Aug 30, 2024

Public workspaceSample Preparation for Elemental Analysis of Auxenochlorella protothecoides (UTEX 250) Cells and Spent Media by Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) and Total Organic Carbon (TOC). V.1

DOI
dx.doi.org/10.17504/protocols.io.14egn69wml5d/v1
  • 1Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA;
  • 2Quantitative Biosciences Institute, University of California, Berkeley, CA 94720, USA
Dimitrios Camacho
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DOI: dx.doi.org/10.17504/protocols.io.14egn69wml5d/v1
Protocol Citation: Dimitrios Camacho, Charles Perrino, Sabeeha Merchant 2024. Sample Preparation for Elemental Analysis of Auxenochlorella protothecoides (UTEX 250) Cells and Spent Media by Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) and Total Organic Carbon (TOC).. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn69wml5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 101627
Keywords: ICPMS, TOC, Organic Carbon, Elemental analysis, Auxenochlorella
Funders Acknowledgement:
National Institutes of Health (NIH): Nutritional Copper Signaling and Homeostasis Grant
Grant ID: GM 042143
National Institutes of Health (NIH): Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts Grant
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
Disclaimer
We would like to thank Dr. Stefan Schmollinger for his mentorship and advice.
Abstract
This protocol describes a method for quantifying the elemental composition of Auxenochlorella protothecoides (UTEX 250) cells and the spent HP medium (Camacho, 2024). Elements measured include C (as total organic C), Na, Mg, P, S, K, Ca, Mn, Fe, Cu, Zn, Se, and Mo. Samples are collected, washed, digested, and analyzed for total organic carbon (Shimadzu) and other elements by ICP-MS/MS (Agilent 8900). Elemental measurements can be normalized to volume of cultures, cell number, sulfur, or total organic carbon. 
Materials
Materials
  1.     Metal free 15 mL tubes - Globe Scientific Inc. Centrifuge, high performance, red screw cap, assembled, polypropylene, printed graduations, sterile, 25 tubes / bag, 20 bags/ case. Catalog No. 6295, with a maximum rating of 17,000 ×g 
  2.      1 L HDPE bottles
  3.      Trace metal grade 1 mM Na2EDTA washing solution
  4.      Ultra-pure ICP-MS grade Milli-Q H2O
  5.      ICP-MS grade Optima 70% HNO3
  6.      Diluted 2.1% HNO3
  7.      Ultra-pure 12 M HCl
  8.      Kimtech KimwipesTM
 
Equipment
  1.      RAININ P1000, P200, P20 pipettes and tips
  2.      10 mL and 25 mL serological pipets
  3.      Fume Hood
  4.      Microscope and Hemocytometer 
  5.      Beckman Coulter Avanti JXN-26 Centrifuge
  6.      Fixed angle centrifuge rotor JA-14.50 with 15 mL adapters
  7.      Eppendorf 5810 R centrifuge 
  8.      Eppendorf A-4-81 swinging bucket rotor with 15 mL adapters
  9.      Liquid waste container
  10.    Solid trash container
  11.    65 °C - 80°C shaker water bath
  12.    Fisherbrand CPX2800 ultrasonic bath 2.8 L with heater
  13.    Agilent 8900 ICP-MS/MS
  14.    Shimadzu Total Organic Carbon Analyzer (TOC-L)
Safety warnings
Nitric acid (HNO3) and hydrochloric acid (HCl) are corrosive and toxic acids. Use a lab coat, closed toe shoes, long pants, gloves, and eye protection when handling HNO3 and HCl. Work with concentrated HCl and HNO3 should be done in the fume hood. Gloves should also be used when handling ultra-pure reagents. Never mix HCl and HNO3. Never mix bleach with ammonia, acids, alcohol, hydrogen peroxide, or other household cleaners.
Before start
  1. Acid wash all stock bottles with fresh (unused) Concentration6 Molarity (M) . Do not use glass containers as glass may leach metals. Use high density polyethylene (HDPE) bottles instead.
 
1.1. Dilute fresh (unused) Concentration12 Molarity (M) HCl to Concentration6 Molarity (M) . Remember to add acid to water and never water to acid.
 
1.2. Add Amount100 mL of Concentration6 Molarity (M) HCl to Amount1 L stock bottles. Swirl Concentration6 Molarity (M) HCl in the bottle to clean every internal surface. Transfer the used Concentration6 Molarity (M) HCl to the next bottle and repeat. 
 
1.3. Rinse with ICP-MS grade ultra-pure H2O at least seven times. See next section for a detailed description of the Milli-Q H2O.
 
1.4. Neutralize the used Concentration6 Molarity (M) HCl. 
 
1.4.1. Add Amount1 L of H2O to a Amount4 L beaker placed in a larger, chemically compatible secondary container, such as an autoclavable polypropylene Amount12 L tray (Cat. no. S37253, Fisher Scientific).

  1.4.2. Slowly and carefully add Amount1 L of Concentration6 Molarity (M) HCl to water. Always add acid to water. The solution may heat up, so take your time. 
 
1.4.3. Neutralize Concentration3 Molarity (M) HCl by adding NaHCO3 (Arm & Hammer pure baking soda) slowly, scoop by scoop until no foam is formed. 
 
1.4.4. Use a pH indicator strip to verify that the acid is safely neutralized (Ph7 ).
 
1.4.5. Neutralized acid may be discarded down the drain.
 
2. The ICP-MS/MS is extremely sensitive and contaminants may confound your measurements. Use trace metal grade reagents and ICP-MS grade ultra-pure Milli-Q H2O. The production of ICP-MS grade ultra-pure Milli-Q H2O relies on a Q-POD‱ Element containing a Quantum‱ ICP filter (REF QTUM00ICP). 
 
2.1. ICP-MS grade ultrapure Milli-Q H2O will be referred to as Milli-Q H2O throughout the rest of this protocol.
 
2.2. Before using the Milli-Q H2O, verify that the resistivity is 18.2 MΩ • cm and the total organic carbon (TOC) reading is <10 ppb.
 
2.3. Keep Amount1 L of Milli-Q H2O in an acid washed Amount1 L high density polyethylene (HDPE) bottle.
 
2.4. Keep the bottle closed to avoid dust from entering. 
 
2.5. Replace your Milli-Q H2O before each experiment.
 
3. Be mindful of metal contamination from tubes, plastic bottles, pipets, and dust.
 
3.1. Ensure all work areas are thoroughly wiped to be free of dust. If a clean room is not available, work can be carried out in a clean fume hood and a room where the paint is not deteriorated.
 
3.2. Keep tubes and reagents closed to avoid metal contamination from dust. Zn is abundant in dust. 
 
3.3. Most Amount15 mL and Amount50 mL conical tubes contain residual metal contamination. We recommend acid washing tubes with Concentration12 Molarity (M) HCl (step 5 in this section). Tube manufacturers may change their sources of materials over time, so make sure to check that the tubes you are using are metal free using the ICP-MS. Record the lot numbers for all tubes.
 
3.3.1. Metal free Amount15 mL tubes - Globe Scientific Inc. Catalog No. 6295 were specially chosen because they consistently contained less metals and can withstand a force of 17,000 ×g, which is needed to collect Auxenochlorella cells. These tubes however contain a large amount of Ca and therefore need to be washed if you are interested in the Ca content of cells and supernatants. 
 
3.4. Disposable Amount10 mL and Amount25 mL serological pipets Cat. No. 13-676-10J and 13-676-10K, respectively, will introduce a detectable amount of Zn contamination to your samples. We advise rinsing each newly opened pipet by pipetting Milli-Q H2O to the maximum volume and disposing the water before pipetting any samples or reagents.
 
4. Pre-label a set of Amount15 mL tubes for cell pellets and another set for spent media (optional). 
 
4.1. Label tubes with the date, your initials, sample name, and experiment ID. 
 
4.2. If measuring - Fe, - Cu, or - Zn media, prepare five Amount15 mL tubes for each supernatant sample. 
 
4.3. Include six or more method blanks for cell pellets and three method blanks for supernatant sample processing. Treat the method blanks as if they contained cells or supernatants, using the same reagents for digestion and dilution. Use a different set of three method blanks for each digestion method.
 
5. Acid-wash all Amount15 mL tubes with Amount5 mL of fresh Concentration12 Molarity (M) in the fume hood. Cap and invert the tube gently. Re-use the Amount5 mL of Concentration12 Molarity (M) HCl for the next 15 tubes. 
 
5.1. Neutralize used Concentration12 Molarity (M) HCl by diluting to Concentration3 Molarity (M) HCl in the fume hood. Refer to steps 1.4 to 1.4.5 but adjust volumes for Concentration12 Molarity (M) instead of Concentration6 Molarity (M) HCl. 
 
5.2. Rinse tubes with ICP-MS grade Milli-Q H2O at least seven times. Rinse the exterior of the capped tube to prevent exposure to residual HCl. It is very important to ensure there is no residual HCl. HCl will discolor some plastics and corrode metals.
 
5.3. Dry the exterior of the tube with a KimwipeTM and centrifuge the empty tubes at Centrifigation3000 x g, 22°C, 00:02:00 using the 5810 R centrifuge fitted with a swinging bucket rotor (rotor A-4-81) and Amount15 mL tube adapters. Rinse the rotors and buckets with Milli-Q H2O when you are finished.
 
5.4. Use a P200 pipette to carefully remove remaining water in the tube. Avoid touching the inner wall of the tube with the pipette. 
 
 6. Prepare the centrifuges, rotors, and adapters.
 
6.1. Set the JXN-26 centrifuge to Centrifigation17000 x g, 22°C, 00:02:00 , rotor A-4-81 with Amount15 mL tube adapters.
 
6.2. Set the 5810 R centrifuge to Centrifigation3220 x g, 22°C, 00:02:00 , rotor JA-14.50 with Amount15 mL tube adapters.
 
7. Determine the density of the culture by counting cells with a hemocytometer (Camacho & Merchant, 2024). Calculate and record the volume of culture required to collect 1 × 108 - 3 × 108 total cells. 
Procedure
Procedure
 Collect 1 × 108 - 3 × 108 cells at Centrifigation17000 x g, 22°C, 00:02:00 in red screw cap Amount15 mL tubes using the JXN-26 centrifuge. Do not disturb the pellet. The pellet will partially adhere to the side of the tube at a 45-degree angle. 

If cells are sticking to the side of the tube and not collecting to the bottom of the tube, centrifuge for an additional Duration00:05:00 - Duration00:10:00 . 

Carefully transfer Amount0.5 mL of supernatant to each prelabeled Amount15 mL tube without disturbing the pellet. If measuring Fe, Cu, or Zn deficient media, then aliquot Amount0.5 mL into each of the five tubes.

Slowly and carefully decant the remaining supernatant into a liquid waste container in one motion. Keep the side with the pellet to the bottom and make sure that no cells are decanted. You may keep about Amount100 µL of liquid to avoid decanting cells.

Do not mix liquid waste with ammonia, acids, alcohol, hydrogen peroxide, or other household cleaners. You will add bleach to the liquid waste later.

Cell pellet is oriented to the bottom while decanting. Some liquid is left to avoid decanting cells.

Pipette Amount1 mL of Concentration1 millimolar (mM) Na2EDTA into the tube containing the pellet. Resuspend the pellet by closing the cap and swinging the tube in a circular motion until the pellet is fully resuspended.

Swinging motion to re-suspend cell pellets in washing solutions.

Centrifuge the mixture Centrifigation17000 x g, 22°C, 00:02:00 and discard the supernatant by carefully decanting, again with one motion and the pellet to the bottom.

Resuspend the cell pellet in Amount1 mL of Milli-Q H2O by closing the cap and swinging the tube in a circular motion.

Centrifuge again at Centrifigation17000 x g, 22°C, 00:02:00 .

Remove the supernatant by slowly decanting in one motion. Do not disturb the pellet.
Transfer the tubes to the swinging bucket rotor on the Eppendorf 5810 R centrifuge and pellet the cells at Centrifigation3220 x g, 22°C, 00:02:00

Carefully remove the supernatant with a P200 pipette. Do not take up any cells. If you accidentally take up cells into the tip, dispense the liquid back into the tube and centrifuge again.

Add bleach to the liquid waste to make a 10% bleach and 90% waste solution. Wait Duration01:00:00 and dispose down the drain. Do not mix bleach with ammonia, acids, alcohol, hydrogen peroxide, or other household cleaners.

(Optional) Freeze the pellet and supernatant samples at Temperature-20 °C until 1 day before the ICP-MS/MS analysis.

Digest cells with Amount143 µL of 70% Optima® grade or similar ICP-MS grade HNO3 and incubate at Temperature65 °C while shaking at 175 RPM (setting 5 on New Brunswick Gyrotory Water Bath Shaker, Model G76) for Duration16:00:00 . Fill the water bath before leaving to ensure that the water does not fully evaporate.

Cell pellets that appear larger than Amount100 µL will require an additional Amount143 µL of HNO3. For instance, fatty mixotrophic or heterotrophic cells grown with 2% glucose are larger in diameter so although the number of cells is the same, the biomass of these cells will be greater than that of photoautotrophic cells. Lipid rich cells are also harder to digest.


Image of 15 mL tubes containing cell pellets. Photoautotrophic (left) and mixotrophic (right) cell pellets and corresponding recommended digestion volume.
The mixture should be clear with absolutely no solids present. If cell pellets are not fully digested, you may take either one (or a combination) of the following actions until the mixture is clear with no precipitates.

1. Incubate for an additional Duration02:00:00 at Temperature80 °C while shaking at 175 RPM.

2. Incubate samples in a Temperature65 °C ultra-sonic water bath for Duration01:00:00 at maximum power. Use floats or a strainer tray to make sure that nothing touches the bottom of the ultra-sonic bath tank. The cavitation effect will be diminished and the transducers may break if an object is placed on the bottom of the tank or if the water level is too low. Ensure that the water level is maintained precisely at the operating level.

3. Add fresh Amount50 µL of 30% hydrogen peroxide to the pellet and incubate at Temperature85 °C for Duration02:00:00 with shaking at 175 RPM.

4. Use a dedicated microwave (800 W) in a fume hood to denature samples. Heat samples in increments of Duration00:00:15 and slightly twist open tubes to avoid fume pressurization. Let the tubes cool before placing back in the microwave. Do not exceed Duration00:01:30 total.

Tubes may leach material and metals as they encounter HNO3, H2O2, are heated, sonicated, and/or microwaved. Use a different set of method blanks for each digestion method or combination of digestion methods. Only analyze blanks that have undergone the same digestion method(s) as the samples.

Fill to Amount5 mL with Milli-Q H2O if Amount143 µL 70% HNO3 was used. Fill to Amount10 mL if Amount286 µL of HNO3 was used. Final HNO3 concentration should be 2%. Record the volume of 70% HNO3 and Milli-Q H2O added.

If filled to Amount5 mL , the dilution factor is 5.

If filled to Amount10 mL , the dilution factor is 10.

ABC
Nitric acid (µL)Fill to (mL)Dilution factor
14355
2861010
Volumes required for the digestion of cell pellets and the resulting dilution factors.


Cell pellet samples are now ready for the ICP-MS/MS analysis.

Samples in capped tubes may be stored at room temperature in a cabinet equipped with an exhaust system to remove HNO3 fumes.
To prepare spent media from cultures grown in replete conditions, add Amount9.5 mL of 2.1% HNO3 to each tube containing Amount0.5 mL of sample, for a final HNO3 concentration of 2%. The dilution factor is 20.

To prepare - Fe, - Cu, or - Zn supernatant samples for measurement, perform a standard addition of Fe, Cu, and/or Zn to each Amount0.5 mL sample.

Prepare 4 acid washed Amount15 mL tubes. Label the tubes standard addition stock (SAS) 1x, SAS 0.1x, SAS 0.01x, and SAS 0.001x.

Aliquot Amount7 mL of Milli-Q H2O into the tube labeled SAS1x and Amount9 mL into each of the remaining tubes.

Pipette Amount1 mL of Concentration2 millimolar (mM) Cu, Concentration20 millimolar (mM) Fe, and Concentration10 millimolar (mM) Zn stocks from the HP medium into the 1x tube.

Execute a serial dilution by transferring Amount1 mL of 1x solution into the tube labeled 0.1x which contains Amount9 mL of Milli-Q H2O. Invert the tube to mix and transfer Amount1 mL of the 0.1x solution to Amount9 mL of Milli-Q H2O in the tube labeled 0.01x. Repeat for 0.001x using the 0.01x solution.

Label the five supernatant samples 0, 1x, 0.1x, 0.01x, and 0.001x

Do not add any standard into the tube labeled 0. Aliquot Amount100 µL of the standard addition stocks into their corresponding supernatant samples. Repeat for all supernatant samples.

Add Amount9.5 mL of 2.1% HNO3 to all supernatant samples without standard additions.

Add Amount9.4 mL of 2.1% HNO3 to all supernatant samples with standard additions.

The dilution factor for all supernatant samples is 20x.





To obtain total organic carbon measurements from ICP-MS/MS cell samples, transfer Amount500 µL of digested 2% HNO3 sample to a TOC vial containing Amount14.4 mL of Milli-Q H2O and Amount135 µL of Concentration3 Molarity (M) HCl. Measure carbon only and include a set of method blanks from step 12.2. Samples are now ready for TOC measurement. The dilution factor for cell pellets is 30 × ICP-MS/MS dilution factor.

For measurement of ICP-MS/MS supernatants from step 15 – 16, transfer Amount500 µL of 2% HNO3 sample to a TOC vial containing Amount14.4 mL of Milli-Q H2O and Amount135 µL of Concentration3 Molarity (M) HCl. The total dilution factor is 600.

If you are measuring ICP-MS/MS supernatants which originally contained 2% glucose, then transfer Amount180 µL of ICP-MS/MS samples to a vial containing Amount14.7 mL of Milli-Q H2O and Amount135 µL of Concentration3 Molarity (M) HCl. The total dilution factor is 1666.67

Protocol references
Dimitrios Camacho, Charles Perrino, Sabeeha Merchant 2024. HEPES-Phosphate Medium, Suitable for Studies of Trace Element Nutrition in Photoautotrophic and Heterotrophic Auxenochlorella protothecoides.. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyzdzl8j/v1

Dimitrios Camacho, Sabeeha Merchant 2024. Determination of Auxenochlorella protothecoides Cell Density With a Hemocytometer. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjk12lx1/v1