Did u check these minimal media? CGYA (Casitone glycerol yeast agar) (Bridson, 1995). I Medium (Atlas, 1993; van Veen, 1973 as cited by Mulder and Deinema, 1981). TYGA (Tryptone yeast glucose agar) (Atlas, 1993).

Which organisms you want to isolate from sludge? Type of media and conditions depends on the organism u want to isolate , for example if the organism is fastidious try using brain heart infusion broth, if the organisms are facultative anaerobes try supplying 10- 15% of carbondioxide during incubation or if they are anaerobes use robertsons cooked meat media and incubate under anaerobic conditions.

Dear Herzog, I agree with some points discussed above and realized you have to mentioned more about nature of sludge sample, source of sludge and target group of organism. Obviously you will get marked difference in DGGE result that is based on direct DNA sample and containing both culturable as well as unculturable microorganism. On the other hand cultivating specific culturable bacteria from sludge is not only depend on media, their are many factor which may affecting your optimum cultivation. These might be source of sludge sample (domestic, Industrial). Second how you are taking the sample for bacterial cultivation from sludge. Because this is most important factor according to my experience. I have tried many method during my work on cultivation of different group of microbes from paper industry sludge (nitrogen deficient sludge). I found first challenge was how to break flock without disturbing microbial population. Due to flocculating nature of flocks, we are not sure about the accuracy of total microbial population. So, I will suggest you, first you should standardized the method by which you can maximum microbial population count from ur desired sludge. Then you can use any of the specific media with different nutrient concentration to get maximum population.

For your reference, one method you can try, but you need to standized it. Also you can try it by modification.

Two ml of sludge sample was taken centrifuged for 10 minute the resulted supernatant was collected in presterilized 30 ml glass bottle, and pellet was resuspended by adding 1 ml of alkaline solution (pH 10), suspension was mixed vigorously with the help of vortexer. Votexed suspension was centrifuged for 10 minute and supernatant was pooled in aforesaid glass bottle. The alkali treated pellet was macerated with help of pestle for 2 minute (Approx stocks 55) washed the centrifuged tube and mortar with sterilized water and collected the clear washing in same glass bottle. The volume was made to 10 ml with sterilized water. This suspension was further break by sonication in 3 cycles at 1µm (Amplitude) for 30 second after 20 second cooling interval in ice bath. Appropriate serial dilution of sludge homogenization was plated on respective medium for microbial counting. Plates were incubated for 24 to 48h at 37⁰C. Following the incubation plates were observed for the bacterial presence or absence.

Good luck

Thanks a lot for all these helpful ideas. My problem is that I have to screen for specific organisms that, in my case, can degrade antibiotics. In diluted sludge and liquid media I find degradation and as I can exclude abiotic factors, I assume that this must be biodegradation. My biomass inside these reactors is already extremely low but still there is biodegradation. If I now try to culture these organisms I only get two different kinds and none of them shows degradation, neither separately nor mixed. In DGGE I can see that there are many other organisms still present that resist cultivation. Therefore I wanted to know if anyone has experience in cultivation of "uncultivable" organisms or if there exists kind of a method to mimic natural conditions to gain pure cultures. First of all I will test your suggestions. Thanks for the moment.

Our lab deals with this on a daily basis! We try to maximize the the diversity of the bacteria the can be cultured. We have developed some specific methods for this.

Do you know what gene is responsible for the degradation of the antibiotics?

Are you interested in just finding out if you have bacteria that degrade antibiotics or do you want the culture in hand, so to speak.

Well, activated sludge contains the zooglea type of organisms. That is the reason that they floc. I have a simple suggestion and that is try cultivating these on a minimal medium containing may be 1% peptone; 0.020% yeast extract and 0.5% NaCl. What you must remember is that these organisms on an artificial medium should produce no slime (if posible). Add agar 2.5% if required.

Read A. Stams et al., Appl Environ Microbiol 1993 59: 1114- and other papers from Stams lab. They work with activated sludge for ages.

Dear Mr. Herzog

I would suggest you to plate serially diluted sample on Soybean-Casein Digest Agar or any similar medium which support the growth wide range of bacterial population.

Now, from your clarification made clear that you are trying to isolate those organisms, which genetic fingerprint you have already seen in DGGE. Even you don’t know whether these are culturable or not. But your effort in this direction clarify that these are not able to cultivable in your previous attempted. While you can see the degradation in liquid medium (sludge & diluted media). According to literature, uncultivable organism cannot be cultivable due to many factor associated with their cultivable condition. Now in your case, we can only suggest hit and trial method. The best suggestion from my side is, rather then using media component for isolating them try natural media for them. It means you can try filter sterilized sludge as media component, try it with adding 1.5% agar as well as liquid with or without addition necessary media component (different concentration). Then isolate each organism then do the same for screening. If you find your desired organism by this method then you can further standardized other media for its growth. The is cumbersome procedure and might be take weeks of bacterial growth, so you should be patients during the experiment.

Secondly you can do this with adding different concentration your desired antibiotics to enrich the desired bacterial population.

Autoclaving the sludge can degrade the important compound or make some toxic compound for those uncultivable organism, in which they are surviving or growing. Sometimes these sludge (Industrial & domestic) sample can coagulate after Autoclaving. On the other hand most of the sludge sample having the pH range around to neutral or slightly basic.