TITLE: Protocol for the double extraction of starch grains and phytoliths from sediments, V.1
AUTHORS: Carlos G. Santiago-Marrero, Jaime R. Pagán-Jiménez

[DESCRIPTION]
Most of the protocols employed for the separation of microbotanical remains from sediments follow the same principles: (1) preparing the sediment samples to be analysed (e.g., weighing, labelling, etc.); (2) deflocculation or particulate dispersal; (3) removing undesired particles (organic matter, carbonates, clay); (4) and extraction of desired remains (e.g., phytoliths, starch grains, fibres or pollen) (Torrence 2006). However, a major concern when applying a combined protocol is the adverse effect that the utilised chemical reagents could have on the proxies intended to be recovered (Pearsall et al. 2004, Torrence 2006, Henry et al. 2016). This is particularly relevant when targeting microbotanical remains with quite different physicochemical properties. For example, protocols for phytolith extraction from sediments typically employ aggressive chemical reagents for the elimination of organic matter, causing severe damage or the full degradation of starch grains (Coil et al. 2003, Piperno 2006, Henry et al. 2016, Le Moyne and Crowther 2021).


Many researchers have addressed the possible ways to extract both microbotanical remains from archaeological sediments in tandem (Pearsall et al. 2004, Torrence 2006, García-Granero et al. 2016, 2018, Henry et al. 2016, Pearsall 2016). One of the most common approaches is the “piggy-back” procedure developed by Chandler-Ezell and Pearsall (2003). This method is based on the gravimetric extraction of lighter starch grains with a density that ranges up to 1.60g/cm³ (Henry et al. 2016) and heavier phytoliths ranging in density up to 2.3g/cm³ afterwards (Pearsall et al. 2004, Piperno 2006). This way, starch grains are not exposed to the reagents employed for removing organic matter and carbonates from sediments. The protocol here presented is a modified version of the protocol for phytoliths and starch grains extraction described by Pearsall 2016 (363 - 364), and the Sodium Polytungstate protocol of phytoliths extraction developed by Madella and colleagues (1998), used at the Universitat Pompeu Fabra Laboratory of Environmental Archaeology. The whole process takes around 7 to 14 days to be completed, although time may vary depending on the amount of residue or sediment to be analysed.

[BEFORE_START]
Lab environment contamination, even cross-contamination of archaeological samples in laboratories, is a constant concern in microbotanical research (Crowther et al. 2014, Henry 2020, Pagán-Jiménez et al. 2016). This is particularly important for starch due to the industrial use of wheat, potato and maize starch in everyday products that may cause false positives (modern starch contaminant) (Fullagar 2006, Crowther et al. 2014, Henry 2020). Once modern starch grains (and phytoliths to a certain extent) are mixed with archaeological sediment samples, it becomes impossible to differentiate between them (Henry 2020). For that reason, it is advisable to follow protocols for avoiding contamination starting in the field, once the archaeological feature or artefact to be analysed has been exposed (Mercader et al. 2017). 


All surfaces and materials in the laboratory should be exhaustively cleaned in advance using a 5% v/v solution of Sodium hypochlorite (Bleach) which hydrolyses and destroys starch grains that may be present in the working area and on equipment (modern contaminants) (Ragheb et al. 1995, Crowther et al. 2014). It is advisable to submerge all equipment (e.g., tubes, vials, spatulas, pipette tips, etc.) in boiling water after cleaning with bleach in order to completely eradicate any potential starch grain contaminant that may have survived the chemical exposure. The ventilation in the laboratory must be turned off, and only one person should be present during the extractions to avoid airborne starch or the air movement and suspension of particles. Hairnets and face masks should be worn all the time, and no gloves should be used during starch grains procedures (even non-powdered globes can contain starch grains). Hands should be constantly washed with a starch-free soapy solution at intervals between sample preparation to avoid any other source of contamination.


Before starting the extraction procedure: label a new sterile pipette tip (100-1000μl) for each sample and place the pipette tip box in a new clean plastic bag. This tip will be used throughout the whole extraction process, leaving at least 4 empty spaces between each tip to avoid cross-contamination. Label a new 50 mL and 2 mL centrifuge tube, and a 10 mL glass vial for each sample, weigh after labelling (with the cap on) and record the weight of each recipient (Figure 1).


 


 
 
Prepare in advance a sample track sheet to record every step in the extraction procedures (Table 1). The format of the sheet may change based on the researcher’s preference, but in general, it should contain five main items related to the protocol’s general steps: (1) Clays deflocculation; (2) Starch grains; (3) Carbonates and organic matter elimination; (4) Phytoliths (5) Microscopy slide mounting (Figure 3).


 
  

References 

-Chandler-Ezell, K. and Pearsall, D.M., 2003. Piggyback microfossil processing: Joint starch and phytolith sampling from stone tools. Phytolitharian Newsletter, 15 (3), 2–8.
-Coil, J., Alejandra Korstanje, M., Archer, S., and Hastorf, C.A., 2003. Laboratory goals and considerations for multiple microfossil extraction in archaeology. Journal of Archaeological Science, 30 (8), 991–1008.
-Crowther, A., Haslam, M., Oakden, N., Walde, D., and Mercader, J., 2014. Documenting contamination in ancient starch laboratories. Journal of Archaeological Science, 49, 90–104.
-Fullagar, R., 2006. Starch on Artefacts. In: R. Torrence and H. Barton, eds. Ancient Starch Research. Walnut Creek, California: Left Coast Press., 177–204.
-García-Granero, J.J., Lancelotti, C., and Madella, M., 2016. A methodological approach to the study of microbotanical remains from grinding stones: a case study in northern Gujarat (India). Vegetation History and Archaeobotany, 26 (1), 43–57.
-García-Granero, J.J., Urem-Kotsou, D., Bogaard, A., and Kotsos, S., 2018. Cooking plant foods in the northern Aegean: Microbotanical evidence from Neolithic Stavroupoli (Thessaloniki, Greece). Quaternary International, 496, 140–151.
-Henry, A.G., ed., 2020. Handbook for the Analysis of Micro-Particles in Archaeological Samples. Cham: Springer International Publishing.
-Henry, A.G., Debono Spiteri, C., Büdel, T., Hutschenreuther, A., Schmidt, S., and Watzke, J., 2016. Methods to isolate and quantify damaged and gelatinized starch grains. Journal of Archaeological Science: Reports, 10, 142–146.
-Le Moyne, C. and Crowther, A., 2021. Effects of chemical pre-treatments on modified starch granules: Recommendations for dental calculus decalcification for ancient starch research. Journal of Archaeological Science: Reports, 35, 102762.
-Madella, M., Powers-Jones, A.H., and Jones, M.K., 1998. A Simple Method of Extraction of Opal Phytoliths from Sediments Using a Non-Toxic Heavy Liquid. Journal of Archaeological Science, 25 (8), 801–803.
-Mercader, J., Abtosway, M., Baquedano, E., Bird, R.W., Díez-Martín, F., Domínguez-Rodrigo, M., Favreau, J., Itambu, M., Lee, P., Mabulla, A., Patalano, R., Pérez-González, A., Santonja, M., Tucker, L., and Walde, D., 2017. Starch contamination landscapes in field archaeology: Olduvai Gorge, Tanzania. Boreas, 46 (4), 918–934.
-Pagán-Jiménez, J.R., Guachamín-Tello, A.M., Romero-Bastidas, M.E., and Constantine-Castro, A.R., 2016. Late ninth millennium B.P. use of Zea mays L. at Cubilán area, highland Ecuador, revealed by ancient starches. Quaternary International, 404, 137–155.
-Pearsall, D.M., 2016. Paleoethnobotany: A Handbook of Procedures. Third. London & New York: Routledge.
-Pearsall, D.M., Chandler-Ezell, K., and Zeidler, J.A., 2004. Maize in ancient Ecuador: results of residue analysis of stone tools from the Real Alto site. Journal of Archaeological Science, 31 (4), 423–442.
-Piperno, D.R., 2006. Phytoliths: A Comprehensive Guide for Archaeologists and Paleoecologists. AltaMira Press.
-Ragheb, A., Abdel-Thalouth, I., and Tawfik, S., 1995. Gelatinization of Starch in Aqueous Alkaline Solutions. Starch - Stärke, 47, 338–345.
-Torrence, R., 2006. Starch in Sediments. In: R. Torrence and H. Barton, eds. Ancient Starch Research. Walnut Creek, California: Left Coast Press., 145–176.




[GUIDELINES]
Recycling of reagents
Instructions for recycling SPT are available on the Sometu website under the “How to use” section of the product description. Link: http://www.sometu.de/howtouse.html.


Disposal of used reagents after use
Used reagents must be discarded following the parameters established by the regulatory entities and university or research regulations of each country.

[STEPS]
SECTION: Clays deflocculation
1. Take a labelled and previously weighed new 50 mL sterile tube and carefully add the corresponding sample to be processed. Cover the mouth of the tube with aluminium foil and poke small holes (Figure 4). This will allow the samples to dry and minimise the chance for airborne contaminants to enter the sample by accident. Put the tube with the sample into the drying oven at a maximum temperature of 32°C to avoid starch gelatinisation. Weigh the tube until it stops losing weight, then the sample will be completely dry. Weigh the tube again with the cap on, and add (up to 45 mL) a 5% v/v solution of Sodium hexametaphosphate (Calgon), close the tube, shake and leave  .                                                                                                 
 

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              

SECTION: Clays deflocculation
2. After 1440 min, shake the samples with Calgon (Figure 5), centrifuge for  3 min at 1500 rpm and discard the supernatant always leaving around 3 to 5 mL of supernatant above the sediment in the tube. Top up with distilled H₂O up to the 45 mL mark and centrifuge again for 3 min at 1500 rpm and discard the supernatant one more time.   Repeat this step at least 4 more times, or until you are able to clearly see through the supernatant in the tube.
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     
SECTION: Clays deflocculation
3. Discard the supernatant once more, leaving around 2 to 3 mL of supernatant in the tube, cover the mouth of the tube with aluminium foil and poke small holes. Repeat the drying process described in step 1, at no more than 32°C. Once dried, weigh the tube with the sample and the cap on, and subtract the original weight of the empty tube to calculate the amount of removed clays.

SECTION: Starch grains extraction
4. Add 5 mL of Sodium polytungstate (SPT) with a specific gravity of 1.8g/cm3. Add more if necessary, it should double the amount of sediment in the tube. Shake gently and centrifuge for  3 min at 1500 rpm.
SECTION: Starch grains extraction
5. Recover all the floating fraction with the corresponding sterile pipette tip and transfer it to the labelled and weighed new 2 mL tube (Figure 6). Do not top up the tube above half its capacity (no more than 1 mL).  
 
  
SECTION: Starch grains extraction
6. SPT removal
Add distilled H2O to the tube with the recovered floating fraction up to the 2 mL mark and centrifuge for 3 min at 7000 rpm. 
SECTION: Starch grains extraction
7. Pipette out the supernatant with the corresponding labelled pipette tip until the 1.5 mL mark, and save the supernatant to recycle the SPT. Top up with distilled H₂O, shake gently and centrifuge for 3 min at 7000 rpm two more times (3 in total). Be mindful that at this point, the density remains high, and starch grains could still be suspended in the middle part of the solution (Figure 7A). Repeat the process two more times until the 1 mL mark, and then two more times until reaching the 0.5 mL mark. Afterwards, do not top up the tube anymore. At this point, the sample should have been washed 7 times in total (Figure 7B).

  
SECTION: Starch grains extraction
8. Starch residue storage
Cover the mouth of the tube with aluminium foil paper and poke small holes (Figure 8). Repeat the drying process described in step 1, at no more than 32°C to avoid starch gelatinisation by heat. Weigh the tube with the dry sample and subtract the original weight of the empty tube to know the amount of the recovered starch sample. Starch samples are ready to be mounted or stored.
 
  


SECTION: Carbonates and organic matter elimination
9. SPT removal from remaining sediments
Add distilled H₂O up to the 45 mL mark to the original 50 mL tube containing the sediment sample, and centrifuge for 3 min at 1500 rpm. Remove the supernatant, always leaving around 3 to 5 mL of supernatant above the sediment in the tube, save the supernatant to recycle the SPT. Top up with distilled H₂O and repeat this step three more times (more if the supernatant is still not clear). 
 

SECTION: Carbonates and organic matter elimination
10. Carbonates elimination                                                                                                                                                                                                                                                                                                                     
Discard the supernatant leaving around 2 to 3 mL  in the tube and add 15 mL of Hydrochloric acid (HCl) at 5% v/v to the 50 mL tubes. This step should be done progressively with a disposable pipette to avoid aggressive reactions. Place the tubes with the samples in a water bath at approximately 40°C until the reaction stops (e.g., no more bubbling occurs when adding HCl). Do not seal the tubes to allow gas to be released.
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                

SECTION: Carbonates and organic matter elimination
11. HCl removal
Top up the 50 mL tube with distilled H₂O up to the 45 mL mark, shake gently and centrifuge for 3 min at 1500 rpm and discard the supernatant always leaving around 3 to 5 mL of supernatant above the sediment in the tube. Repeat this step at least three more times (more if the supernatant is still not clear).
SECTION: Carbonates and organic matter elimination
12. Organic matter elimination
Discard the supernatant leaving around 2 to 3 mL of supernatant above the sediment in the tube and add 15 mL of Hydrogen peroxide (H₂O₂) at 33% v/v. Place the tube with the sample in a water bath at approximately 40°C until the reaction stops (e.g., no more bubbling occurs when adding a drop of H₂O₂). Do not seal the tube to allow gas to be released. 

SECTION: Carbonates and organic matter elimination
13. H₂O₂ removal
Top up the 50 mL tube with distilled H₂O up to the 45 mL mark, shake gently and centrifuge for 3 min at 1500 rpm and discard the supernatant leaving around 3 to 5 mL of supernatant above the sediment in the tube. Repeat this step at least three more times (more if the supernatant is not clear). 

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
SECTION: Carbonates and organic matter elimination
13. 
SECTION: Phytoliths extraction
15. 
Add  15 mL  of SPT with a specific gravity of 2.35g/cm3. Adjust the amount of added SPT if necessary, it should double the amount of sediment in the tube. Shake gently and centrifuge for 3 min  at 1500 rpm. 
SECTION: Phytoliths extraction
16. Recover the floating fraction (residues of interest or silicates) with the corresponding labelled pipette tip and transfer it to a new labelled 50 mL tube. The entire floating fraction has to be recovered. 
 

SECTION: Phytoliths extraction
17. SPT removal
Top up the tube with the recovered floating fraction with distilled H₂O until the 45 mL mark and centrifuge for 3 min at 2000 rpm. Discard the supernatant until the 20 mL mark, leaving the residue at the bottom. Save the supernatant and recycle the SPT. 
SECTION: Phytoliths extraction
19. Phytoliths (silicates) storage
Use the corresponding pipette tip to transfer the remaining H₂O and residues at the bottom of the tube to the corresponding labelled and weighed 10 mL glass vial and let it dry in the drying oven at 60-70°C following the drying process described in step 1. After drying, weigh the vial with the residues (silicates) and subtract the original weight of the vial to know the amount of extracted silicates. The phytolith sample is ready to be mounted on a microscopy slide or stored.
SECTION: Mounting microscopy slides - starch grains
20. Wash a new microscopy slide and coverslip with a 5% v/v solution of bleach. Rinse first with distilled H₂O, and then with ethanol. Let it dry and label the microscopy slide. 
SECTION: Mounting microscopy slides - starch grains
21. With a new sterilised pipette, add a few drops of the mounting medium (a solution of 50% glycerine and 50% distilled H₂O) to the 2 mL tube containing the dried extracted starch sample, mix and let it hydrate for 10 min. Always use a new sterilised pipette per sample. 
 


SECTION: Mounting microscopy slides - starch grains
22. Add a few drops of the solution from the 2 mL tube to the corresponding clean and labelled microscopy slide and cover with a clean cover slip. The slide is ready to be studied under the microscope.
 

SECTION: Mounting microscopy slides - starch grains
23. Once the scanning of the slide is done, store it in a flat position in a microscopy slide folder or microscopy slide storage box.
 

SECTION: Mounting microscopy slides - phytoliths
24. Wash with ethanol, label and weigh a new microscopy slide.
SECTION: Mounting microscopy slides - phytoliths
25. Place around 1 mg of the corresponding extracted silicates on the slide and weigh the slide with the silicates to know the exact weight of the sample on the slide.  
SECTION: Mounting microscopy slides - phytoliths
26. Add a few drops of the mounting medium (in this case, Entellan New®) with a new sterile disposable pipette. Mix the Entellan New® and silicates on the slide with a stainless-steel micro spatula. Wash the spatula with ethanol very well before and after mounting a new slide. 
 

SECTION: Mounting microscopy slides - phytoliths
27. Place a clean cover slip on the sample and leave the slide in a flat position in the fume hood to let the sample sit and eliminate noxious fumes. After a few hours, the slide is ready to be studied under the microscope. 
28. After finishing with the slide scanning, leave the slide for two weeks in the fume hood to let the Entellan New® dry. Then, store the slide in a microscopy slide folder or microscopy slide storage box.
SECTION: Phytoliths extraction
18. Top up with distilled H₂O until the 45 mL mark, shake gently and centrifuge for  3 min at 2000 rpm three more times and discard the supernatant until the 5 mL mark, leaving the residue at the bottom. The supernatant of the last two washes can be discarded. 
SECTION: Carbonates and organic matter elimination
14. Discard the supernatant leaving around 2 to 3 mL of supernatant above the sediment in the tube, cover the mouth of the tube with aluminium foil and poke small holes. Dry in a drying oven at 60-70°C following the drying process described in step 1. Weigh the tube with the dry sample again with the cap on and subtract the original weight of the empty tube to obtain the weight of the remaining sediment sample after the carbonates and organic matter elimination, also known as the Acid Insoluble Fraction (AIF).