Have you characterized that bacterium?

I recommend the culture plate of MacConkey agar.

If would you have any question, reply me.

I am currently busy with the isolation and description of the symbiotic bacteria from various species of entomopathogenic nematodes. I have found that tryptic soy broth works much better than nutrient broth. Are you working with Xenorhabdus or photorhabdus?

Your problem may be due to 'lonely cell syndrome' ; try preparing a starter culture in a small volume of broth say 2ml in a universal bottle, inoculate from a single colony from an agar plate and incubate overnight in a shaking incubator. Then use this to inoculate a shake flask. I can confirm that TSB is a better medium than Nutrient Broth it yields more biomass and favours the primary phase variants of these organisms.

I have also isolated symbiotic bacterium from my native EPN isolates and successfully culturing in MacConkey, NA and NB media. In case of these bacteria, if olated from Haemolymph mehtod, many chances of getting other flora already existing in wax moth larvae. So you first of all be very sure about the target bacteria. After that culture it in NBTA or MacConkey agar media. In MacConkey agar media, if bateria belongs to the Enterobacteriacae, will produe red to pink rounded colonies with clear zone arrond them.

Wish you good luck.

I have the same problem as yours!!!!!!!!!!!!

Your problem may be due to the growth of other unwanted species supressing your target organism,try using selective media as maconkey,it ll prove satisfactory

Also verify the strain and check for other organisims growin in NB

thanks for ur commands. Can i use tryptic soy broth with phenol red for culturing this bacteria. vinod kumar, as u said u have isolated bacteria . can u plain the method? how do u recognize this bacteria during isolation.

I have not yet characterized the bacteria. Yes i am working with xenon/phto-rhabduds bacteria.

Puspa first of all you please tell about the group of your EPN (Steinernema/Heterorhabdtis). Then any body will be in stage to guid well.

K vinod. Based on color of dead cadaver and catalase test my bacteria seems to be steinernema. Here i am facing subculturing problem means they are not multiplying in large quantitiy.

If you are getting an overwhelming level of contaminants when culturing the free bacteria from the haemolymph, try harvesting the nematodes from the insect host and extracting the bacteria direct from them. You will need to surface sterilize them with hypochlorite, then homogenise aseptically in a minimal volume of water to release the bacteria. Finally plate out the undiluted homogenate.

Stephen I liked your comments and would try this. Vinod is working with me. We would try out your method of homogenated insect host. But will it not bring more contaminants from other body parts of the insects.

Don't homogenise the insect! Instead disect it immersed in a small volume of water to release the nematodes, then pipette them up and transfer to a micro-centrifuge tube. Centrifuge them, discard the supernatent, then wash in hypochlorite. Recentrifuge and resuspend in sterile water. Homogenise the nematodes in a small glass tissue homogeniser (the manual sort, consisting of a thick walled glass tube and a close-fitting glass plunger). To grow the bacteria, streak out onto a rich agar medium such as Blood Agar Base or Tryptone Soy Agar. Good Luck!

stephen as per your comment bacteria will be isolated from Nematodes it self right. homogenizing is important step. can u explain in better way of homogenizing nematodes(how to break open nematodes to release the bacteria)

I think we can go for spread plate method while isolating.

i also would like to discuss the most interesting part, the primary and secondary phase of Entomopathogenic bacteria.

Miss pushpa.... from my experience, in working with earthworms fibrinolytic enzyme, I prefer U to put the lump of nematodes in sterile distilled water at luke worm temperature, bcoz of heat, the body fluid content will released into the environment. then collected the fluid and proceed with your bacteria isolation procedure. Meantime I agree with Mr. Stephens Comments. All The very Best!

I recommend the Xenorhabdus culture plate of NBTA agar