Currently, I am testing Pst DC3000 cfu assay on Arabidopsis Col-0. DC3000 was grown on KB (Rif) media, and I infected with OD600=0.001 density by hand syringae infiltration.
0 day cfu result looks fine (near 10 to the 4th cfu / ml), but 3 and 5 days cfu results are quiet lower (10 to the 5 th, 3 and 5 days results are similar like this) than I exptected (10 to the 7th or 8th).
This is just preliminary to set up DC3000 cfu work in the lab.
I am wondering that for 3 and 5 days cfu, should I try to spread on the KB media without antibiotics?
I used short day condition, 22 celcius, 60-70 % humidity. After inoculation, plants were covered with cover to keep relatively high humidity for 1 day.
I tested DC3000, DC3118 (cor-) and DC3000 AvrB. All the results were similar.
Hi Daesung,
Sounds like your protocol is pretty good actually.
I don't think that the antibiotics are the issue. I encountered a similar problem earlier. Two things helped to largely fix it:
1. Reisolating DC3000 colony for glycerol stock following plant infection to recover fully virulent bacteria
2. Increasing the number of leaf discs used for isolation and increasing grinding solution volume (and adjusted for in CFU calculations)
It might also help to drop the OD a little to around 0.0001 - 0.0005 to see if you can recover a better final result.
Good luck!
Hi Jay,
Good to see your comment here!!
I will re-isolate and also try to get new strain from other group.
Thanks for your recommendation, helpful^^
Happy New Year~~!
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