viability assays

Richard Winder rwinder at PFC.Forestry.CA
Thu Sep 16 10:53:28 EST 1993


In article <1993Sep13.073232.221 at infodev.cam.ac.uk>, mjgh at mbfs.bio.cam.ac.uk (Martin Hughes (Bioc)) writes:
>>In article <26lcir$n6j at bigboote.WPI.EDU> eeyore at wpi.WPI.EDU ( eeYORE ) writes:
>>>Subject: viability assays
>>
>
>[about wanting a viability assay for roots
>
>>>I have tried FDA (fluorescein diacetate) stain, but this is not quantitative
>>>or reliable for plant tissue.  Anyone have any ideas or experience?
>>>                                                                        Melissa
>
>I would also like to add a request.  I have been using FDA to assay
>for viability in protoplasts, which is a "normal" recomended method.
>I am trying (as a control) to kill off protoplasts.  Unfortunately,
>follwing a "death" treatment (30 min in 0.1% NaAzide, or 0.3% KCyanide)-
>which I imagine should be pretty fatal to the cells, the FDA stain
>still shows positive (this is also the case with even higher toxin
>levels).  Any suggestions for a more sensitive method of viability 
>staining would be appreciated.
>Thanks
>-- 
>Martin J. G. Hughes
>Department of Biochemistry, University of Cambridge, Cambridge, UK.
>Email: mjgh at mbfs.bio.cam.ac.uk


If I remember correctly, FDA is not quantitatively reliable in all
circumstances because it is essentially testing the activity of certain
enzymes, and not respiration per se.  Its sole advantage is ease of
measurement.  Tests for membrane integrity are a little more reliable, because
loss of integrity is the usually a consequence (or a cause) of cell death.
Loss of integrity may not happen immediately, so the numbers using an 
integrity stain will still flutter a bit.  Regardless, I've had good results
with Evan's Blue (q.v. Gaff, D. F. and O. Okong'o-ogola.  1971. The use of 
nonpermeating pigments for testing the survival of cells.  Journal of Exp. 
Bot. 22:756-758). I've used this stain sucessfully to probe the activity of 
phytotoxins in root cap cells accoring to the method of Hawes (q.v. Hawes, 
Martha C. 1983. Sensitivity of isolated oat root cap cells and protoplasts
to victorin. Physiol. Plant Path. 22:756-758.)  95% confidence limits on mean
viability  ended up ranging about +- 5% viability.   -RSW

  RICHARD WINDER                    Title: Visiting Fellow
  Forestry Canada                   Phone: (604) 363-0600
  Victoria, B.C.                    Internet: RWINDER at A1.PFC.Forestry.CA



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