As Dick notes in the article below, Calcofluor is effective in labeling cellulose, but not starch. If the microalgae of interest are chlorophytes, then Calcofluor will stain the cell wall (though the mucilaginous capsule on some might diminish dye uptake). Calcofluor has been used in vivo as I recall, to label the growing cellulose portion of red algal cell walls. In the other microalgae that store starch, you may see Calcofluor wall labeling in some dinoflagellates, but not in cryptomonads. Iodine staining will not be very dense in the so-called Floridean starch (a more glycogen-like polysaccharide) of red algae. I've never looked at storage products in the other microalgae, so I don't know. With green algae you may be able to distinguish starch deposition within the chloroplast using confocal microscopy and no added dye. Starch should appear as dark areas surrounding the pyrenoid within fluorescing chloroplast (488nm excitation and fluorescence after, say a 650nm long pass filter should work well to image chlorophyll a fluorescence.) If you have sufficient spatial resolution to get several sections per cell, you may get an estimate of the cellular volume occupied by starch. But find an expert in confocal microscopy. Dave ---------------- David M. Coder, Ph.D. Consultant in cytometry email: d_coder@msn.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Richard Haugland" <richard.haugland@probes.com> To: cyto-inbox Sent: Thursday, February 07, 2002 2:04 PM Subject: Re: Starch in microalgae > > No warranties on this one but Calcofluor White M2R (Fluorescent brightener > 28, Sigma) may work but staining may not be very specific. Lectins are > another possible alternative. > > Biotech Histochem 1992 Mar;67(2):88-97 > > Block-surface staining for differentiation of starch > and cell walls in wheat endosperm. > > Glenn GM, Pitts MJ, Liao K, Irving DW. > > Western Regional Research Center, USDA-ARS, Albany, California 94710. > > A staining technique for differentiating starch > granules and cell walls was developed for computer-assisted studies of starch > granule > distribution in cells of wheat (Triticum aestivum L.) > caryopses. Blocks of embedded caryopses were sectioned, exposing the > endosperm tissue, and stained with iodine potassium > iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining > was > avoided by using stains prepared in 80% ethanol and > using short staining times. The IKI quenched background fluorescence which > facilitated the use of higher concentrations of > Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor > staining > combination compared to Calcofluor alone. The high > contrast between darkly stained starch granules and fluorescent cell walls > permitted computer assisted analysis of data from > selected hard and soft wheat varieties. The ratio of starch granule area to > cell area > was similar for both wheat classes. The starch granule > sizes ranged from 2.1 microns 3 to 22,000 microns 3 with approximately 90% > of the granules measuring less than 752 microns 3 (ca. > 11 microns in diameter). Hard wheat samples had a greater number of small > starch granules and a lower mean starch granule area > compared to the soft wheat varieties tested. The starch size distribution > curve > was bimodal for both the hard and soft wheat > varieties. Three-dimensional starch size distribution was measured for four > cells near the > central cheek region of a single caryopsis. The > percentage of small granules was higher at the ends than at the mid-section > of the cells > > > > Adelina de la Jara wrote: > > > I am a new list member and use to look up this e-mail archive to solve my > > questions. Now I would like to ask the list if anyone can suggest a good > > fluorescent dye for monitoring starch accumulation in microalgae? > > > > Many thanks. > > > > Adelina de la Jara Valido > > Instituto Tecnologico de Canarias > > Dpto. de Biotecnologia > > Pozo Izquierdo 35119 Santa Lucia, Gran Canaria, Spain > > Tfno.: 0034 928 133290 > > Fax: 0034 928 132830 > >
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