- Pipette 50μl of the resuspended culture strains into wells
containing the different media. Prepare three to five replicates
for each strain and media. - Cover the plate with the lid and seal with paraffin to avoid
excessive evaporation. - Incubate the plates in incubator at optimal growth temperature
(seeNote 9). - Remove carefully the supernatants with a pipette. Avoid dis-
ruption of attached cells at the base of the wells. - Gently wash twice the wells with 200μl of sterile water.
- Allow fixation of the surface-attached bacteria by placing the
plates at 60C for 20 min. - Add 120μl of 0.1% crystal violet solution in water to each well
of the multidish. - Incubate the plates at room temperature for 1 h.
- Gently rinse the wells three times with 200μl distilled water.
- Let the plates to air-dry for at least 1 h.
- Solubilize the crystal violet in each well by adding 1 ml of 95%
ethanol. - Briefly pipet the content of each well and transfer to 1.5 ml
tubes. - Dilute 1:10 with sterile water.
- Transfer the solutions to cuvette and measure the absorbance
with spectrophotometer at 595 nm.
4 Notes
- To investigate the role of QS system in virulence and move-
ment of plant pathogenic bacteria knockout mutants inrpfF
andrpfCshould be generated. Any knockout or insertional
mutagenesis protocol can be used [15]. - Pathogenicity and colonization tests depend on the pathogen
and its host. For example, in the case of vascular pathogens, as
presented here withXhp, the inoculation will be performed by
puncturing the stem. In foliar diseases, bacterial inoculations
will be performed by spraying the leaves with the pathogen
[16]. It is recommended to do the tests with young plants
which are more sensitive to the pathogens. - To study movement in planta, label the strains with a plasmid
carrying thegfpgene. In the case ofXhp, the vector pKT-Cm
[9] carrying the constitutively expressedgfpgene is used. Any
other plasmids drivinggfpexpression can be used. In that case,
250 Shulamit Manulis-Sasson and Laura Chalupowicz