Before use, the SG06 Special Blend needs to be thoroughly mixed using the supplied magnetic stirrer. This step is facilitated by the very similar density/specific gravity values of marker grains and medium, which also prevents centrifugal sorting from occurring.

A suitable amount of the homogeneous suspension is then added to the volume- or weight- measured sediments. Ideally, in order to obtain absolute pollen concentration measurements with sufficient precision, the number of added Palynospheres™ grains should be greater than the number of fossil pollen grains within the sample. The standard procedure for pollen extraction can then be applied without any modification.

Palynospheres™ can be used with all types of chemicals typically utilized in microfossil extraction. Ideally, the recovery rate of fossil pollen grains, as well as that of the Palynospheres™, should not vary according to the extraction methods. This, however, can be quantitatively checked by the recently proposed standard sample method for microfossils [1]. Palynospheres™ can be used for any analyses of microfossils which are of a corresponding size to the marker grains.

The absolute pollen concentration can be calculated using the following equation:

pollen concentration per unit volume: *c* (grains/cm^{3})

*c* = *p*/*a* × *dv*/*g*

pollen concentration per unit mass: *c´* (grains/g)

*c´* = *p*/*m* × *dv*/*g*

where *p* is the number of pollen counts (grains), *a* is the volume of sediment sample (cm^{3}), *d* is the concentration of the marker grain suspension (grains/cm^{3}), *v* is the added marker grain suspension volume (cm^{3}), *g* is the number of marker grains counted with pollen grains (grains), and *m* is the sample mass (g).

When the sediment accumulation rate *s* (cm/yr) is known, the pollen flux *N* (grains/yr·cm^{2}) can be calculated using the following equation:

*N* = *c*×*s*

If two sizes of marker grains mixed at a known ratio were used (which is typically the case when using SG06 Special Blend, but can also be achieved by using a customized mixture prepared by users from dry powders), then one can check whether sorting (preferential recovery of large or small pollen) occurred during sample preparation.

**References:**

- Nakagawa T, Kitagawa H, Payne R, Tarasov P, Demske D (2013) A standard sample method for controlling microfossil data precision: A proposal for higher data quality and greater opportunities for collaboration. Quaternary International 290–291: 239–244.
- Nakagawa T. & Kitaba, I. (2017) Black ceramic spheres as marker grains for microfossil analyses, with improved chemical, physical, and optical properties. Quaternary International 455: 166–169.