TITLE: Isotope sample preparation of diatoms for paleoenvironmental research
AUTHORS: George Swann, Andreasnelling

[DESCRIPTION]
Isotopes in diatoms are increasingly used in palaeoenvironmental studies in both lacustrine and marine settings, enabling the reconstruction of a range of variables including temperature, precipitation, salinity, glacial discharge, carbon dynamics and biogeochemical cycling. This protocol details an optimised methodology for extracting diatoms for isotope analysis from sediment samples, using a range of chemical and density separation techniques that minimise sample loss and avoids the need for expensive equipment. Whilst designed for the extraction of diatoms for oxygen, silicon and carbon isotope analysis, additional stages are outlined for the analysis of other isotopes that are of increasing interest to the palaeo community (e.g., boron and zinc). The protocol also includes procedures for assessing sample purity, to ensure that analysed samples produce robust palaeoenvironmental reconstruction. Overall, the method aims to improve the quality of palaeoenvironmental research derived from isotopes in diatoms by maximising sample purity and the efficiency of the extraction process.

[GUIDELINES]
This protocol has been designed and tested with a range of different aged marine and lacustrine sediments that have either been "wet" or freeze dried before use. The technique is suitable for all lab users, including masters and PhD students. Whilst the order of Steps 1-7 can be changed, the order listed here is the most efficient in terms of time and maximising the amount of diatom frustules recovered from any sample.

The protocol has been routinely used for diatom δ13C, δ18O, δ30Si analysis and can be extended (as outlined in Stage 9.5) for other isotopes including δ11B and δ66Zn. However, this protocol is not fully compatible with accepted diatom isotope protocols for δ15N. Instead, samples for diatom δ15N should be prepared following Robinson et al. (2004) and Studer et al., (2018). Caution should be exerted when applying this, or indeed any, protocol to living/cultured diatom frustules due to the potential for post‐mortem oxygen isotope exchange (Tyler et al., 2017).

Although not designed for this purpose, the protocol can also be used to extract other forms of biogenic silica, (e.g., siliceous sponges and radiolaria).

[STEPS]
SECTION: Disaggregation of samples
1. 
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.
SECTION: Disaggregation of samples
1.1. Place up to 1 cm3 of freeze dried sediment sample in 10-12 ml centrifuge tube labelled with “A” suffix.
 


SECTION: Disaggregation of samples
1.2. Add 0.5 mL of 30 % volumeH2O2 at Room temperature in a fume cupboard and allow to react for 30 min
 



SECTION: Disaggregation of samples
1.3. Add a further 0.5 mL of 30 % volumeH2O2 at Room temperature in a fume cupboard and allow to react for 180 min 

 


 


SECTION: Disaggregation of samples
1.4. 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.
 


SECTION: Disaggregation of samples
1.5. Tighten lids and 1500 rpm, 5 min

SECTION: Disaggregation of samples
1.6. Syphon off solution/suspended material.
 

SECTION: Disaggregation of samples
1.7. 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.



SECTION: Disaggregation of samples
1.8. 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.
 

SECTION: SPT: 1st separation
2. 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.
 

SECTION: SPT: 1st separation
2.1. 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.

 

SECTION: SPT: 1st separation
2.2. Add 4 mLof SPT solution to each centrifuge tube and shake to combine, ensuring that all of sample is in suspension.2500 rpm, 20 min 
 

SECTION: SPT: 1st separation
2.3. Label a second centrifuge tube with the suffix ‘B’
SECTION: SPT: 1st separation
2.4. 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.

 



SECTION: SPT: 1st separation
2.5. Add4 mLof SPT solution to the "A" centrifuge tube  and shake to combine, ensuring that all of the sample is in suspension.

 

SECTION: SPT: 1st separation
2.6. 2500 rpm, 20 min 

SECTION: SPT: 1st separation
2.7. Repeat step 2.4
SECTION: SPT: 1st separation
2.8. 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. 1500 rpm, 5 min 
 

SECTION: SPT: 1st separation
2.9. Syphon liquid suspension in "B" centrifuge tube into SPT waste container #1 for recycling (see Section 9).
SECTION: SPT: 2nd separation
3. The SPT separation is repeated for a second time with minor differences to the first SPT separation.
SECTION: SPT: 2nd separation
3.1. Add 4 mLof SPT solution and 0.15 mL deionised water (or equivalent) to "B" centrifuge tube and shake to combine. Ensure all of sample is in suspension.2500 rpm, 20 min 

 

SECTION: SPT: 2nd separation
3.2. Label a second centrifuge tube with the suffix ‘C’
SECTION: SPT: 2nd separation
3.3. 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.
SECTION: SPT: 2nd separation
3.4. 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. 1500 rpm, 5 min 
 

SECTION: SPT: 2nd separation
3.5. Syphon liquid suspension in "C" centrifuge tube into SPT waste container #1 for recycling (see Section 9).
SECTION: SPT: 3rd separation
4. The SPT separation is repeated for a third time with minor differences to previous SPT separations.
SECTION: SPT: 3rd separation
4.1. Add 4 mLof SPT solution and 0.3 mLdeionised water (or equivalent) to "C" centrifuge tube and shake to combine. Ensure all of sample is in suspension.2500 rpm, 20 min 

 

SECTION: SPT: 3rd separation
4.2. Label a second centrifuge tube with the suffix ‘D’
SECTION: SPT: 3rd separation
4.3. 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.

SECTION: SPT: 3rd separation
4.4. 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. 1500 rpm, 5 min 
 

SECTION: SPT: 3rd separation
4.5. Syphon liquid suspension in "D" centrifuge tube into SPT waste container #1 for recycling (see Section 9).
SECTION: SPT: 3rd separation
4.6. First wash: fill "D" centrifuge tube to top with deionised water (or equivalent). Shake to combine, ensuring all material is in suspension.1500 rpm, 5 min.
SECTION: SPT: 3rd separation
4.7. Syphon off solution/suspended material.
SECTION: SPT: 3rd separation
4.8. Repeat steps 4.6 and 4.7 for a second wash.
SECTION: SPT residue clean-up
5. 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.
SECTION: SPT residue clean-up
5.1. Add 10 mL 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.1500 rpm, 5 min

SECTION: SPT residue clean-up
5.2. Syphon liquid (including material in suspension) into the SPT waste container #1 for recycling (see Section 9).
SECTION: SPT residue clean-up
5.3. Repeat steps 5.2 and 5.3 twice
 

SECTION: Organics removal
6. 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 (see Tyler et al., 2007) or even hydrogen fluoride, we elect to use H2O2 as it is typically sufficient for removing all organic matter and minimises the risks of attacking the diatom frustules.
SECTION: Organics removal
6.1. Add 10 mLH2O2 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 10080 min (1 week), topping up centrifuge tubes with H2O2 where necessary.
 

SECTION: Organics removal
6.2. Allow samples to cool toRoom temperature. Tighten lids and 1500 rpm, 5 min.

SECTION: Organics removal
6.3. 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. 1500 rpm, 5 min.

SECTION: Organics removal
6.4. Repeat Step 6.3 twice.

SECTION: Organics removal
6.5. Syphon off solution.
SECTION: Carbonate removal
7. 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.
SECTION: Carbonate removal
7.1. 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.
 

SECTION: Carbonate removal
7.2. Syphon off solution.
SECTION: Carbonate removal
7.3. Fill centrifuge tube to top with deionised water (or equivalent). Shake to combine, ensuring all material is in suspension.1500 rpm, 5 min.
SECTION: Carbonate removal
7.4. Repeat step 7.2 and 7.3 twice for second and third washes.

SECTION: Carbonate removal
7.5. Syphon off solution.
SECTION: Purity assessment
8. 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.
SECTION: Purity assessment
8.1. 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.
SECTION: Purity assessment
8.2. 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 1500 rpm, 5 min 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. 
SECTION: Purity assessment
8.3. 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).
SECTION: Purity assessment
8.4. 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.
SECTION: SPT filtering
9.  This protocol can generate significant amounts of SPT waste. However SPT can be easily recovered and purified for future use.
SECTION: SPT filtering
9.1. 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.
 


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


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


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