Stain-Clearing Methods for Cytological Analysis of Ovule Development

G. L. Hodnett and D. M. Stelly


Stain-clearing is an effective method to prepare specimens for brightfield cytological analysis. It is less laborious than preparation of sectioned specimens, and preserves three-dimensional relationships. Large numbers of specimens can be prepared simultaneously using simple lab equipment. We have developed stain-clearing methods well suited to analysis of ovule development and fertilization in cotton. These methods allow for a range of refractive indices and stain intensities, making them suitable for direct viewing, as well as image processing. The aromatic oils methyl salicylate (MS), dibutyl phthalate (DBP) and benzyl benzoate (BB) were used as clearing agents. These oils can be mixed to change the refractive index from that of dibutyl phthalate (RI=1.50) to that of benzyl benzoate (RI=1.57). Cotton ovules were fixed in FAA(50) for at least 24 hr after which the nucellus was excised and stained at least overnight in water or an ethanol solution buffered to a pH of 4, 7 or 9. The stains used were either azure C, toluidine blue or methylene blue. Destaining occurred in the presence of alcohol but not in the pure clearing medium. Dehydrated samples were cleared by methyl salicylate infiltration. The refractive index was adjusted when necessary by adding either dibutyl phthalate to decrease the refractive index, or benzyl benzoate to increase it. Staining was much darker in the aqueous solutions. A lighter stain gave a much clearer image when using digital image processing, providing a much more detailed image than could be obtained by direct observation in the microscope. Stain-clearing methods were flexible in terms of stain contrast and refractive index, allowing optimization for direct observation or the use of image processing. These stain-clearing techniques can be applied to other cytological studies, e.g. plant pathology.

Reprinted from Proceedings of the 1994 Beltwide Cotton Conferences pg. 702
©National Cotton Council, Memphis TN

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Document last modified Sunday, Dec 6 1998