Perhaps a vive-versa attempt could help: try a dead cell stain...

Trypan Blue od Evan's Blue stain dead cells in blue

(and both stains showdark red fluorescence as an alternative readout).

Though the spore walls could block the dead cell stain as efficiently as a live cell stain - just try ;-)

@Mr Decken:

Thank you very well for the proposed staining with evans blue. I just tried staining of fresh material and could not detect any dead cells. The positive control - fixed material (ethanol:glacial acedic acid (3:1)) - showed clear colouring of the cells by staining with 1% evans blue in destilled water ...

So it seems as if the exine blocks the staining solution quite well.

You might want to try cell impermeable fluorescent dyes-- Hoechst 33258, Propidium iodide, etc.

Dear Dr. Ralph Heinrich,

hope you will have found reasons for the not staining of your microspores...

Not working in your field (plants, spores) nevertheless I would like to add some information here (you might already know but if not, perhaps could be useful):

Apologize if my post is too long but I prefer to include here not only names of stains/dyes/solutions but some excerpt out from the text also so you can see whether something of dyes not mentioned by the earlier postings could fit for your preparations...

(1) @ http://www.ncbi.nlm.nih.gov/pubmed/1703673

Stain Technol. 1990; 65(5): 251-258. (I know: old one!)

Vital fluorescent staining technique for microspores of Brassica napus.

Swanson E.B., Yarrow S.A., Coumans M.P., and Erickson L.R.From the abstract:

“…The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores.”

(2) @ http://journal.ashspublications.org/content/132/6/777.full

Journal of the American Society for Horticultural Science

JASHS November 2007 vol. 132 no. 6 777-78

Abnormal Microspore Development Leads to Pollen Abortion in a Seedless Mutant of ‘Ougan’ Mandarin (Citrus suavissima Hort. ex Tanaka)

Zhiyong Hu, Min Zhang, Qigen Wen, Jie Wei, Hualin Yi and Xiuxin Deng

„...fixed overnight in 5 formalin : 5 acetic acid : 90 alcohol [FAA (by volume)] at 4 °C…. were squashed and stained with 1% acetocarmine in 45% acetic acid to observe microsporocyte morphology.”

(3) @ www.ib.uj.edu.pl/abc/pdf/46/20_vizi.pdf Acta Biol. Cracoviensia Series Botanica 46, 177–183, 2004

In vitro manipulation of Cucumber (Cucumis sativus L.) pollen and microspores: Isolation procedures, viability tests, germination, maturation.

By: Vizintin L & Bohanec B

From the text: “….... Five common methods for estimating the viability of fresh or damaged microspores or pollen grains of cucumber were tested. Two methods (acetocarmine according to Jahier, 1996; p-phenylenediamine according to Rodriguez-Riano and Dafni, 2000) were excluded as not suitable for cucumbers by preliminary testing (data not shown). The methods examined in detail were aniline blue in lactophenol, FCR and MTT.

.... Aniline blue in lactophenol completely stained both fresh (Fig. 2a) and damaged microspores, but clearly distinguished between undamaged and aborted mature pollen… FDA sufficiently discriminated putatively viable and non- viable microspores and pollen grains (Fig. 2 b,e) and MTT similarly showed very good contrast between putatively viable and non-viable microspores and pollen grains..”

(4) @ http://www.scielo.br/scielo.php?pid=S1516-89132006000500002&script=sci_arttext

Brazilian Archives of Biology and Technology, Print version ISSN 1516-8913

Braz. arch. biol. technol. vol.49 no.4 Curitiba July 2006

@ http://dx.doi.org/10.1590/S1516-89132006000500002

Isolation and culture of soybean (Glycine max L. Merrill) microspores and pollen grains.

Rodrigues L.R., de Camargo Forte B., Bodanese-Zanettini M.H.

From Text:„...Microspore viability was determined by fluorochromatic reaction to FDA in samples at 0 and 28 days of incubation. Microspore responses during culture were analyzed under the microscope at 0, 14, 28 and 42 days of incubation in samples fixed in Farmer's solution (100% ethanol: glacial acetic acid, 3:1) and stained with propionic-carmine. Fifty four samples were collected from BRSMT Uirapuru suspension before culturing and analyzed simultaneously by fluorochromatic reaction to mithramicin according to Coleman and Goff (1985) and propionic carmine staining. The normality test, ANOVA and correlation analysis were performed.

….

Comparing fluorochromatic reaction to mithramycin and stainability to propionic-carmine, a total of 26,236 microspores and pollen grains were analyzed. The fluorochrome allowed significant better characterization of the morphology of typical and atypical nuclei.”

Best wishes and good luck,

Wolfgang

Salzburg, Austria