Overview

These media recipes will facilitate growth of Volvox and other volvocales, including ChlamydomonasVolvox media contains biotin, vitamin B12 and thiamine, all of which Volvoxrequires for growth. The carbon source in Volvox media is bicarbonate, since many Volvocales cannot use acetate as a carbon source like Chlamydomonas. For optimal and synchronous growth, cultures should be bubbled with air or 0.5% CO2. Note the Volvox uses a mobile sex inducer to induce its sexual program, thus all glassware for Volvocales species that respond to a sex inducer should be baked prior to preparing media. All glassware used to make Volvox media should be dedicated to this purpose.

SVM media is called “standard Volvox media, because it is a good starting point for growing Volvoxand most other volvocales. Once strains are established in the lab, they can be tested with simpler medias such as those used for Chlamydomonas. Note that most volvocales require vitamin supplementation of Chlamydomonas medias.

SVM is made as either a 1X or 10X solution from media stocks. Whenever possible, the highest grade of each chemical should be purchased. When possible, it is noted the catalog numbers and suppliers of chemicals that work well in our hands. The most difficult part of making SVM is preparing stock solutions and ensuring that the PIV metals are fresh. If any growth problems occur when using SVM media, the first suspect is always the PIV metals.

Testing for Contamination

Volvocales strains should be routinely tested for contamination. To do this, about 1 ml of a well growth culture should be plated on a TAP plate with yeast extract. The best time to do this is when strains are being transferred for maintenance. The TAP-Y plate should be grown under similar conditions as the volvocales strains or on a light shelf for algae plates. Should contamination be detected, the strains should be decontaminated as soon as possible.

SVM Stocks

Chemical Supplier and Cat# Stock concentration Notes
Ca(NO3)2•4H20 23.6 g/200 mL autoclave, store at 4°C
MgSO4•7 H2O 8.0 g/200 mL autoclave, store at 4°C
Na2glycerophosphate•5 H2O 14.0 g/200 mL autoclave, store at 4°C, use 10.0 g/200 mL if using anhydrous glycerophosphate
KCl 10.0 g/200 mL autoclave, store at 4°C
Na2CO3 2.0 g/100 mL filter sterilize, store at 4°C
urea 3.0 g/100 mL filter sterilize, store at 4°C
HEPES, free acid, pH 8.0 with NaOH 60.0 g/L autoclave, store at 4°C
Biotin 200 μg/mL store at -20°C
Vitamin B12 2.5 mg/mL store at -20°C
Biotin/Vitamin B12 Working Stock 9.9 mL H2O, 125 μL biotin stock + 6 μL Vit B12 stock prepare from frozen stocks above, store at 4°C
Thiamine 200 mg/200 mL filter sterilize, store at 4°C
CaCl2 110.1 g/200 mL For N-free SVM, Use highest possible grade of CaCl2 or else it may precipitate
1000X NaAcetate 200 g/1 L For SVMA media, autoclave, store at 4°C

PIV Metals

Note: PIV metals should be made monthly and stored at room temperature. The best practice is to make a new batch of PIV metals before a previous batch expires!

 

100 ml of Zn, Co, Mo, Se stock

Prepare fresh and discard unused portion.

Chemical Supplier and Cat# Stock concentration Notes
ZnCl 50 mg
CoCl2•6 H2O 20 mg
Na2MoO4•2 H2O 40 mg
Na2SeO3 14 mg

PIV Metal Stock

Chemical/Step Supplier and Cat# Stock concentration Notes
To 1L of MQ-H20
Na2EDTA 0.75 g Dissolve completely with vigorous stirring
FeCl3•6 H2O 97 mg Let dissolve for 15 minutes before proceeding
MnCl2•4 H2O 41 mg
CuSO4•5 H2O 42 mg
Zn, Co, Mo, Se stock From above 10 mL
Filter sterile and store at RT in the dark

10X SVM

Chemical Stock Volume Notes
Ca(NO3)2•4 H2O Stock 10 mL
CaCl2 Stock 10 mL
MgSO4•7 H2O Stock 10 mL
Na2glycerophosphate•5 H2O 10 mL
KCl Stock 10 mL
Na2CO3 10 mL
Urea Stock 10 mL
HEPES-NaOH pH 8.0 100 mL
Biotin/B12 Working Stock 1 mL
Thiamine Stock 10 mL
PIV Metal Stock 60 mL

1x SVM

Note, Volvox will use the sodium carbonate and CO2 from the air as a carbon source. Some volvocales such as Chlamydomonas and Gonium can use acetate as a carbon source. If increased growth of acetate utilizing strains is desired, NaAcetate can be added to 200 mg/L from a 1000X stock. Note, that acetate in stocks can lead to rapid contamination of the media if not autoclaved (even at 4°C). Therefore, NaAcetate should only be added to 1X stocks of SVM immediately before autoclaving.

Chemical Stock Volume Notes
Ca(NO3)2•4 H2O Stock 1 mL
CaCl2 Stock 1 mL
MgSO4•7 H2O Stock 1 mL
Na2glycerophosphate•5 H2O 1 mL
KCl Stock 1 mL
Na2CO3 1 mL
Urea Stock 1 mL
HEPES-NaOH pH 8.0 10 mL
Biotin/B12 Working Stock 0.1 mL
Thiamine Stock 1 mL
PIV Metal Stock 6 mL

10X N-free SVM

Chemical Stock Volume Notes
CaCl2 Stock 10 mL
MgSO4•7 H2O Stock 10 mL
Na2glycerophosphate•5 H2O 10 mL
KCl Stock 10 mL
Na2CO3 10 mL
Urea Stock 10 mL
HEPES-NaOH pH 8.0 100 mL
Biotin/B12 Working Stock 1 mL
Thiamine Stock 10 mL
PIV Metal Stock 60 mL

10X Urea-free SVM

Chemical Stock Volume Notes
Ca(NO3)2•4 H2O Stock 10 mL
CaCl2 Stock 10 mL
MgSO4•7 H2O Stock 10 mL
Na2glycerophosphate•5 H2O 10 mL
KCl Stock 10 mL
Na2CO3 10 mL
HEPES-NaOH pH 8.0 100 mL
Biotin/B12 Working Stock 1 mL
Thiamine Stock 10 mL
PIV Metal Stock 60 mL