The isolation of nematode parasitic bacteria or some other arthropod soil must be to cure the infected externally specimen and deposited this on a specific medium for the nutrition of this group of bacteria . For bacillus are anaerobic , which can be introduced on the environment. Not sure if both bacteria cleave chitin, but if this component is included in the medium, the dilution medium reveal the presence of this .

Dear Tabassum

There are the most common and successful methods for symbiotic bacterial isolation. I used them in my researches. 

Isolation and culturing of bacterial strains

1- Grinding method was used to isolate the nematode symbiotic bacteria Photorhabdus spp. or Xenorhabdus spp. according to Goetsch et al. (2006). Infective Juveniles (IJs) were surface sterilized by immersing in 1% sodium hypochlorite for 1 min and then washed three times with sterile distilled water. 50 µl of the nematodes suspension (about 100 IJs) was placed in a sterile 5 ml Petri dish, followed by 100 µl of sterile Luria-Bertani Miller (LB) broth (Difco). Sterile nematode (5-7 IJs) was picked and submerged in a sterile 100 µl of LB broth supplemented with 2.5% ampicillin. Using a sterile pestle, the nematodes were ground for 1 min on ice. Nutrient bromothymol blue agar (NBTA) media (2.3% “Difco” nutrient agar, 0.0025% “Merck” bromothymol blue, 0.004%, 2, 3, 5- triphenyltetrazolium) plates were incubated at 29°C, and single bacterial colonies were subcultured onto new NBTA plates until no contamination was detected. A single pure colony was subcultured into LB broth at 29°C for 24 h in a shaking incubator at 200 g.

2- About 20 sterilized

infective juveniles (Ijs) of Entomopathogenic nematodes (EPNs) were infected of a greater wax moth larvae (Galleria mellonella). After 24h, a leg of surface sterilized G.mellonella larvae was removed and the released haemolymph was streaked on Nutrient-Agar (NA) plates containing 8% nutrient broth (LAB) and 1,2% agar. Plates were incubated at 25°C for 3 days. Then, the characteristic of bacterial colonies developed (Thomas & Poinar, 1979); these were repeatedly subculture until a pure culture was

obtained. Pure cultures were transferred to nutrient broth slants. Incubated at 25°C for 3 days. Stored at 6°C.and subculture at least once a month to maintain viability.

Bromothymol Blue (BTB) 0.025g and 0.04g Triphenyl tetrazolium Chloride (TTC) were added to NA plates as an indicator.

Hope it can help you. Please don't hesitate to ask for any explanation.

Good luck

Hanan

Thank you so much Dear Hanan and Roberto. i will be followed your procedue.  

Dear all,

I hope you are well,

actually, I followed the same process of Hanan for making the medium of bacteria ( Photorhabdus spp ), but unfortunately, after 10min the medium was destroyed, and was solid after adding the TTC.

I dont Know the problem!

Please, help me to solve this problem

Best regards

Dr. AL-Zaidawi Jawad

Sir thanks i am following these methods for isolation of bacteria from soil nematodes

I wonder if it is possible to add bromophenol blue instead of bromothymol blue into NA?

Dear Tabassum, I first need to know that what nematodes you are trying to isolate the bacteria from.