plant surface microbiology.pdf

26 Quantifying the Impact of ACC Deaminase-Containing Bacteria on Plants 497
chilled mortar and pestle, suspended in 2.5 ml of 0.1 M sodium acetate pH 5.5
and kept on ice for 15 min. The contents of the mortar are scraped into a 15ml
glass centrifuge tube and the mortar and pestle are rinsed with 0.5 ml of
the same buffer. The ground tissue suspension, together with the rinses, is
centrifuged in an SS34 rotor at 17,500xg in a Sorvall R5C/B centrifuge for
15 min at 4 °C to remove cell debris. The supernatant is collected and clarified
by centrifugation in a Beckman L8–70 ultracentrifuge at 100,000¥g in a 70.1
Ti rotor for 1 h at 4 °C and then, if necessary, by an additional centrifugation
at 100,000xg for 15 min. The clarified supernatant is collected and distributed
into 1-ml aliquots, some of which are stored at –80 °C in glass vials for ACC
determination by HPLC, and the remainder stored in 1.5-ml microcentrifuge
tubes at 4 °C for protein determination.
6.3 Protein Concentration Assay
The protein concentrations are measured according to a protocol based on
the method of Bradford (1976) and BSA (bovine serum albumin) is used as
the standard protein. Each point on the standard curve and all of the samples
are assayed in triplicate.
6.3.1 Protein Concentration Assay of Bacterial Extracts
The 100-ml aliquots of toluenized cell suspensions, which have been set aside
and stored at 4 °C during the preparation of crude bacterial cell extracts, are
each mixed with 100 ml of 0.1 N NaOH and incubated for 10 min at 100 °C.
After the mixtures have cooled, between 20 and 50 ml of each sample is transferred
to a clean glass test tube (100x13 mm); the volume is adjusted to 100 ml
with 0.1 M Tris-HCl pH 8.5, and 5 ml of the diluted dye reagent is added to the
tube. The contents of the tube are vortexed and incubated for 5–20 min at
room temperature. The absorbance of the samples is measured at 595 nm.
6.3.2 Protein Concentration Assay of Plant Extracts
Aliquots of the plant extracts, set aside and stored at 4 °C, are each transferred
to clean glass test tubes (100x13 mm) and the volume is adjusted to 100 ml
with 0.1 M sodium acetate pH 5.5. Varying amounts of the different extracts
are transferred to the tubes, depending on the concentration of the extract:
routinely, 30 ml of seed extract and 100 ml of root extract are used. Sufficient
buffer is added to each tube to bring the volume up to 100 mL.After 5 ml of the
diluted dye reagent are added to each test tube, it is vortexed and incubated at
room temperature between 5 and 20 min. The absorbance of each sample is
measured at 595 nm.