TITLE: Dung Spherulite Analysis and pH Testing
AUTHORS: bubest, natalia.vanzojais, dejonge, Kali R Wade

[DESCRIPTION]
The dung spherulite and pH testing methods used for sediment samples from Yavne, Israel, based upon the methods from Smith et al. 2019. 

[STEPS]
SECTION: Fail-Safe Practices
1. Wiping down all equipment, surfaces, and tools used with soap and water followed by acetone (beginning and end of every day of the project).
SECTION: Fail-Safe Practices
2. Plating one slide of Cargille Immersion Oil (Type B) and viewing them under microscopy to ensure no contamination.
SECTION: Fail-Safe Practices
3. Calibrating analytical balances.  
SECTION: Sample Preparation - pH testing
4. Washed 20 x 150 mL beakers and labeled them 1-20. 
SECTION: Sample Preparation - pH testing
5. Added 10.00 g of sample from each sample bag in their respective numbered beaker. I then wrote down the weight of the actual weights of the 10 g in an Excel sheet. 

SECTION: Sample Preparation - pH testing
6. Added 25mL of water into each of the beakers. I ensured there was enough water to insert the pH probe without it touching sediment after it settled. 
SECTION: Sample Preparation - pH testing
7. Left samples to sit overnight.
SECTION: pH Testing
8. Using the Vernier software (the Vernier software that we will be using is the same as the pH meter and has a USB cable which allows us to see the pH values on our computer); https://www.vernier.com/downloads/graphical-analysis/.  The bottom part of the probe that measures the pH must be completely submerged in the water but cannot touch the sediment that settles at the bottom. Ensure there is enough distilled water to place the probe in the proper place. 
SECTION: pH Testing
9. The same pH meter is being used for all 20 samples. After each sample the equipment must be carefully rinsed with distilled water to avoid contamination.
SECTION: Dung Spherulite Analysis
10. Labeled every slide “dung spherulites - Yavne sample name” 
SECTION: Dung Spherulite Analysis
11. Placed the slide with the drop of Cargille Immersion oil (Type B, code 1248) on the slide into the analytical scale, the slide was placed on a plastic cap to make it more easier to take out and put in the slides in the scale. Everything was then zeroed/tared. 
SECTION: Dung Spherulite Analysis
12. ~0.0020g of sample was placed on the slide in the Immersion oil. The weight was recorded. 
SECTION: Dung Spherulite Analysis
13. Removed the slide from the analytical balance. Spread the sample and immersion oil together with the very edge of the coverslip to ensure no sample loss from the slide. Dispersed the sediment as evenly as possible and placed the coverslip on top of the slide. 
SECTION: Microscopy Analysis
14. Plug in the microscope, green button turns on, and light button is another separate button says "hal 100" above it. Turn that on as well.
SECTION: Microscopy Analysis
15. Start at low magnification and increase to 400x, to view dung spherulites, with cross-polarizing filter on.  
SECTION: Sample Preparation - pH testing
6. Added 25 mL of water into e.ach of the beakers. I added 5 mLmore than stated in the protocol because there needed to be enough water to insert the pH probe without it touching sediment after it settled. 
Protocol mentioned above: Smith, A., Proctor, L., Hart, T.C. et al. The burning issue of dung in archaeobotanical samples: a case-study integrating macro-botanical remains, dung spherulites, and phytoliths to assess sample origin and fuel use at Tell Zeidan, Syria. Veget Hist Archaeobot 28, 229–246 (2019). https://doi.org/10.1007/s00334-018-0692-9.

SECTION: Sample Preparation - pH testing
7. Leave the pH samples   to settle

SECTION: pH Testing
8. Using the Vernier Software, we imported pH measurements into an Excel. 
The Vernier software that we used is the same company as the pH meter and has a USB cable which allows us to see the pH values on our computer.  Software is downloadable here: https://www.vernier.com/downloads/graphical-analysis/
SECTION: pH Testing
9. Held the probe under the top of the water and above the sediment, 20 s, to get an accurate reading. 

SECTION: pH Testing
10. The same pH meter is being used for all 20 samples. After each sample the equipment must be carefully rinsed with distilled water to avoid contamination.
SECTION: Sample Preparation - Dung Spherulite Analysis
11. Weighed ~ 2.00 g of crude samples into a small weight boat. I did not record the actual weight because they are just going to be sifted and we will lose weight. We were aiming for ~1.00 g of material after being shifted (why I weighed ~ 2.00 g of crude). 

SECTION: Sample Preparation - Dung Spherulite Analysis
12. Crushed all the samples using a mortar and pestle; using 10 mortars and pestles with a separate one for each sample in order to avoid contamination. After the first ten samples, I washed the 10 mortar and pestles with laboratory soap, rinsed with distilled water,  and crushed the remaining samples. 
SECTION: Sample Preparation - Dung Spherulite Analysis
13. Sieved samples through a 120 micrometer mesh; we had 2 screens and to prevent as little contamination as possible, I dumped the sample in a little section of the screen. Each sample was sieved individually with the mesh being cleaned between every sample. To clean the meshes, pressurized clean air was blown through the screen, tapped, and brushed with a clean toothbrush. 

SECTION: Sample Preparation - Dung Spherulite Analysis
14. Samples were placed in a labelled, small weigh boat after sieving, weighed, and the weight recorded. 
SECTION: Sample Preparation - Dung Spherulite Analysis
15. All microscopy slides were labelled with “dung spherulites Yavne sample name” 
SECTION: Sample Preparation - Dung Spherulite Analysis
16. Placed the slide with the drop of Cargille Immersion Oil (Type B, code 1248) on the slide into the analytical scale. The slide was placed on a plastic cap to make it easier to take out and put in the slides in the scale. Everything was then zeroed/tared. 
SECTION: Sample Preparation - Dung Spherulite Analysis
17. 0.0020 g of sample was placed on the slide in the Immersion oil. The weight was recorded. 

SECTION: Sample Preparation - Dung Spherulite Analysis
18. Used the cover slide to mix the Immersion oil with the sample, to try to evenly disperse the sediment/sample across the slide, then placed the same coverslip onto the slide. 
SECTION: Microscopy Analysis - Dung Spherulites
19. Plug in the microscope, green button turns on, and light button is another separate button says "hal 100" above it. Turn that on as well. Knob on the right side above the on switch is how you turn up and turn down the light.
SECTION: Microscopy Analysis - Dung Spherulites
20. Start at low magnification and increase to 400x
SECTION: Microscopy Analysis - Dung Spherulites
21. Have setting at 5 dic and have little lens by the difference sizes of lenses be roman numeral 2 and match the knob on the condenser to roman numeral 2 as well
SECTION: Microscopy Analysis - Dung Spherulites
22. Scan the slide at 400x, noting presence of any dung spherulites viewed under cross polarized light. Cover slips were gently tapped to rotate bodies within the mounting agent. Mechanical stage is necessary for this type of analysis.