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Quantification and Identification of Active Microorganisms in Microbial Plant Protection Products

Appendix A

Separation of Bacillus thuringiensis spores in microbial plant protection products

The separation of Bt spores prior to spread plating was optimized through testing of different methods (Table a.1), using chemical, enzymatic and physical treatments. One ml of a ten-fold dilution of the Bt product Bactimos L was tested for each treatment. The number of individual and aggregated spores as well as the number of CFU determined after the separation treatments showed that the treatment with proteinase, SDS and Dowex followed by mixing yielded the highest fraction of individual cells and the highest number of CFU (Table a.2). Subsequently, this treatment was applied to all the Bt products.

Table a.1: Treatments for separation of Bt spores prior to CFU counts

Treatment Cellulase
(5 mg ml-1) 		protein-ase
(5 mg ml-1) 		Na2-EDTA (3.72 mg ml-1) Tween80 (20 µl
ml-1) 		SDS
(3 drops 10%) 		Dowex (82.5 mg ml-1) mixing time (h)
A1 + - - - + + rotary shaker 4
B1 - + - - + + rotary shaker 4
C - + - - - - - 4
D + - - - - - - 4
E - - - - - - - 4
F - - - - - - - 4
A2 - - - - + + rotary shaker 4
B2 - + - - + + rotary shaker 4
G - - - - - - magnetic bar 4
H - - + + - - 600 rpm 0.25

+: treatment added; -: treatment not added

Table a.2: Number of individual and aggregated spores and CFU

  Spores CFU
Treatment Total 1 cell 2 cells 3 cells >4 cells CFUml-1
A1 402 83.58% 13.68% 1.74% 1.00% nd
B1 403 85.36% 11.41% 1.24% 1.99% nd
C 475 60.84% 14.95% 5.47% 18.74% nd
D 428 54.91% 13.55% 3.50% 28.04% nd
E 381 58.27% 16.01% 4.99% 20.73% nd
F 397 72.80% 12.59% 6.80% 7.81% 6.20 x 109
A2 402 83.58% 10.70% 2.49% 3.23% 5.40 x 109
B2 392 86.73% 9.95% 2.55% 0.77% 6.37 x 109
G 436 70.87% 17.43% 3.90% 7.80% 4.83 x 109
H 426 72.07% 10.56% 5.63% 11.74% nd
H 392 76.02% 13.01% 3.32% 7.65% nd
H 400 73.00% 11.25% 3.25% 12.50% nd

nd: not determined

 

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Version 1.0 February 2005, © Danish Environmental Protection Agency