Diatom isotope sample preparation for palaeoenvironmental research

This step breaks up aggregated sediment using non-alkaline chemicals so that diatoms can be successfully extracted. Full removal of external organic matter occur later in Step 6 of the protocol.

Place up to 1 cm³ of freeze dried sediment sample in 10-12 ml centrifuge tube labelled with “A” suffix.

Add 0.5mL of 30% volumeH₂O₂at Room temperature in a fume cupboard and allow to react for 0h 30m 0s

Add a further 0.5mL of 30% volumeH₂O₂ at Room temperature in a fume cupboard and allow to react for 3h 0m 0s

Add deionised water (or equivalent) to top of centrifuge tube and shake to combine, ensuring that all of the sample is in suspension. Leave in a fume cupboard.

Tighten lids and 1500rpm

Syphon off solution/suspended material.

Second wash: fill centrifuge tube to top with deionised water (or equivalent) and shake to combine, ensuring that all of the sample is in suspension. Repeat steps 1.5 and 1.6.

Third wash: fill centrifuge tube to top with deionised water (or equivalent) and shake to combine, , ensuring that all of the sample is in suspension. Repeat steps 1.5 and 1.6.

This step aims to separate diatoms from clay/aluminosilicates. A range of heavy density liquids are available, however this protocol uses sodium polytungstate (SPT) which:

• is a non-toxic high density agent;
• creates a non-alkaline solution when dissolved in water;
• can easily be recovered and recycled for future use.
  Safety information

  Ensure appropriate dust mask is worn when handling SPT in dry form to avoid inhalation.

Make up heavy liquid SPT solution to achieve a specific gravity of 2.25-2.30. Ensure SPT powder is fully dissolved before use - solution should be transparent.

Add 4mLof SPT solution to each centrifuge tube and shake to combine, ensuring that all of sample is in suspension.2500rpm

Label a second centrifuge tube with the suffix ‘B’

Add a small amount of deionised water (or equivalent) to the top of the "A" centrifuge tube and use a 3 ml Pasteur pipette to extract the supernate and as much SPT solution as possible into the ‘B’ centrifuge tube without disturbing the precipitate.

Add4mLof SPT solution to the "A" centrifuge tube and shake to combine, ensuring that all of the sample is in suspension.

2500rpm

Repeat step 2.4

Fill "B" centrifuge tube to the top with deionised water (or equivalent) and shake to combine, ensuring that all of the sample is in suspension. 1500rpm

Syphon liquid suspension in "B" centrifuge tube into SPT waste container #1 for recycling (see Section 9).

The SPT separation is repeated for a second time with minor differences to the first SPT separation.

Add 4mLof SPT solution and 0.15mL deionised water (or equivalent) to "B" centrifuge tube and shake to combine. Ensure all of sample is in suspension.2500rpm

Label a second centrifuge tube with the suffix ‘C’

Add a small amount of deionised water (or equivalent) to the top of the "B" centrifuge tube and use a 3 ml Pasteur pipette to extract the supernate and as much SPT solution as possible into the ‘C’ centrifuge tube without disturbing the precipitate.

Fill "C" centrifuge tube to the top with deionised water (or equivalent) and shake to combine, ensuring that all of the sample is in suspension. 1500rpm

Syphon liquid suspension in "C" centrifuge tube into SPT waste container #1 for recycling (see Section 9).

The SPT separation is repeated for a third time with minor differences to previous SPT separations.

Add 4mLof SPT solution and 0.3mLdeionised water (or equivalent) to "C" centrifuge tube and shake to combine. Ensure all of sample is in suspension.2500rpm

Label a second centrifuge tube with the suffix ‘D’

Add a small amount of deionised water (or equivalent) to the top of the "C" centrifuge tube and use a 3 ml Pasteur pipette to extract the supernate and as much SPT solution as possible into the ‘D’ centrifuge tube without disturbing the precipitate.

Fill "D" centrifuge tube to the top with deionised water (or equivalent) and shake to combine, ensuring that all of the sample is in suspension. 1500rpm

Syphon liquid suspension in "D" centrifuge tube into SPT waste container #1 for recycling (see Section 9).

First wash: fill "D" centrifuge tube to top with deionised water (or equivalent). Shake to combine, ensuring all material is in suspension.1500rpm.

Syphon off solution/suspended material.

Repeat steps 4.6 and 4.7 for a second wash.

Material left in the "A", "B" and "C" centrifuge tubes will contain non-diatom contaminants. Residue SPT liquid in these samples should be recovered for recycling.

Add 10mL of deionised water (or equivalent) to "A", "B", and "C" centrifuge tubes and shake to combine, ensuring that all of the sample is in suspension.1500rpm

Syphon liquid (including material in suspension) into the SPT waste container #1 for recycling (see Section 9).

Repeat steps 5.2 and 5.3 twice

At this point the remaining sample material in the "D" centrifuge tube should be mainly comprised of diatoms that are potentially covered in external organic matter. This step removes this organic matter using H2O2. Whilst others have used stronger chemicals such as nitric acid, perchloric acid or even hydrogen fluoride, the use of H2O2 minimises the risks of attacking the diatom frustules and is typically sufficient for removing all organic matter. ₂O₂. Whilst others have used stronger chemicals such as nitric acid, perchloric acid or even hydrogen fluoride, the use of H₂O₂ minimises the risks of attacking the diatom frustules and is typically sufficient for removing all organic matter.

Add 10mLH₂O₂ to “D" centrifuge tube and shake to combine all material. Loosen lids and place in heat block/water bath at75°C in a fume cupboard. Leave for 168h 0m 0s (1 week), topping up centrifuge tubes with H₂O₂ where necessary.

Allow samples to cool toRoom temperature. Tighten lids and 1500rpm.

Syphon off solution and fill centrifuge tube to the top with deionised water (or equivalent). Shake centrifuge tube to combine, ensuring that all of the sample is in suspension. 1500rpm.

Repeat Step 6.3 twice.

Syphon off solution.

Samples at this point will typically contain no/minimal amounts of carbonate. However, this step remains important to ensure that all trace levels of carbonate are fully removed.

Add 5% volume HCl to top of centrifuge tube. Shake to combine, ensuring all material is in suspension. Leave at Room temperature in a fume cupboard.

Syphon off solution.

Fill centrifuge tube to top with deionised water (or equivalent). Shake to combine, ensuring all material is in suspension.1500rpm.

Repeat step 7.2 and 7.3 twice for second and third washes.

Syphon off solution.

At this point in the protocol, an assessment of sample purity is recommended to identify what additional work is required. This also allows the diatom frustules to be checked for evidence of dissolution, diagenesis or other processes that might have caused isotope fractionation.

Initial assessment: Check sample purity under a light microscope (using a smear slide) or inverted microscope [in a plastic petri dish] at ≥ x400 magnification. Contamination can be assessed by eye and/or through quantitative counts using a grid graticule (see Morley et al., 2004).

• If sample is "contaminated", go to Step 8.2.
• If sample appears clean, go to Step 8.3 for further assessment of sample purity.

If sample is contaminated, conduct further clean-up steps based on the type of contaminant in the sample.

Clay/aluminosilicate contamination: complete the sub-steps below in the order listed. Perform an "initial assessment of sample purity" (Step 8.1) between each sub-step to check whether the contaminants have been removed.

• Repeat 3rd SPT wash ( )

• Differential settling (see Morley et al. (2004))

• Sieve sample. In addition to removing contaminants, sieving samples can be useful to remove other forms of biogenic silica and/or separate diatom frustules/species which have different sizes. It is recommended that: 1) prior to sieving, careful visual analysis of the sample under a microscope is carried out to identify what size sieve/sieve cloth is used; and 2) to avoid loss of material, all sieving is done over a 0.45 μm cellulose nitrate membrane filter. This filter can be rolled up and place into the top of a centrifuge tube, filled with deionised water (or equivalent) and 1500rpm to recover the material off the filter. Organic matter

• Repeat organic removal ( ) Other biogenic silica (e.g., radiolaria, sponges)

• Sieve sample (see above)

After further cleaning . If visual inspections show sample can not be purified, either:

• disregard for isotope analysis;
• consider separating diatoms/contaminants using a micro-manipulator (Snelling et al., 2013). As the use of a micro-manipulator is time consuming, it is recommended that this only be done on critical samples.

Further assessments of sample purity: There are a number of options for this and it is suggested that at least one type of assessment is carried out.

• Light microscope (x1000 magnification) using a grid graticule to quantify contamination (Morley et al., 2004).
• XRF - this was originally done by Brewer et al. (2008) who used the data to mass-balance the isotopic impact of non-diatom contamination in samples. We now suggest that XRF is simply used to check sample purity (rather than as the basis for mass-balancing) with contaminated samples either undergoing further purification or disregarded (see Step 8.2). At the University of Nottingham we analyse samples in XRF as a loose powder (not pressed pellet) to ensure material can be recovered afterwards for isotope analysis. As XRF provides a "whole sample" perspective of sample purity, we predominantly use it to check for clay/aluminosilicate contamination. Taking into account the presence of naturally occluded Al within diatom frustules, we require samples to have a Al/Si ratio of at least ≤0.03 and ideally ≤0.02 to be considered "clean".
• Scanning electron microscope (SEM) - this can identify micro-contaminants within a sample (e.g., Brewer et al. (2008)).
• SEM with Energy-Dispersive-X-ray Spectroscopy (EDS) - Chapligin et al. (2012).
• Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) - Chapligin et al. (2012).
• Fourier Transform Infrared (FTIR) - see Swann and Patwardhan (2011).

If samples are free of non-diatom contamination, samples can be analysed for δ¹³C, δ¹⁸O δ³⁰Si. For the analysis of other isotopes that have begun to be developed for diatoms, further steps may be required to remove trace organics. These additional steps are outlined in the citations below, but the need for these (or modification to them) may change as the analysis of these isotopes becomes more widespread.

• δ¹¹B: Donald et al., 2020.
• δ⁶⁶Zn: Andersen et al., 2011.

This protocol can generate significant amounts of SPT waste. However SPT can be easily recovered and purified for future use.

Sieve SPT from waste container #1 through a 5 μm cellulose membrane and place into SPT waste container #2. Use a vacuum pump to expedite this process, although a hand pump can instead be used.

Sieve SPT from waste container #2 through a 1 μm cellulose membrane and place into SPT waste container #3.

Sieve SPT from waste container #3 through a 0.45 μm cellulose membrane and place in into SPT waste container #4.

Place SPT solution into evaporating bowl and into drying cabinet (max temperature should be <105^oC. Once dry, SPT can be ground and reused.