Oct 12, 2016

Public workspaceASP12A Recipe for culturing Aureococcus anophagefferens V.2

DOI
dx.doi.org/10.17504/protocols.io.f3ybqpw
  • Eric Gann
  • Protist Research to Optimize Tools in Genetics (PROT-G)
  • The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Ashley A Humphrey
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DOI: dx.doi.org/10.17504/protocols.io.f3ybqpw
External link: http://www.nefsc.noaa.gov/nefsc/publications/crd/crd0517/crd0517.pdf
Protocol Citation: Eric Gann 2016. ASP12A Recipe for culturing Aureococcus anophagefferens. protocols.io https://dx.doi.org/10.17504/protocols.io.f3ybqpw
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
Created: October 12, 2016
Last Modified: June 11, 2019
Protocol Integer ID: 3928
Abstract
Defined Medium for Aureococcus anophagefferens
Guidelines
Anhydrous Salts
Constituent Final Conc. Molar Mass g/1.0L g/0.75L g/0.5L g/0.25L
NaCl 4.28E-01 58.44 25.01232 18.75924 12.50616 6.25308
KCl 1.10E-02 74.5513 0.8200643 0.615048225 0.410032 0.205016
Na2SO4 1.00E-02 142.04 1.4204 1.0653 0.7102 0.3551
NaHCO3 2.00E-03 84.007 0.168014 0.1260105 0.084007 0.042004
Hydrous Salts
Constituent Final Conc. Molar Mass g/1.0L g/0.75L g/0.5L g/0.25L
CaCl2·2H2O 1.25E-02 147.02 1.83775 1.3783125 0.918875 0.459438
MgCl2·6H2O 4.00E-02 203.3027 8.132108 6.099081 4.066054 2.033027
Macronutrients
Constituent Final Conc. Molar Mass g/1.0L g/0.75L g/0.5L g/0.25L
NaNO3 1.47E-03 84.9947 0.124942209 0.093706657 0.062471 0.031236
Ca2·glycerophosphate 2.00E-05 210.14 0.0042028 0.0031521 0.002101 0.001051
K2HPO4 4.00E-05 174.2 0.006968 0.005226 0.003484 0.001742
Other
Constituent Final Conc. Molar Mass g/1.0L g/0.75L g/0.5L g/0.25L
Tris Base 4.13E-03 121.14 0.5003082 0.37523115 0.250154 0.125077
Stock Solutions
Solution Final Conc. g/L mL/1.0L mL/0.75L mL/0.5L mL/0.25L
Fe-EDTA sodium Salt 1.79E-06 0.65702 1 0.75 0.5 0.25
TSM - I   1 0.75 0.5 0.25
TSM - II   1 0.75 0.5 0.25
TSM - III   1 0.75 0.5 0.25
S3 Vitamin Solution   1 0.75 0.5 0.25
Trace Metal Solution - TMS - I
Constituent Final Conc. Molar Mass g/1.0L 1000X Stock    
Na2EDTA·2H2O 8.53E-05 372.24 31.752072    
FeCl3·6H2O 1.79E-06 270.3 0.483837    
H3BO3 2.77E-04 61.83 17.12691    
MnCl2·4H2O 9.10E-06 197.91 1.800981    
ZnSO4·7H2O 1.07E-06 287.56 0.3076892    
NaMoO4·2H2O 5.21E-06 218.9779 1.140874859    
CoCl2·6H2O 2.21E-07 237.93 0.05258253    
Trace Metal Solution - TMS - II
Constituent Final Conc. Molar Mass g/1.0L 1000X Stock    
KBr 1.25E-04 119.002 14.87525    
SrCl2 · 6H2O 3.42E-05 266.62 9.118404    
RbCl 2.34E-06 120.92 0.2829528    
Trace Metal Solution - TMS - III
Constituent Final Conc. Molar Mass g/1.0L 1000X Stock    
Na2SeO3 2.00E-08 172.94 0.0034588    
NaF 5.26E-05 41.98871 2.208606146    
NaAsO2 4.00E-08 129.91 0.0051964    
Al2(SO4)3·18H2O 1.11E-06 666.43 0.7397373    
NH4VO3 2.00E-08 116.98 0.0023396    
S3 Vitamin Solution
Constituent Final Conc. Molar Mass g/1.0L 1000X Stock    
Thiamine HCL 2.96E-07 337.27 0.09983192    
biotin 4.10E-09 244.31 0.001001671    
vitamin B12 7.40E-10 1.36E+03 0.0010064    
Prepare Trace Metal Solutions I - III, Fe-EDTA Sodium Salt Solution, and Vitamin Solution. Add constituents to 750 mL MilliQ water, shake to dissolve. Add MilliQ water to 1.0 L. Filter sterilize. Trace Metal Solutions and Fe-EDTA Sodium Salt Solution are stored at 4ºC in 15 - 50 mL aliquots, while the Vitamin solution is stored at -20ºC in 5 - 10 mL aliquots.
Add anhydrous salts, hydrous salts, macronutrients and Tris Base to 75% desired MilliQ water while stirring to dissolve. Add MilliQ water to final volume, autoclave.
Once cooled, add in 1mL of the Trace Metal Solutions, Fe-EDTA Sodium Salt Solution, and Vitamin Solution per 1L of media made.