Most palynologists have probably had an occasional uncomfortable sense that the slide they were working on was not homogeneous, or discovered that two slides of the same sample produced somewhat different results. I have always been struck by one apparent anomaly - that sometimes no specimens of a rare taxon would appear for many traverses and then two would appear nearly adjacent. Other palynologists to whom I have spoken have remarked on the same phenomenon. Knowing palynology's ability to produce biologically and geologically reasonable results, and also knowing how much effort would be required to substantiate suspicions of non-homogeneity, I have always ignored the problem.

When I joined the GSC to work on Tertiary palynology I experimented with an adaptation of the Quaternarist's exotic spike technique to biostratigraphy. The results, which led in an unexpected direction, have been reported in White (1988). A short summary follows.

Palynomorphs observed in pre-Quaternary samples are often brown to black as a result of heating during burial. Initially I wondered if the specific gravity of the palynonomorphs might change during diagenesis, so that an exotic spike such as recent Lycopodium, might not settle homogeneously with the fossil palynomorphs.

Measuring the specific gravity of palynomorphs is not a simple undertaking. The experimental technique finally arrived at was to mix a stock suspension containing three different batches of palynomorphs: alder pollen collected from trees near Calgary; medium maturity Carboniferous spores from the Horton Bluff Formation, Nova Scotia, and very mature Cenozoic palynomorphs from the White Lake Formation, B.C. The suspension was homogenized in a vortex mixer while equal aliquots were withdrawn and put into 11 test tubes. Solutions of water and zinc bromide of specific gravities 1.0, 1.1, 1.2, 1.3, ....to 2.0 were added to the test tubes, the samples stirred, and let sit. The experiment was performed twice with slightly different techniques. The ring of floated material formed at the top of each test tube was recorded, and the float fraction and some sink fractions removed for analysis of their composition.

The results were similar for both experiments. Almost no palynomorphs floated at sp. gr. 1.0 (distilled water), and few floated at 1.1 or 1.2. At sp. gr. 1.3 and above a distinct ring of palynomorphs formed around the top of each test tube. The width of the ring increased with each increasing step in specific gravity. There were still small fractions which sank to the bottom of the test tube even at sp. gr. 2.0. Analysis of slides made from the float fractions showed that high maturity palynomorphs tended to be more abundant in the high specific gravity samples. However, low and medium maturity palynomorphs could be found in the sink fraction at high specific gravities. Although I found it intuitively difficult to accept that such a broad range of specific gravities could exist amongst palynomorphs, the observations were hard to ignore. I could not satisfactorily determine whether the specific gravity range existed in the original palynomorphs (a possibility which would have implications for pollination ecology), or whether it is somehow a product of the processing, perhaps of the heavy liquid solution itself.

Stokes' Law allows an approximation of the terminal velocity of a palynomorph during settling assuming that the palynomorph is a solid sphere. R² (radius squared) is the most important term in Stokes' equation. If one generates a matrix including a range of specific gravities from 1.1 to 1.9 and particle radii from 6 to 50 microns, at one gravity, about three orders of magnitude difference in theoretical terminal velocity can be demonstrated. Although the results are surely too extreme for many reasons, they are sufficient to emphatically dispel one's illusion that palynomorphs are setting homogeneously in the test tubes during settling or centrifugation.

Why are non-homogeneous results not more obvious on the slides? Possibly because when the sample is stirred and resuspended throughout the test tube column, the arrival order of palynomorphs at the bottom is randomized. Possibly also because one's traverse pattern on a slide transects, rather than follows, the smear pattern of slide preparation.

One of the fascinating questions is the putative adjacent appearance of rare types. Could differential sorting explain such a phenomenon? My mentor in fluid mechanics, B. Karney (now at U. of T.), advised me that if two particles did not begin in close proximity, sorting processes would not bring them together. Perhaps the phenological pattern of a plant results in juxtaposition of its pollen or spores in sediment, and some of this pattern is not eradicated by bioturbation or sample processing. However, the question has not been resolved whether this phenomenon is real or only a product of selective memory of a few rare events? A plausible answer to these questions would be interesting to many palynologists.

While it is not possible to avoid differential settling, it may be possible to assess the non-homogeneity it may induce. The possibility of testing the effectiveness of mixing during sample processing emerges from Stokes' Law; particle size is a more important term than the difference in specific gravity between the liquid and the particle. As micro-spheres are available in different sizes, two suspensions of different sized microspheres could be made and added to a sample before processing. A comparison of their ratios in the original suspensions and final slide would be a measure of sample bias caused by differential settling. This analysis could be more important to palynologists practising high resolution Quaternary palynology than those involved in stratigraphic palynology. Possibly some of the noise typical of influx diagrams could be eliminated by a measure of sample bias.

Reference

White, J.M. 1988. Methodology of the exotic spike: differential settling of palynomorphs during sample preparation. Pollen et Spores 30:131-148.

---

Notes added in April, 2003

On rereading Benninghoff 's (1962) paper, I noticed that he suggested using particles of different size to test for differential settling.

Ogden (1986) documented the high resistance of NEN microspheres to most standard palynological preparation techniques, except for strong oxidation. However, months after leaving a suspension of NEN microspheres stirring in glycerine on a vortex mixer, we checked the suspension, and found that the microspheres had disappeared! A NEN product representative speculated that the stirring bar had eventually eroded the coating on the microspheres, and the contents had dissolved. From his perspective, this was potentially a useful technique for recovering valuable radioisotopes from bad batches of microspheres! Obviously, anybody interested in using microspheres would need to develop a different method of mixing a homogeneous suspension, or be assured of their stability by the supplier. We reverted to using Lycopodium tablets as a spike because of the routine convenience of the technique.

Biostratigraphers do not normally use an exotic spike in a geological study of continental rocks because it is unusual to have any fine control of sedimentation rate, so that useful influx estimates are unobtainable. However, in the analysis of the Mallik 2L-38 gas hydrate research borehole in the Mackenzie River delta, I have found palynomorph concentration estimates to be a useful tool for differentiating different sedimentary units, even if the changes were due to different pollen productivity or different sedimentation rate.

References

Benninghoff, W. S. Calculation of pollen and spores density in sediments by addition of exotic pollen in known quantities. Pollen et Spores, IV, 2, 332-333.

Ogden, J. G. III, 1986. An alternative to exotic spore or pollen addition in quantitative microfossil studies. Canadian Journal of Earth Sciences 23:102-106.

---

Note: This item orginally appeared in CAP Newsletter 12(2):28-30, 1989. It has been slightly updated.