INQUA Working Group on Data-Handling Methods

Newsletter 9: January 1993


Edward J. Cushing
Department of Ecology, Evolution & Behavior
University of Minnesota
1987 Upper Buford Circle,
St. Paul, MN 55108.

One of the advantages of silicone oil as a mounting medium for pollen analysis is its stability over time. Its volatility is very low, so slides may be left unsealed to permit greater control over turning grains for examination, measurement, and photomicrography. For reference slides that receive much handling, however, sealing the coverglass is desirable to prevent leakage of the oil. Unfortunately, many common compounds used for sealing slides react over time with pollen exines mounted in silicone oil. The experience in our laboratory with various compounds is summarized here, followed by our current recommendations.

Fingernail polish was the first sealing compound we used. Pollen grains in slides sealed with nail polish begin to deteriorate noticeably after several months to several years. We first called it "nail-polish disease," but now I suggest "pollen pox," because the first symptoms are pits on the ektexine surface that resemble the pock marks of chicken pox and smallpox. These are especially noticeable on grains with fine sculpturing and thick exines, and they often appear first on the grains of Corylus that we add as a control to our reference preparations, following the recommendation of Faegri & Iversen (1964). With time, the exine softens and becomes plastic, as may be demonstrated by squashing a grain between coverglass and slide. Fine sculptural and structural details disappear. Ultimately the exine becomes completely amorphous, apertures disappear, and the grains round up into hollow or solid spheres. Early in the process the grains begin to adhere to the slide or coverglass and will no longer move when pressure is applied to the coverglass, which provides a useful test of quality. Most slides we sealed with nail polish deteriorated within three years, although a few are still serviceable after 30 years.

We have since tested a variety of sealants, including various brands of fingernail polish, epoxy cements, silicone rubber cements, casein glues, varnishes, lacquers, waxes, and paints. Most have failed for various reasons, of which pollen pox is but one. An ideal sealing compound for pollen slides in silicone oil should (1) adhere firmly to glass, (2) be sufficiently fluid to flow under the coverglass and displace any air between the silicone oil and the edge of the coverglass, (3) be immiscible with silicone oil, (4) harden within a few hours with little shrinkage, (5) have no ingredients that will dissolve in silicone oil when the sealant is fluid and later crystallize out in the oil, (6) have no ingredients that will react with sporopollenin (as in pollen pox), (7) be stable over many years, (8) resist, after hardening, objective immersion fluids (immersion oil, anisol) and other solvents (water, xylene) used to clean slides, (9) be easy to mix and apply. No compound that we have tested meets all these criteria. One proprietary brand of epoxy cement, no longer available, was very satisfactory. Other epoxy formulations failed, but experimentation with this group of compounds should be fruitful.

The cause of pollen pox remains uncertain, although circumstantial evidence points to dibutyl phthalate, a plasticizer commonly used in nail polishes, glues, and cements. Dibutyl phthalate does soften and dissolve exines experimentally. A mixture of dibutyl phthalate and silicone oil forms two phases, and pollen grains in the mixture occur preferentially in the dibutyl phthalate phase. In sealed slides, I presume the dibutyl phthalate diffuses from the sealant through the oil to the pollen. Other factors are involved, too. Sporopollenin varies in its resistance to pollen pox; in general, gymnosperm pollen and pteridophyte spores are less susceptible than angiosperm pollen. Slides containing many pollen grains, or large grains, may be little affected, presumably because the concentration of dibutyl phthalate (if that is the causative agent) is insufficient in relation to the quantity of sporopollenin. Grains that are hydrated (i.e., that have a high water content) are less susceptible than grains that are thoroughly dehydrated, perhaps because water molecules occupy the sporopollenin sites that the dibutyl phthalate would bind to.

At present I recommend either of two imperfect sealants. Paraffin, applied under the coverglass, makes a neat and effective seal. However, paraffin wax when molten is soluble in silicone oil, and platy crystals of paraffin are likely to appear over time in the slide, sometimes obscuring the pollen grains. This can be minimized by heating the slide only just enough to melt the wax. We place small fragments of paraffin under the edge of the coverglass when it is placed on the slide and melt these over a very small gas flame, with care not to heat the silicone oil directly. Cool the slide quickly. Paraffin waxes with low melting points may be the best, but I have not tested this variable systematically.

We are now using with success a commercial brand of latex enamel paint (Tru-Test latex gloss enamel). It has the proper viscosity, shrinks little on drying, and seals well. The paint is water-based, so that pollen in slides sealed with it is fully hydrated. However, the seal dissolves slowly in anisol and xylene and softens in water, so care in cleaning slides is necessary. Slides sealed with this paint four years ago remain in excellent condition, with no deterioration of pollen or extraneous crystals in the preparation. Unfortunately, the composition of the paint is proprietary and may vary from brand to brand and over time within a brand. The risk is that one may make thousands of slides with a particular sealant only to find, after several years, that they are deteriorating.

If others have experience with other kinds of sealants, I would be glad to hear about them.

Copyright © 1993 Edward J. Cushing
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