I have a culture of Ralstonia mixed with Bacillus, I need to isolate Ralstonia, but I couldn't have clean my isolated, I use SMSA but nothing works, what I could do?
Angela Rivera Some chemicals supress growth of many Gram(+) bacteria (and some gram(-):
Conc. %
Brilliant Green 0,0015
Sulphapyridine 0.1
Na Citrate 0,5
Na Deoxycholate 0.1
Fucsin Basic 0,04
Na Sulphite 0,25
Eosin Y 0,04
Methylen Blue 0,0065
Malachite Green 0.04
Cristal violet 0.0002
The references were quite old, like Kristensen, M., V. Lester, and A. Jurgens. 1925. On the use of trypsinized casein, bromthymol blue, bromcresol purple, phenol red and brilliant green for bacteriological nutrient media. Br. J. Exp. Pathol. 6:291.
Simply dilute the culture to max possible and streak on media specific to each bacillus and other for ralstonia... Collect and subculture single colonies and reassess ur isolate.
Angela Rivera Ralstonia spp. is very specific pathogen.
It is often isolated with other bacteria (Pectobacterium spp., Advenella spp., Stenotrophomonas spp., etc.).
It was reported that Advenella can produce positive PCR reaction with some classic primers for Rs. If you have a stable association of Rs + Bacillus, the bacterial cell can stick together due to EPS capsules, and it is better :
1) to use non-selective medium like YDC,
2) use long shake of bacterial suspension in water with low concentration (10E3 CFU/ml).
3) keep plates at least 7-10 days before transfer of single colonies to new plate - you will see contamination and bacteria on YDC live longer that on other media.
4) Try to inoculate susceptible plant and re-solate Rs from the infected plant stem without secondary infection - 10-20 cm above the first wilted leaves.
Dear Angela Rivera,
You can use both technics( the dilution and a bacteriocide). You can use a very strong gram+ antibiotic to get rid of bacillus.
Good luck
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