Quaternary International 455 (2017) 166e169

Contents lists available at ScienceDirect

Quaternary International
journal homepage: www.elsevier.com/locate/quaintBlack ceramic spheres as marker grains for microfossil analyses, withimproved chemical, physical, and optical properties
Ikuko Kitaba a, b, c, *, Takeshi Nakagawa c, baResearch Center for Inland Seas, Kobe University, Kobe 657-8501, Japan
Research Centre for Palaeoclimatology, Ritsumeikan University, Kusatsu 525-8577, Japanc
Department of Geography, University of Newcastle, Newcastle upon Tyne NE1 7RU, UKba r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 January 2017
Received in revised form
15 August 2017
Accepted 22 August 2017
Available online 5 September 2017Marker grain method is commonly used by palynologists to quantify absolute pollen concentration in
sediments. DuPont NEM plastic microbeads and Lycopodium spore tablets are two of the most commonlyused marker grains. The DuPont NEM series has an obvious advantage over Lycopodium tablets because
Lycopodium is not guaranteed ‘exotic’ in many ecosystems (especially in the past). Moreover, NEM also
has high visibility, easily identiﬁable spherical shape, and availability in different size ranges. However,the production of NEM has been discontinuous since more than a decade, and palynologists of the world
are using the precious remainders of their own stock in very small (¼suboptimal) quantities. Here we
propose a solution. Microspheres of black ceramic can be used as marker grains for a wide range of
microfossil analyses (in this paper, however, we mainly discuss the use of the material for pollen analysis
because of the expertise of the authors). The ceramic spheres are tolerant to all chemicals commonlyused during pollen extraction processes and is extremely resistant to physical stresses. The particles arematt black, spherical, available in a wide range of size clusters, have a density very close to that of fossil
pollen grains (1.424 g/cm3 vs. 1.494 g/cm3 in average), and don't change color and density over a longstorage in acidic liquids. The behavior of ceramic spheres was closer to the pollen grains than the
Lycopodium spores regardless of the pollen concentration and composition in sediments. In combination,these properties make the black ceramic spheres an even better solution for palynologists than DuPont
NEM microbeads or Lycopodium spore tablets. The absolute pollen concentrations estimated fromceramic spheres and those estimated from volume method were indistinguishable within errors. On theother hand, when the samples were relatively poor in fossil pollen grains, the concentrations calculatedby Lycopodium method tended to be signiﬁcantly overestimated. The ceramic microbeads are available
either as a dry powder in different size ranges, or as a mixture of two size ranges blended in a deﬁned
ratio and dispersed in a buoyancy-neutral liquid, ready to be added to sediment samples. Mixing two
different size ranges in a known ratio serves to detect any laboratory failures that differentially favor
recovery of larger or smaller pollen types.
© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

Keywords:
Marker grains
Microbeads
Black ceramic spheres
Absolute pollen concentration
Pollen analysis
Microfossil analysis

1. Introduction
The absolute quantity of microfossils is a very useful parameter(Bonny, 1972 and references therein). For instance, with quantitative data we can discuss pollen productivity, presence or absence ofsorting or ultraviolet decomposition of organic matter and biomassin sparse vegetation (e.g. boreal forest or arid zone). Because the* Corresponding author. Research Centre for Palaeoclimatology, Ritsumeikan
University, Kusatsu 525-8577, Japan.
E-mail address: i-kitaba@fc.ritsumei.ac.jp (I. Kitaba).

absolute pollen concentration is independent from frequencies ofpollen grains of other taxa, it often reﬂects environmental changes
more directly than percentage values. Adding known-amount ofmarker grains, such as Lycopodium spores and DuPont NEM plastic
microbeads series was making those scientiﬁc approaches possible(Stockmarr, 1971; Ogden, 1986).
The DuPont NEM microbeads series has been, however, sorely
missed since its production was discontinued. The microbeads
method was ﬁrst proposed in 1986 (Ogden, 1986) and, since thattime, the DuPont product has attracted a number of enthusiastic
users (e.g. Wang and Geurts, 1993; Turetsky et al., 2004; Fedotov

http://dx.doi.org/10.1016/j.quaint.2017.08.052
1040-6182/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

I. Kitaba, T. Nakagawa / Quaternary International 455 (2017) 166e169

167

et al., 2012; Sugaya et al., 2016). Unfortunately, since the DuPont
NEM series was discontinued, no alternative marker grain productof comparable quality has been introduced. The DuPont NEM
microbeads were supplied in various size ranges and had highvisibility under the microscope due to their uniform sphericalshape and matt black color. With an average density of 1.369 g/cm3,
they were also close to that of fossil pollen grains (1.494 g/cm3 inll and Everitt, 1996). The microbeads were also inert
average; Regne
and tolerant of the chemical treatments used in conventional
palynological procedures (HCl, KOH, ZnCl2, acetolysis, and evenconc. HF). The synthetic microbeads from DuPont NEM therefore
offered what seemed to be an ideal marker grain for palynologicalresearch and so the discontinuation of the product line sent shockwaves throughout the palynology community.
Here we report a high quality alternative to the DuPont NEM
microbeads. As well as being tolerant of all palynological processing
chemicals, including HF, this novel marker grains does not decolorize or swell after lengthy storage in liquid, which was somethingof an issue with the DuPont NEM materials. Its speciﬁc gravity isalso closer to that of fossil pollen grains than the DuPont
microbeads, which ensures more coherent movement of pollen andmarker grains during pollen extraction. This paper aims to assess
the performance of this new product in comparison with the conventional Lycopodium spore tablets.
2. Materials and methods
We tested the black ceramic spheres (PALYNOSPHERES;
Fig. 1aeb) obtained from a UK-based company called Palynotech(http://www.palynotech.com/). The density of the microbeads is1.424 ± 0.008 g/cm3 (2s) (Table 1), which, according to our measurements by Archimedes method, does not depend on their size
clusters. The ceramic spheres are available in ﬁve size ranges
centered at ca. 23 mm (coded “Orange”), 27 mm (“Blue”), 34 mm(“Green”), 38 mm (“Yellow”) and 46 mm (“Red”) (Table 2). (N.B. The
color codings are simply for convenience; the actual color of thepowder is matt black in all size ranges). Two marker grains, coded“Red” and “Orange”, are easily distinguishable by sizes under the
microscope (Fig. 1a, b, d). Using the mixture of small and large
marker grains enables to check the size dependency of the microfossil recovery rate after the treatment of sediments.
In this research, PALYNOSPHERES SG06 Special Blend (Batch no.
SG0605; Fig. 1a and b) was used for the experiment. The SG06
Special Blend is a mixture of two easily distinguishable size clustersof ceramic microbeads (coded “Orange” and “Red” e see above)
suspended in a buoyancy-neutral carrying media. The Orange/Red
mixing ratio is ca. 2.9 (exact ratio is variable across batches; theproduct label provides more precise and lot speciﬁc value), whichcounterbalances the higher visibility of larger grains. The carrier
media (sodium bromide; NaBr) has speciﬁc gravity of 1.40 ± 0.05
(measured at 20  C; temperature dependence unknown). This
media density has been chosen to be quasi-buoyancy neutral withthe marker grains so that re-dispersal is easily achieved. The media
contains non-ionic detergent, Tween 20. The marker grain concentrations differ to a small degree from lot to lot, but are generally
in the region of 1.8 ± 0.1  104 grains/ml and 6.2 ± 0.4  103grains/
ml for the small and large grains, respectively. Exact concentrations
to 3 decimal places are provided by the manufacturer for eachproduction lot and are detailed on the product label. The lot No.
SG0605, the one that we used in this experiment, contains
6.17 ± 0.42  103 large (‘Red’) grains and 1.78 ± 0.13  104 small(‘Orange’) grains per ml, according to the lot-speciﬁc product label.
The number of grains in 1 ml of the formulated material is equivalent to approximately two Lycopodium tablets manufactured by
Lund University (Stockmarr, 1971), which makes it easy to convert

Fig. 1. Photos of (a, b) the black ceramic spheres. (c) Amorphous (i.e. non-spherical)
grains. (d) The marker grains and Lycopodium spores added to the sediment, treatedand extracted by a normal preparation protocol (Nakagawa et al., 1998) along withfossil pollen grains. L denotes the Lycopodium spore. (eef) New and conventional
microbeads after storage in suspension liquid for long-term. DuPont NEM microbeads(f) are loosing its color after storage in suspending solution for 13 years. The marker
grains proposed in this paper (e) were developed very recently and hence such lengthy
stability check has not been carried out. However, the oldest available lot, which has
been stored in the buoyancy neutral medium described in this paper for almost 5
years, is not showing any recognizable changes in optical properties. Scale bars
represent 100 mm and 20 mm in (a) and (bee), respectively. (For interpretation of thereferences to colour in this ﬁgure legend, the reader is referred to the web version ofthis article.)

Table 1Comparison of density.
Density (g/cm3)
Fossil pollen grainsa
Black spherical ceramic powderb
DuPont NEM series
Lycopodium sporec

1.494 ± 0.028
1.424 ± 0.008
1.369 ± 0.013
1.377 ± 0.016

ll and Everitt (1996).
After Regne
The product (PALYNOSPHERES) discussed in this paper.
c
Lycopodium tablet (Batch no. 3862) made by Lund University.

abTable 2Available size clusters of black ceramic spheres.
Color code

Particle size (mm)OrangeBlue
Green
Yellow
Red

22.6 ± 2.0
27.2 ± 3.6
33.7 ± 3.7
38.0 ± 3.2
45.5 ± 3.86

The color codes are simply for convenience. The color of
microspheres is matt black in all size clusters.

between Lycopodium tablets and PALYNOSPHERES in the laboratory
procedures.
The pollen analysis was carried out to compare the behavior of
the black ceramic spheres and Lycopodium spores. The DuPont NEM

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I. Kitaba, T. Nakagawa / Quaternary International 455 (2017) 166e169

series was excluded from the comparison simply because theproduct is no longer available. The SG12 core from Lake Suigetsu,
Japan (35 350 0800 N, 135 520 5600 E, Suzuki et al., 2016) were used forthe analysis. Based on the preliminary result of pollen analysis of
the core (unpublished data), six 1-m long sections (7.7 m, 12.5 m,
18.5 m, 25.8 m, 33.0 m, 38.9 m), which contain most of typicalpollen taxa in Japan (Gotanda et al., 2002), were chosen. The
rejected materials from each target depth were homogenized into abatch of “standard sample” (Nakagawa et al., 2013). Then four
samples of 1.50 ± 0.05 wet g were taken from six standard samples.
We then treated those 24 samples in an identical procedure as
follows: two Lycopodium tablets and 1 ml of well mixed PALYNOSPHERES SG06 Special Blend were added to all samples (24 samples
in total); pollen slides were prepared by the “dense-media separation” method of Nakagawa et al. (1998), i.e. 10% HCl treatment(room temperature for 12 h); 10% KOH treatment (90  C for
10 min); washing with water for 7 times; washing with 10% HCl;dense media separation (ZnCl2 of density 1.90, 1800 rpm for
10 min); acetolysis treatment (sulfuric acid: acetic anhydride ¼ 1:9v/v, 90  C for 10 min); washing with water twice; ethanol treatment
twice; mounting with 100% glycerin. The method is widely used by
the palynologists of the world as it allows preparing remarkably
clear pollen slides with relatively small effort, with no signiﬁcantdistortion of pollen composition data (Campbell et al., 2016). At
least 400 pollen grains were counted under the optical microscopeat 400  magniﬁcation. The pollen grains were classiﬁed into twogroups: pollen-L and pollen-S. Pollen-L means large grains with airsacs, i.e., Abies, Picea and Tsuga. Pollen-S type contains all other taxa.
Spores, Lycopodium spores and black ceramic spheres were alsocounted at the same time.
Absolute pollen concentration and its error were calculated
based on the volume method and two marker grain methods using
black ceramic sphere and Lycopodium tablet. The absolute pollen
concentration by volume method was calculated using thefollowing equation:c ¼ p=m  a=a0  v=v0 ;(1)where c denotes pollen concentration per unit mass (grains/g); p is
the number of pollen grains counted (grains), a is the area of coverglass (cm2), a’ is the observed area (cm2), v is total volume of residue (cm3), v’ is the volume of mounted residue (cm3), and m is the
sample mass (g).
The pollen concentration by marker grain method was calculated using following equation:

c0 ¼ p=m  dc=g;(2)where c’ denotes pollen concentration per unit mass (grains/g); p is
the number of pollen grains counted (grains), d is the concentrationof the marker grain suspension (grains/cm3), c is the volume of theadded marker grain suspension (cm3), g is the number of marker
grains counted with pollen grains (grains), and m is the sample
mass (g).
3. Results and discussion
3.1. Usability of PALYNOSPHERESDuring the experiment, we observed a total of 17,321 black
ceramic grains under microscope. The product that we tested
exhibited the following characteristics.
3.1.1. Chemical and physical properties
We did not observe any chemical or physical degradations of theceramic spheres through treatments by acids and alkalis. After thetreatment, the black ceramic spheres were not decolorize anddamaged (Fig. 1d). It was not even possible to squash the spheres byﬁngers between slide and cover glasses, which was not the casewith the discontinuous DuPont NEM series.
Amorphous (i.e. non-spherical) marker grains are occasionallyfound amongst more healthy ceramic spheres (Fig. 1c). The frequency of such amorphous grains which were not clearly distinguishable from organic matter was <4%. Presence of the amorphousgrains, however, does not cause distortion of pollen concentration
data because they are not included in the marker grain concentration speciﬁed on the product label.
3.1.2. Optical properties
The black spherical ceramic powder exhibits a high level ofvisibility and a distinct character under the microscope (Fig. 1d),being matt black and spherical. The grains do not suffer from edge
diffraction artifacts, nor do they decolorize over time (Fig. 1e).
3.2. Comparison of pollen concentration between different methods
The absolute pollen concentrations were calculated from threemethods, i.e. volume method and two different microbeads
methods using ceramic spheres and Lycopodium tablet, respectively. The result of comparative experiment is shown in Table 3 andFig. 2. The error bars in Fig. 2 denotes standard deviation amongfour analysed samples deriving from the same ‘standard’ batch. The
absolute pollen concentrations estimated from ceramic spheres
and those estimated from volume method were indistinguishable
within errors, regardless of the pollen composition and sample
depth. By contrast, the quantiﬁcation by Lycopodium tablets werevery variable depending on the pollen composition. Using thepollen-rich samples, which contain more than 98% of Pollen-S type(7.7 m and 12.5 m), the absolute pollen concentration obtained byvolume and Lycopodium methods were relatively close. The absolute pollen amounts estimated from Lycopodium is only 1.3e2.1
times larger than that from volume method. When the samples
were relatively poor in fossil pollen grains, however, the concentrations calculated by Lycopodium method tended to be signiﬁcantly overestimated. The absolute pollen concentration estimatedby Lycopodium method is by 16e164 times larger (18.5 m and25,8 m). This indicates that Lycopodium spores were preferentiallylost in the process of pollen extraction. Relatively low density of
Lycopodium spores, when compared with fossil pollen grains and
PALYNOSPHERES is the most likely cause of the loss (Table 1).
PALYNOSPHERES, by contrast, has almost identical behavior withthe true fossil pollen grains because their densities are remarkably
similar (Table 1).
4. Advantages over alternative/conventional products
4.1. Comparison with DuPont NEM microbeads
We believe that when used as marker grains, the black ceramic
spheres are not only a quality alternative to the now discontinued
DuPont NEM microbeads, but that they actually offer a number ofphysical, chemical and optical advantages over the historic product.
The black ceramic spheres do not break, dissolve, swell or decolorize, maintaining good visibility over a long period of storage. This
was not necessarily the case with the DuPont NEM microbeads,
which faded in color on prolonged exposure to liquid (mixture of 1%
HCl solution, approximately 30% ZnCl2 and a small amount of washup liquid) (Fig. 1f). The speciﬁc gravity of the black ceramic spheresis closer to that of fossil pollen grains than the DuPont NEM mall and Everitt, 1996) (Table 1), making them a better
terial (Regne

I. Kitaba, T. Nakagawa / Quaternary International 455 (2017) 166e169

169

Table 3Pollen concentrations calculated using three different methods.
Method7.7 m

12.5 m18.5 m25.8 m33.0 m38.9 mVolume method
PALYNOSPHERESa
Lycopodiumb2.12 ± 0.19  105
2.77 ± 0.85  105
3.38 ± 0.47  105

1.16 ± 0.15  105
1.42 ± 0.10  105
1.97 ± 0.20  105

6.96 ± 1.40  103
8.65 ± 1.74  103
1.68 ± 0.34  105

3.38 ± 0.46  103
4.25 ± 0.69  103
3.61 ± 1.12  105

1.35 ± 0.23  104
1.50 ± 0.19  104
7.16 ± 1.55  105

6.26 ± 2.27  103
8.66 ± 3.32  103
5.18 ± 1.36  105ab

PALYNOSPHERES SG06 Special Blend (Batch no. SG0605).
Lycopodium tablet (Batch no. 3862) made by Lund University.

closer density to fossil pollen grains, increased chemical andphysical durability, and availability in either format as a dry powder
or formulated suspension in a buoyancy-neutral liquid with
controlled concentration. The ceramic spheres are not lost signiﬁcantly during the course of the dense-media separation treatment,
which was not necessarily the case with Lycopodium spores when
the sample is pollen-poor.

Acknowledgements
We are very grateful to Dr. Tom Johnston for meeting all requests
with regard to the required specs of the product, as well as for
improving the manuscript. This study was supported by grants
from the United Kingdom Natural Environment Research Council(NERC) (grant NE/D000289/1 to T. Nakagawa), as well as a German
DFG project (grant TA-540/3-1 to P. Tarasov) and a Japanese
KAKENHI projects (grant 24840028 to I. Kitaba; grant 23221002 to
R. Tada; grant 211001002 to H. Yonenobu).
Fig. 2. Comparison of the pollen concentrations quantiﬁed by three different methods.
The error bars are 1s. The pie charts show the pollen and spore composition of eachsample. The identiﬁed palynomorphs were classiﬁed into three groups: pollen-S,
pollen-L and spores. Pollen-L group contains Abies, Picea and Tsuga, the pollen grainsof which are relatively large and have air sacs. Pollen-S contains all the other taxa.
Spores include both monolete and trilete spores.

microfossil surrogate for extraction control.
4.2. Comparison with Lycopodium spore tablets
The dense color, spherical shape and uniformity of black ceramic
spherical marker grains (Fig. 1a, b, d, e) offers overwhelming advantages to operators compared to the Lycopodium tablets produced by Lund University (Stockmarr, 1971). In contrast to
Lycopodium spores, the artiﬁcial marker grains do not exist in nature and thus can be used for all types of naturally derived samples
from any location (for example, Lycopodium spontaneously grows
in Japan). Microscopic examination is made much easier courtesy of
their uniform shape and high visual contrast, allowing even inexperienced users to signiﬁcantly reduce their analysis time (Fig. 1d).
Interestingly, the density of the black ceramic spheres is closer tothat of raw fossil pollen grains than that of the commerciallyavailable acetolysed Lycopodium spores (Table 1). The supply of theblack ceramic spheres is stable whereas that of the Lycopodium
spore tablet essentially relies on the volunteer spirit of the Lund
University.
5. Conclusion
Black ceramic marker grains substitute discontinuous DuPont
NEM marker grains. They inherit all essential properties of theconventional product, and have added advantages such as even

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