Yeast Methods

Richard Haugland richard.haugland at probes.com
Thu Jul 5 22:50:47 EST 2001


I suggest they take a look at the FUN-1 stain, which is metabolized only by
viable yeast to a red-fluorescent structure (see pictures in the link):

http://www.probes.com/servlets/product?region=USA&item=7009

Here  are seven references on this

http://www.probes.com/servlets/bib?item=7009

including the paper

Appl Environ Microbiol 1997 Jul;63(7):2897-905

                       Development of the FUN-1 family of fluorescent probes for
vacuole labeling and viability testing of
                       yeasts.

                       Millard PJ, Roth BL, Thi HP, Yue ST, Haugland RP.

                       Molecular Probes, Inc., Eugene, Oregon 97402, USA.
paulm at probes.com

                       A new family of fluorescent probes has been developed for
assessing the viability and metabolic activity of yeasts. This class of
                       halogenated unsymmetric cyanine dyes is exemplified by
the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-
                       methylidene)-1-phenylquinolinium iodide] stain, a
membrane-permeant nucleic acid-binding dye that has been found to give rise
                       to cylindrical intravacuolar structures (CIVS) in
Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts
                       yielded CIVS that were markedly red shifted in
fluorescence emission and therefore spectrally distinct from the nucleic
                       acid-bound form of the dye. The formation of CIVS
occurred under both aerobic and anaerobic conditions and was highly
                       temperature dependent. Treatment of yeasts with the
nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP
                       levels approximately 6-fold and completely inhibited CIVS
formation. Under aerobic conditions, the formation of CIVS was
                       abrogated by the cytochrome oxidase inhibitors azide and
cyanide; however, the H+ transport uncoupler carbonyl cyanide
                       m-chlorophenylhydrazone inhibited CIVS formation under
both aerobic and anaerobic conditions. Depletion of cellular thiols,
                       including glutathione, with millimolar concentrations of
N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited
                       CIVS production. Marked reduction in the formation of
CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione
                       S-transferase, suggested that dye processing can involve
enzyme-mediated formation of glutathione conjugates. The
                       conversion of FUN-1 by S. cerevisiae was studied
quantitatively by using several techniques, including fluorometry, flow
                       cytometry, and wide-field and confocal laser scanning
fluorescence microscopy.


A search of our bibliography for "yeast" will give you almost all the other dyes
that have been used for staining yeast, including all the others that you
mention except acriflavin, which we don't make:

http://www.probes.com/servlets/bibsearch/




Jan Hesketh wrote:

> I have had an enquiry from someone in the brewing industry regarding flow
> cytometry methods for yeasts. They are specifically interested in Cell cycle
> analysis with PI, Viability with PI/ Fluorescein diacetate, Intracellular
> pH, Neutral lipids with Nile Red?, Glycogen with acriflavin, Mitochondria
> with Rhodamine 123, flocculation with ConA-FITC lectin.
>
> I have had no experience with any of these areas and was hoping that someone
> out there may be able to point me in the right direction. Any tips or
> references would be much appreciated.
>
> Thanks,
>
> Jan Hesketh
> Molecular Medicine
> University of Auckland
> phone 09 373 7599 ex 4492
> fax   09 373 7492




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