Citrus Systemic Pathogen Research

(MIEF) for Xylella fastidiosa in Plant Tissue

 

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Finely chop plant tissue*, usually leaf midribs and petioles, with a razor blade.  Collect chopped tissue and place in a 12 x 75 mm test tube and add 2 ml of 0.02 M Tris pH 8.2.  Parafilm the tube and vortex the suspension.  Allow the materials to settle. * (NOTE:   dried material should be rehydrated in buffer for 2 hours to overnight before chopping)

Assemble the membrane filter tower and attach 10 ml syringe.

membrane filter tower

Upper membrane: 5.0 micron plain

Lower membrane: 0.2 micron black polycarbonate shiny side up

 

Pipet supernatant from above into syringe, push down plunger and force liquid through the filter tower.  It may require more than one syringe full of air to accomplish this.  Remove syringe from filter tower before pulling the syringe = s plunger out.  Catch the waste liquid in a petri dish.  Disassemble the tower and remove the lower 0.2 micron black polycarbonate filter.  Place filter, sample side up, in a 35 x 10 mm petri dish.

For direct labeling: overlay the filter with 3-4 drops of fluorescent labeled X. fastidiosa antibody diluted 1/20 with antibody buffer (0.02 M Tris pH 8.2 + 0.9% NaCl + 1% gelatin + 0.1% BSA).  Incubate 30 minutes to 1 hour. 

For indirect labeling: overlay the filter with 3-4 drops of X. fastidiosa antibody diluted 1/20 with antibody buffer.  Incubate 30 minutes to 1 hour.  Flood the filter with a pipette full of antibody buffer, let stand for 1 minute and pipet buffer off.  Overlay the filter with 3-4 drops of goat anti-rabbit fluorescent labeled IgG diluted 1/20 with antibody buffer and incubate 30 minutes to 1 hour.

At the end of the antibody incubation period, flood the membrane with a pipette full of antibody buffer, let stand for 1 minute and pipet the buffer off.  Mount the membrane, sample side up, on a glass microscope slide using 1-2 drops of Aquamount or another non-fluorescing mounting media and cover with a 22 x 30 mm coverslip.  While slides can be examined immediately, care should be taken not to disturb the membrane.  After drying overnight, slides should be sealed with clear fingernail polish to preserve them and kept in the dark afterwards to prevent fluorescent quenching.

Notes:

25mm Swin-Lok Filter Holders


 

Nuclepore Corp.
7035 Commerce Circle
Pleasanton, CA
9 4566-3294
800-882-7711

25mm Black Polycarbanate Filters
0.2 micron                                                   

25mm Plain Polycarbanate Filters
5.0 micron

 

Poretics Corp
111 Lindberg Ave.
Livermore, CA 94550
800-922-6090

Reference:

Brlansky, R. H., R. F. Lee, and E. L. Civerolo.  1990.  Detection of Xanthomonas campestris pv. citrumelo and X. citri from Citrus Using Membrane Entrapment Immunofluorescence.  Plant Dis.  74:863-868.