pre-treatments. Briefly; filter the water sample through 0.22 micron cellulose ester filter paper and place the filter paper on suitable media with the right mix of antibiotics. Ideally a good combination of anti-fungal agents and naldixic acid alongside Trimethoprim should be good enough. Filamentous actinobacteria can be observed passing through the filterpaper in a weeks time and can be observed and picked once, removing the filter paper after 2-3 weeks depending on the density. The method is particularly useful for water samples as the density of actinobacteria in free water is less when compared to invertebrates or sediments. I personally was benefited using this method for isolation of micromonospora genera from intertidal waters.

This is a slight varient method and the original method can be found via the link provided: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC239490/

If you have suitable facilities, density gradient centrifugation would be the best alternative.