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Effectiveness of Cleaning Methods for Investigating Diatoms
M. Webber, J. van Dam and E.C. Humphrey*
* Advanced Microscopy Facility, University of Victoria, Canada
Diatoms are routinely chemically cleaned of all organic matter in order to study the silicious wall
(frustule) ultrastructure and to make accurate taxonomic determinations. Since the 1800‘s many
methods have been developed for cleaning organic matter from diatoms [2, 6, 7]. Currently most
techniques use concentrated acids, hydrogen peroxide or bleach. Recent developments in electron
microscopy facilities include instruments for cleaning such as the plasma cleaner, UV (Zone) cleaner
and sputtering by focused ion beam (FIB).
Although chemical cleaning methods remain useful, they are harsh, not often highly selective, nor
well controlled. Diatoms have complex wall structures, with as many as 50 components impregnated
with 10-72% amorphous silica [6] and a complex organic coating lines the inside and outside of the
wall [6, 2]. Chemical cleaning can destroy some features of diatoms useful for taxonomy or other
studies, especially those diatoms constructed of thin or delicate silicious-organic frustules, processes
or with chitin strands. Researchers wanting to study 3D frustule structure [4], the organic casing [6],
energy dispersive x-ray spectroscopy (EDS) and structures of mineral-organics [4], polysaccharide
and chitin secretions, structural and connective relationships with epiphytic, symbiotic or parasitic
organisms, such as bacteria or diatoms [6], may require other techniques for gentle and controlled
cleaning. Thus we researched different chemical methods and plasma, UV and FIB instruments to
determine their effectiveness in a wide range of applications that require gentle, staged and
controlled removal of organics or cutting and cross-sectioning of diatom cell structures.
Most current cleaning techniques use concentrated acids (nitric and sulphuric), 30% hydrogen
peroxide (H2O2) or bleach (5% sodium hypochlorite), often in combination with oxidizers (most
commonly: potassium permanganate and potassium dichromate) [1, 2, 5, 6]. We first reviewed the
effectiveness of seven commonly used cleaning techniques for marine diatoms, via light microscopy
and SEM examination: 1) concentrated and diluted nitric and sulphuric acids (in combination and
singly), 2) 5% sodium hypochlorite (household bleach), 3) 30% hydrogen peroxide, 4) 30%
hydrogen peroxide and hydrochloric acid, 5) 30% hydrogen peroxide and nitric acid, 6) above
chemical methods with acetone, 7) Hydrogen peroxide with potassium permanganate and potassium
dichromate. Today, many researchers favor using 30% H2O2and a hot-water bath because it does
not require concentrated acids, strong oxidizers (with potential violent reactions and Cr toxicity) a
fume-hood and careful safety precautions. The chemical cleaning of diatoms is partly a technical art,
with each genus requiring modifications of the technique. We report on a generally excellent, quick
and relatively safe chemical method that also cleared organics and salts: hydrogen peroxide, nitric
acid, hexane or acetone, followed by washes with distilled water. This method can be varied
somewhat to control the removal of organics.
We demonstrate, with marine diatoms, that plasma and UV cleaning can be highly effective,
especially for the gentle and controlled removal of layers of organics (sometimes only 10 nm thick),
keeping delicate frustules intact (see figs. 1 and 2) and studying environmental samples. Their
limitations and considerations of use, especially when combined with FIB are discussed. And we
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doi:10.1017/S1431927611002200 Microsc. Microanal. 17 (Suppl 2), 2011
© Microscopy Society of America 2011
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show that these EM cleaning and cutting/ablation techniques provide novel opportunities for a wide
range of research avenues previously mentioned.
References
[1] Carr, J., et al. (1986). Am. Microscopical Soc. Vol. 105, No. 152-157.
[2] Hasle G. R. and Fryxell, G.A. (1970) Transactions of the American Microscopic Society 89: 469-
474.
[3] Hecky, R.E., et al. (1973). Mar. Biol. 19, 323 –331.
[4] Hildebrand, M., et al. (2009\. J Struct Biol. 166 (3): 316–328.
[5] Ma, J .C.W. and Jeffery, L.M. (1978). Journal of Microscopy. vol.112. pp. 235-238.
[6] Round, F.E., et al. (1990) The Diatoms, Biology & Morphology of the Genera. Cambridge
University Press, Cambridge, UK.
[7] Van Heurck, H. (1896) A Treatise on the Diatomaceae. Baxter Ltd. London.
FIG 1. UV Zone cleaning. 1a: 0 minutes; 1b: 20 minutes; 1c: higher power to show details of 1b.
FIG. 2. EtOH/acetone first then Plasma cleaning. 2a 0 minutes plasma cleaning, 2b after. 20 minutes
plasma cleaning. Shows how the cleaning is gentle and even (arrows). Further cleaning removes
most of the organics including the bacteria.
Microsc. Microanal. 17 (Suppl 2), 2011 267
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