I need some help from people who have experience in using DAPI as a vital
staining!!

I'm studying the nuclear cycle of some particular Zygomycetes. For this
doing I incorporate a certain concentration of DAPI to the culture medium,
and, after a certain period of fungal growth, I observe the features of
nuclei along the fungal hyphae. I've observed that some nuclei stain more
brightly than other, and I'm wondering about the reason of this.

I have several questions I'd really appreciate to be answered...

- Do some of you have any experience on possible interferences of DAPI with
the nuclear cycle? It's generally considered as a "vital stain",
semi-permeant (??) and used for cell-cycle studies (Molecular Probes'
Handbook, 6th ed.); however, and although some authors have stated that it
do not reduce the viability of cells (Toda et al., 1981) I still wonder if
it could disturb/interfere:

                                        - DNA replication
                                        - Chromosome condensation
                                        - Mitosis
                                        - RNAs viability

- Kulik and Dery (1995) stated that DAPI reacts only with LIVING nuclei
(!!); Would that meant that it would not stain nuclear debris or DNA
suspensions? What is the real meaning of "vital stain" (in this case,
applied to DAPI)?

- Do you guess any reason for the increased DAPI's fluorescence intensity
in certain nuclei of my cultures compared to others? Could I expect an
increase in fluorescence when the nucleus is endommaged (as in this
situation the DNA would be in a more decondensated/degrading state,
therefore more accesible to the dye)?

- Barcellona et al. (1990) stated that DAPI interacts REVERSIBLY with DNA.
Do any of you know/guess the nature of this reversibility?

- Any hints about possible DAPI degradation/elimination once inside a
living cell?

Please, reply directly to my email address!

Many thanks for your cooperation,

Berta.

Dr. Berta Bago
Centre de Recherche en Biologie Forestiere
Pavillon C.-E. Marchand
Universite Laval, Quebec
G1K 7P4,Canada