TITLE: HEPES-Phosphate Medium, Suitable for Studies of Trace Element Nutrition in Photoautotrophic and Heterotrophic  Auxenochlorella protothecoides.
AUTHORS: Dimitrios Camacho, Charles Perrino, Sabeeha Merchant

[DESCRIPTION]
This protocol describes a method for preparing a defined medium for phototrophic and heterotrophic Auxenochlorella protothecoides (UTEX 250). It is adapted from the commonly used TAP medium for Chlamydomonas reinhardtii (Goodenough, 2023, Chapter 11), with special attention to trace element supplementation (Kropat, Hong‐Hermesdorf, et al., 2011) and buffer composition. The Tris-acetate buffer is replaced with a HEPES buffer to control acidification of the growth medium as cells consume glucose. As a starting point, we determined the elemental composition of laboratory-grown Auxenochlorella protothecoides cells by ICP-MS/MS (Hui et al., 2022) and calculated how much of each essential element would be required in the growth medium to support growth of a culture with an initial density of 105 cells / mL to stationary phase (2.4 x 108 cells / mL). The elemental composition of the medium before and after growth of cells to stationary phase was also measured to ensure that each element was provided in excess (2- to 5-fold) to accommodate variation in elemental quotas in response to the external conditions. All elements provided in the medium were measured except for H, C, N, O, and Cl. High purity chemicals (>99.999% pure with certificate of analysis indicating the level of contamination with other salts) are required for successful elemental deficiency and all glassware and plasticware are freshly washed with 6 M HCl (Quinn & Merchant, 1998). 

[BEFORE_START]

 
Obtain only high purity chemicals which are usually labelled as “metals basis,” “extra pure,” or “>99.999%.” More importantly, check the “Specifications” tab of each chemical to verify that it has been assayed for the elements that you are interested in. Choose brands that have the lowest allowance for the elements that your lab is interested in. Contact the chemical’s manufacturers to get the certificate of analysis (CoA) for each chemical. Specify the lot number you have chosen so that the correct item is shipped.

When chemicals arrive, record each lot number and label the bottles with the date received, date opened, and quantity (e.g. 1 of 5). High purity trace metal grade chemicals typically have a higher cost than normal chemicals, so it is recommended that they be labeled and their use restricted for trace metal work. Avoid inserting spatulas into containers; instead, carefully shake or pour chemicals out. Chemicals brought out of the original container should not be returned into the container. Use plastic, non-metal, spatulas to handle chemicals.

Navigate to the supplier’s website and enter the lot number in the “Certificates” search tab. Download and review the certificate of analysis (CoA) of the specific batch. For example, you may find the certificates search tab for Millipore Sigma at this link: https://www.sigmaaldrich.com/US/en/documents-search?tab=coa You can view an example CoA for potassium dihydrogen phosphate by entering 1.05108 as the product number and B1642708 as the batch number. Record the values of each element you are interested in studying and calculate the potential concentration of contaminating metals expected in the final medium. See an example of this calculation for potentially up to 0.005 ppm of Cu contamination from potassium dihydrogen phosphate in equation 1. These estimates will provide insight into potential contamination, but ultimately, you should measure the final medium.

 
The final medium may have as much as 117 picomolar (pM) of Cu from KH2PO4 alone. Total potential Cu contamination in the medium will be the sum of potential Cu contamination from all chemical components. See table 2 for potential contamination from macronutrient stocks if 1 ppm of each metal is present in each chemical.


 

Use only ICP-MS grade ultrapure Milli-Q H2O, which can be sourced for example, from the Milli-Q® Advantage A10 Water Purification System (Model: Z00Q0V0T0). This system is internally outfitted with an A10 UV lamp (Cat# ZFA10UVM1), a Q-GARD® T2 pack filter (REF QGARDT2X1), and a Quantum® TIX Ultrapure Cartridge (REF QTUM0TIX1). An additional filtration step using a Q-POD® Element with a Quantum® ICP filter (REF QTUM00ICP) ensures ppt (parts per trillion) to sub ppt levels of trace elements. Before using the Milli-Q H2O, verify that the resistivity is 18.2 MΩ.cm at 25 °C and the total organic carbon (TOC) reading is <10 ppb.

[GUIDELINES]
Download all Safety Data Sheets (SDS) for each chemical you will purchase. Review the proper handling and disposal guidelines from your institution for each chemical. Review your municipal wastewater drain disposal guidelines before disposing any chemicals down the drain. Wear approved personal protective equipment (PPE) such as a lab coat, closed toe shoes, eye protection, and gloves.

To avoid precipitation in the stock solutions and the final medium, it is important to add the chemicals in the specified order. Various methods can be used for mixing solutions. To mix macronutrient stock solutions, we recommend slowly adding chemicals to the acid washed 1 L bottles that are prefilled with the specified volumes of Milli-Q H2O and mixing by shaking the tightly capped bottle vigorously. Alternatively, you may slowly add solutes to 1 L bottles containing an acid washed PTFE coated 55 mm magnetic stir bar (VWR 76264-442), placed on a magnetic stirrer at  . Minimize exposure of solutions to dust particles by closing bottles while mixing. For micronutrient solutions, we recommend adding chemicals slowly to the 250 mL bottle filled with the specified volume of Milli-Q H2O and placing the tightly capped bottle on a rocker for 30 min. Some chemicals may not dissolve completely unless titrated to the proper pH. To avoid metal contamination from a pH probe, we recommend using metal free pipette tips to transfer 30 µL of the solution onto an MQuant pH-indicator strip (non-bleeding) pH 5.0 – 10.0 (Supleco 1.09533.001). Do not insert the pH strip into the solutions. 
 
For hygroscopic chemicals, we recommend ordering the smallest amount that will meet your needs and reconstituting the entire bottle into solution immediately after opening. Chemicals labeled as anhydrous, ultra-dry, dehydrated, or hygroscopic will have different molecular weights than chemicals containing hydrates. Most ultra-pure chemicals are only available in the hygroscopic form, so be sure to verify the molecular weights and the masses required to achieve the concentrations in this protocol. When hygroscopic chemicals are exposed to atmospheric moisture, they will gain mass, so it is important to process the entire vial to generate a solution of known concentration immediately after opening. Search for the chemical’s solubility in water and obtain an appropriately sized acid washed HDPE bottle with a pre-drawn line indicating the fill level. Fill the HDPE bottle halfway with Milli-Q H2O. Add the chemical to the HDPE bottle containing water. Rinse the vial with water and pour the water from the rinse into the HPDE stock bottle. Repeat rinse three times, transferring each rinse to the HDPE bottle. Fill the bottle to the fill line. Mix the solution well. Label the solution with the estimated concentration and store at 4 °C. The actual concentration can be verified by ICP-MS/MS measurement. 

[STEPS]
SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
2. Do not use glass containers to store stock solutions as glass will leach metal contaminants. Use new (previously unused) wide-mouth polypropylene / translucent high-density polyethylene (HDPE) bottles. For macronutrient stock solutions, prepare five 1 L bottles (Nalgene 2104-0032). For micronutrient stock solutions, prepare ten 250 mL bottles (Nalgene 2104-0008) and one bottle for 2 Molarity (M) NaOH. Keep the bottles capped as much as possible to avoid dust from entering the bottles.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
4. Discard the Milli-Q H2O used in step 3 and add 250 mL of fresh (unused) 6 Molarity (M) HCl to one bottle. Cap bottle and swirl to wash the interior. Pour into the next bottle and repeat for all stock solution bottles. Rinse bottles with Milli-Q H2O at least 7 times.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
3. For each bottle, fill with 100 mL, 250 mL, or 1 L, of Milli-Q H2O to mark the fill line. This can be achieved by weighing the bottle with the appropriate amount of water. Repeat for all bottles.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
7. Rinse each flask with Milli-Q H2O seven times (Quinn & Merchant, 1998). Rinse the exterior of the flasks as well so that there is no HCl residue. 6 Molarity (M) HCl will corrode work surfaces. Leave cleaned flasks covered with parafilm so that no dust particles in the air (potentially with contaminating metals) enter the flasks.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
5. To clean culture flasks, perform an initial 6 Molarity (M)HCl wash by filling a 250 mL culture flask with 100 mL 6 Molarity (M) HCl and swirl. Pour used HCl into the next flask and repeat for all flasks.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
6. For the secondary wash, add 200 mL of 6 Molarity (M) HCl to each flask. Cover each flask with parafilm and leave in the fume hood for 1440 min or more. Ensure that the parafilm is properly adhered to the flasks so that they do not get sucked into the fume hood intake vents. HCl should never be left in the fume hoods uncovered. It is corrosive and may destroy the hoods.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
8. Re-use or discard used HCl. The 6 Molarity (M) HCl from the secondary rinse of culture flasks can be re-used up to 5 times or until a color change is observed (whichever comes first). HCl used for the first round of culture flask and stock solution bottle washing should not be reused and should be neutralized safely before discarding.

SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
9. To neutralize 1 L of 6 Molarity (M) HCl, fill a 4 L beaker with 1 L water and put it in a secondary container within a fume hood. Slowly add 1 L used 6 Molarity (M) HCl to dilute to 3 Molarity (M) HCl. Neutralize 2 L of 3 Molarity (M) HCl by adding NaHCO3 (Arm & Hammer pure baking soda) slowly, scoop by scoop (< 25 g) until no foam is formed. Use a pH indicator strip to verify that the acid is safely neutralized (pH 7). Please refer to your institutional and municipal guidelines for disposing liquids with high concentrations of NaCl.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
10. Make 1 Molarity (M) HEPES Stock (1 L)

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
10.1. Dissolve 238.3 g HEPES in approximately 500 mL Milli-Q H2O and fill to 1 L. Mix well until dissolved. 

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
10.2. Do not adjust pH.
SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
10.3. Store at 4 °C.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
11. Make 2 Molarity (M) NaOH Stock (100 mL)

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
11.1. Add 8 g of NaOH and fill to 100 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
11.2. Store at Room temperature in a properly designated cabinet for strong bases.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
12. Make 100× Phosphate Solution (1 L).

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
12.1. Dissolve 153.77 g of KH2PO4 in approximately 600 mL of Milli-Q H2O. Then add 38.15 g of KOH and fill to 1 L using Milli-Q H2O. Do not adjust the pH. Mix well until dissolved

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
12.2. Store at 4 °C.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
13. Make 40× Macronutrient Solution (N, Ca, Cl, Mg, and S) (1 L). 

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
13.1. Dissolve 1.5 g of fresh anhydrous CaCl2 in approximately 300 mL of Milli-Q H2O. Alternatively, use a liquid stock of CaCl2.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
13.2. In a separate acid washed bottle, dissolve 11.55 g MgSO4·7H2O and 16.05 g NH4Cl in approximately 500 mL of Milli-Q H2O completely.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
13.3. Mix slowly with CaCl2 solution and fill to 1 L with Milli-Q H2O. Mix well until dissolved.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
13.4. Filter-sterilize the solution, using a   pore syringe filter with proper sterile technique in a sterile hood.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
13.5. Store at 4 °Cand only open the bottle in the sterile hood.
 

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
14. Make 2 millimolar (mM) (1000×) thiamine stock solution. 

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
14.1. Dissolve 675 mg of thiamine hydrochloride in approximately 800 mL of Milli-Q H2O.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
14.2. Fill to 1 L. Mix well until dissolved.

SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
14.3. Filter-sterilize the solution using a   pore syringe filter in a sterile hood using proper sterile technique.
SECTION: Make the macronutrient, thiamine, and buffer stock solutions on a bench near balances, rockers, and/or stirrers. Perform the filter sterilization (steps 13.4 and 14.3 ) in a sterile hood. Record the actual mass of chemicals added to each solution.
14.4. Store at 4 °C. Open bottle in the sterile hood only.

SECTION: Make the preliminary concentrated stock solutions of trace elements.
15. Make Pre 1 - 125 millimolar (mM) Na2EDTA concentrate (250 mL)

SECTION: Make the preliminary concentrated stock solutions of trace elements.
15.1. Dissolve 11.63 g of Na2EDTA·2H2O in 180 mL of Milli-Q H2O. Note pH needs to be adjusted to completely dissolve Na2EDTA. See next step. 

SECTION: Make the preliminary concentrated stock solutions of trace elements.
15.2. Titrate to pH 8 with trace element grade2 Molarity (M) NaOH solution (from step 11). Use 43 mL – 47 mL of 2 Molarity (M) NaOH. Measure the pH by pipetting 30 µL of solution onto a pH-indicator strip. Record the volume of 2 Molarity (M) NaOH used.

SECTION: Make the preliminary concentrated stock solutions of trace elements.
15.3. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make the preliminary concentrated stock solutions of trace elements.
15.4. Store at 4 °C

SECTION: Make the preliminary concentrated stock solutions of trace elements.
16. Make Pre 2 - 285 micromolar (µM)(NH4)6Mo7O24 (250 mL)
SECTION: Make the preliminary concentrated stock solutions of trace elements.
16.1. Dissolve 88 mg of (NH4)6Mo7O24·4H2O in Milli-Q H2O and fill to 250 mL. Mix well until dissolved.
SECTION: Make the preliminary concentrated stock solutions of trace elements.
16.2. Store at 4 °C

SECTION: Make the preliminary concentrated stock solutions of trace elements.
17. Make Pre 3 - 1 mM Na2SeO3 (250 mL)

SECTION: Make the preliminary concentrated stock solutions of trace elements.
17.1. Dissolve 43 mg of Na2SeO3 in Milli-Q H2O and fill to 250 mL. Mix well until dissolved.

SECTION: Make the preliminary concentrated stock solutions of trace elements.
17.2. Store at 4 °C .

SECTION: Make individual metal · EDTA stock solutions (1000×)
18. 
 

Make 25 millimolar (mM) Na2EDTA (250 mL).

SECTION: Make individual metal · EDTA stock solutions (1000×)
18.1. Add 50 mL of Pre 1 (125 millimolar (mM) Na2EDTA concentrate from step 15). 
SECTION: Make individual metal · EDTA stock solutions (1000×)
18.2. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
18.3. Store at 4 °C .
SECTION: Make individual metal · EDTA stock solutions (1000×)
19. Make 28.5 micromolar (µM) (NH4)6Mo7O24 (250 mL).

SECTION: Make individual metal · EDTA stock solutions (1000×)
19.1. Add 25 mL of Pre 2 (285 micromolar (µM) (NH4)6Mo7O24 from step 16). 

SECTION: Make individual metal · EDTA stock solutions (1000×)
19.2. Fill to 250 mL  with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
19.3. Store at 4 °C .

SECTION: Make individual metal · EDTA stock solutions (1000×)
20. Make 0.1 millimolar (mM) Na2SeO3 (250 mL).

SECTION: Make individual metal · EDTA stock solutions (1000×)
20.1. Add 25 mL of Pre 3 (1 millimolar (mM) Na2SeO3 from step 17).

SECTION: Make individual metal · EDTA stock solutions (1000×)
20.2. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
20.3. Store at 4 °C.

SECTION: Make individual metal · EDTA stock solutions (1000×)
21. Make Zn·EDTA stock solution (250 mL).

SECTION: Make individual metal · EDTA stock solutions (1000×)
21.2. Add 720 mg of ZnSO4·7H2O.

SECTION: Make individual metal · EDTA stock solutions (1000×)
21.1. Add 22 mL of Pre 1 (125 millimolar (mM) Na2EDTA concentrate from step 15)

SECTION: Make individual metal · EDTA stock solutions (1000×)
21.3. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
21.4. Store at 4 °C.

SECTION: Make individual metal · EDTA stock solutions (1000×)
22. Make Mn·EDTA stock solution (250 mL).

SECTION: Make individual metal · EDTA stock solutions (1000×)
22.1. Add 24 mL of Pre 1 (125 millimolar (mM) Na2EDTA concentrate from step 15).
SECTION: Make individual metal · EDTA stock solutions (1000×)
22.2. Add 594 mg of MnCl2·4H2O.
SECTION: Make individual metal · EDTA stock solutions (1000×)
22.3. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
22.4. Store at 4 °C.

SECTION: Make individual metal · EDTA stock solutions (1000×)
23. Make Fe·EDTA stock solution (250 mL) (does not use Pre1). 

SECTION: Make individual metal · EDTA stock solutions (1000×)
23.1. Combine 2.05 g Na2EDTA·2H2O with 580 mg of Na2CO3 and dissolve completely in 100 mL of Milli-Q H2O

SECTION: Make individual metal · EDTA stock solutions (1000×)
23.2. If using fresh anhydrous FeCl3 powder, add 811 mg of anhydrous FeCl3 to the solution only after the previous two components are completely dissolved. Alternatively add trace metal grade FeCl3 liquid stock to a final concentration of 20 millimolar (mM).

SECTION: Make individual metal · EDTA stock solutions (1000×)
23.3. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
23.4. Store at 4 °C.

SECTION: Make individual metal · EDTA stock solutions (1000×)
24. Make Cu·EDTA stock solution (250 mL).

SECTION: Make individual metal · EDTA stock solutions (1000×)
24.1. Add 4 mL of Pre 1 (25 millimolar (mM) Na2EDTA concentrate from step 15).

SECTION: Make individual metal · EDTA stock solutions (1000×)
24.2. Add 67 mg of fresh anhydrous CuCl2 or add liquid stock solution (see note above) to a final concentration of 2 millimolar (mM).

SECTION: Make individual metal · EDTA stock solutions (1000×)
24.3. Fill to 250 mL with Milli-Q H2O. Mix well until dissolved.

SECTION: Make individual metal · EDTA stock solutions (1000×)
24.4. Store at 4 °C.

 

 

SECTION: Making 1 L of HP medium
25. Close all stock bottles tightly and invert several times to mix.
SECTION: Making 1 L of HP medium
26. Fill an acid washed 1 L graduated cylinder to 700 mL with ICP-MS grade Milli-Q H2O.

SECTION: Making 1 L of HP medium
27. Add 10 mL of 1 Molarity (M) HEPES stock (from step 10). 

SECTION: Making 1 L of HP medium
28. Add 10 mL of 100x phosphate buffer (from step 12).

SECTION: Making 1 L of HP medium
30. Use parafilm to tightly cover the graduated cylinder. Hold the parafilm in place with one hand and mix well by inverting the graduated cylinder at least 10 times. Alternatively, use an acid washed stir bar and an acid washed 1 L bottle to mix.
SECTION: Making 1 L of HP medium
29. Add 1 mL of each trace element solution (from steps 18 to 24). 

 

SECTION: Making 1 L of HP medium
31. Add 2 Molarity (M) trace metal grade NaOH (from step 11) to bring the pH to pH 7 – pH 7.5. Record the volume of 2 M NaOH used. 

SECTION: Making 1 L of HP medium
32. For photoautotrophic growth, fill to 975 mL. For growth with 2% glucose, fill with Milli-Q H2O to 935 mL.

SECTION: Making 1 L of HP medium
31.1. Measure the pH by pipetting 30 µL onto the MQuant pH-indicator strips (non-bleeding pH 5.0 – 10.0, Supleco 1.09533.001).

SECTION: Making 1 L of HP medium
33. Use parafilm to tightly cover the graduated cylinder. Hold the parafilm in place with one hand and mix well by inverting the graduated cylinder at least 10 times. Alternatively, you may mix in an acid washed 1 L bottle with a pre-labeled 975 mL or 935 mL fill line.

SECTION: Making 1 L of HP medium
35. Cover flasks with a double layer of 3 in. x 3 in. aluminum foil (Kirkland Signature Reynolds foodservice foil RK611 item 31680).
SECTION: Making 1 L of HP medium
34. For photoautotrophic growth, aliquot 97.5 mL into 250 mL flasks using an acid washed 100 mL graduated cylinder. For mixotrophic or heterotrophic growth, add 93.5 mL.

SECTION: Making 1 L of HP medium
36. Put flasks in an autoclave bin and fill the bin with 200 mL of H2O. Autoclave using the liquid setting. Cool to Room temperature.

SECTION: Making 1 L of HP medium
40. Store at Room temperature and use culture flasks within two weeks.

SECTION: Making 1 L of HP medium
39. For mixotrophic or heterotrophic growth, add 4 mL of filter sterilized 50% glucose to each flask. For photoautotrophic growth, skip this step.

SECTION: Making 1 L of HP medium
38. Add 100 µL of filter sterilized 1000 x (2 millimolar (mM) stock) thiamine (from step 14) to each 100 mL flask in a sterile hood

SECTION: Making 1 L of HP medium
37. Add 2.5 mL of filter sterilized 40 x macronutrient (N, Ca, Cl, Mg, and S) solution (from step 13) to each 100 mL flask or 25 mL to 1 L bottle using sterile technique in the sterile hood.

SECTION: Making 1 L of HP medium
41. Please refer to your institutional and local rules and guidelines for proper and safe disposal of media and stock solutions.
SECTION: Wash all culture flasks, stock solution containers, and graduated cylinders with 6 M HCl
1. Dilute pure (certified ACS plus) 12 Molarity (M) HCl to 6 Molarity (M).  To dilute 1 L of 12 Molarity (M) HCl to 2 L of 6 Molarity (M) HCl, add 1 L of Milli-Q H2O to an empty 12 Molarity (M) HCl bottle that has only previously contained unused 12 Molarity (M) HCl (or use a dedicated mixing bottle). Slowly add 1 L of 12 Molarity (M) HCl to 1 L of Milli-Q H2O. Always add acid to water and never add water to acid. The bottle may heat up as the acid is diluted, so let it cool before capping and mixing.