May 28, 2020

Public workspaceStandard Volvox Medium

This protocol is a draft, published without a DOI.
Standard Volvox Medium
  • Raymond Goldstein1,
  • Kyriacos Leptos1
  • 1University of Cambridge
Kyriacos Leptos
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Raymond Goldstein, Kyriacos Leptos 2020. Standard Volvox Medium. protocols.io https://protocols.io/view/standard-volvox-medium-bedyja7w
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: March 31, 2020
Last Modified: May 28, 2020
Protocol Integer ID: 34968
Keywords: standard volvox medium recipe, standard volvox medium, protocol for the execution, modification of provasoli, protocol, medium
Abstract
This is a protocol for the execution of a Standard Volvox Medium recipe. Starr's (1969) modification of Provasoli & Pintner's Medium (1959), as further modified by Starr, Kochert and Kirk at various times.
Guidelines
Description

Class of ChemicalsStock SolutionStock ConcentrationFinal Concentration
Macro minerals, N-source, and S-sourceCa(NO3)2500 mM0.5 mM
MgSO4160 mM0.16 mM
KCl670 mM0.67 mM
Na2CO3190 mM0.19 mM
N-sourceUrea500 mM0.5 mM
P-sourceNa2Glycerophosphate163 mM0.16 mM
pH RegulatorHEPES250 mM2.5 mM
Trace mineralsNa2EDTA4.47 mM13.4 µM
FeCl3717 µM2.15 µM
MnCl2414 µM1.24 µM
ZnCl273 µM0.22 µM
CoCl217 µM0.05 µM
Na2MoO438.8 µM0.10 µM
VitaminsThiamine (Vitamin B1)1 mg/mL1 µg/mL
Biotin (Vitamin B7)2.5 µg/mL0.25 ng/mL
Cyanocobalamin (Vitamin B12)1.5 µg/mL0.15 ng/mL
Description of the SVM Medium compounds and their final concentration.

Molar Composition

  • Concentration2.2 mM Na+
  • Concentration670 µM K+
  • Concentration160 µM Mg2+
  • Concentration500 µM Ca2+
  • Concentration679 µM Cl-
  • Concentration160 µM SO42-
  • Concentration2.5 mM [HEPES]-
  • Concentration1 mM NO3-
  • Concentration0.5 mM Urea
  • Concentration0.16 mM [Glycerophosphate]2-
  • Concentration13 µM EDTA4-
  • Concentration2.2 µM Fe2+
  • Concentration0.22 µM Zn2+
  • Concentration1.2 µM Mn2+
  • Concentration0.1 µM MoO42-
  • Concentration0.05 µM Co2+
Materials
Required Materials:

  • Stock solutions (see below)
  • NaOH
  • 1 x 2 L Beaker
  • 1 x 1 L Measuring cylinder
  • 2 x 1 L Bottles


Stock solutions:

Macro minerals, N-source, and S-source

  • Concentration500 mM Ca(NO3)2
  • Concentration160 mM MgSO4
  • Concentration670 mM KCl
  • Concentration190 mM Na2CO3

N-source

  • Concentration500 mM Urea

P-source

  • Concentration163 mM Na2Glycerophosphate

pH Regulator

  • Concentration250 mM HEPES

Trace Minerals ("P IV Metal solution" 2X)

  • Concentration4.47 mM Na2EDTA
  • Concentration717 µM FeCl3
  • Concentration414 µM MnCl2
  • Concentration73 µM ZnCl2
  • Concentration38.8 µM Na2MoO4

Vitamins

  • Concentration1 mg/mL Thiamine (Vitamin B1)
  • Concentration2.5 µg/ml Biotin (Vitamin B7)
  • Concentration1.5 µg/ml Cyanocobalamin (Vitamin B12)

Note
Storage and Shelf Live of Stock Solutions

TemperatureRoom temperature
  • Na2CO3
  • P IV Metal Solution

Temperature4 °C
  • Ca(NO3)2
  • MgSO4
  • KCl urea
  • Phosphoglycerate HEPES
  • Thiamine (wraped in foil)

Temperature-20 °C
  • Biotin
  • Cyanocobalamin (in dark e.g. a cryobox)


Additional Notes:

  1. All stocks except biotin and cyanocobalamin should be made fresh at least every 2 months.
  2. The trace metal stocks should be made fresh every month, and sooner if there is any problem with the cultures that cannot be explained.

Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
The amounts in this protocol are shown for Amount1 L SVM but the protocol can be scaled up or down as desired if more or less SVM is needed.

Adding Solvent
Add Amount950 mL Initial MilliQ water to a 2L beaker.

Adding Macro minerals, N-source, and S-source
Add Amount1 mL Ca(NO3)2 .
Pipetting
Add Amount1 mL MgSO4 .

Pipetting
Add Amount1 mL KCl .

Pipetting
Add Amount1 mL Na2CO3 .

Pipetting
Adding N-source
Add Amount1 mL Urea .

Pipetting
Adding P-source
Add Amount1 mL Na2Glycerophosphate .

Pipetting
Adding pH Regulator
Add Amount10 mL HEPES .

Pipetting
Adding Trace Minerals
Add Amount3 mL "P IV Metal solution" 2X .


Pipetting
Adjusting pH and final volume
Adjust the pH to Ph7.8 with NaOH.
Note
Make sure this is done under mild mixing.

Mix
Adjust the final volume to Amount1000 mL .

Autoclaving
Dispense the mixture as 500 ml batches into desired 1L bottles.

Autoclave the medium.
Keep the medium at Temperature4 °C until you are ready to use it.

Pause
Adding Vitamins (Just before use)

Note
Always add vitamins fresh and keep the medium at Temperature4 °C .

Safety information
Be sure to work under a laminar flow hood of this step.

Using pre-plugged sterile tips mix the following as a cocktail in a 1.5 ml sterile microcentrifuge:

  • Amount1000 µL Thiamine (Vitamin B1)
  • Amount100 µL Biotin (Vitamin B7)
  • Amount100 µL Cyanocobalamin
Add Amount1000 µL Thiamine (Vitamin B1) .

Pipetting
Add Amount100 µL Biotin (Vitamin B7) .


Pipetting
Add Amount100 µL Cyanocobalamin .

Pipetting
Mix.
Mix
Add the vitamins to the SVM-vit.
Pipetting