Overview

These media recipes will facilitate growth of most strains of Chlamydomonas. In order to synchronize Chlamydomonas to light-dark cycles you will need to utilize a media lacking acetate, since most strains of Chlamydomonas will grow in the dark in the presence of acetate (REF). However, since media lacking acetate requires fixation of carbon from gaseous CO₂, cells will not grow as densely as in media containing acetate. For routine strain maintenance on plates, it is generally a good idea to grow strains on acetate containing media such as TAP.

At the heart of all these media recipe’s is the preparation of Hutner’s trace elements. Hutner’s trace elements is stored at -20C and last practically indefinitely. However, Hutner’s trace elements need to be prepared at least a week or two in advance. Hutner’s trace elements can be purchased from the Chlamydomonas stock center by using this link.

HSM and HSMA (with acetate) are much more economical that their tris buffered counterparts TP and TAP, respectively. However, because the cells utilize the phosphate for growth, the buffering capacity of HSM based media will gradually decrease. This is especially important when cells are being bubbled with high concentrations of CO2.

Agar Plates

In order to make agar plates, 15 g/L of Bacto Agar should be added to the media prior to autoclaving. Note that Chlamydomonas is very sensitive to the quality of agar used. Whenever possible, Bacto brand agar should be used for Chlamydomonas growth. If you wish to try less expensive agar brands, they should be compared side-by-side to Bacto brand agar.

Testing for Contamination

Chlamydomonas strains should be routinely streaked in parallel onto an agar plate containing yeast extract (4 g/L) and grown side-by-side with the strain on a regular TAP or HSM plate. This encourages the growth of microbial contaminants that otherwise may not be obvious. Contaminated strains should be immediately decontaminated. Strains should never be maintained on media containing yeast extract as these plates can be easily contaminated.

Vitamin Supplementation

Chlamydomonas does not require any additional vitamins or trace elements other than what these recipes dictate. However, some volvocales can be successfully grown on HSM or TAP media as long as the media is supplemented with vitamins. In these cases, individual vitamins can be tested individually, or 1 mL/L of 100 mg/mL thiamine, 0.1 mL/L of the biotin/vitamin B12 working stock can be added.

Hutner’s Trace Elements

Hutner’s trace elements are used to make HSM and TAP media. The original reference for this method is Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170. However, more recently Sabeeha Merchant’s group at UCLA has defined the trace element requirement of Chlamydomonas more strictly and their results are reported in Merchant et al. (2006) Biochim. Biophys. Acta 1763, 578-594, a formulation they call “special-K”.For Hutner’s trace elements, each of its constituents are prepared as separate solutions and then mixed in the order given below. Before mixing each of the components, make sure they are dissolved completely. Following oxidation in open air or by bubbling, there should be no obvious sign of precipitate and should a a deep rich purple color.

Before an existing stock of trace elements is used up, a new batch should be prepared and thoroughly tested.

First, prepare the EDTA solution.

Chemical	Supplier and Cat#	Amount	MQ-H₂O	Notes
EDTA, free acid		39.26	250 mL	Dissolve in about 25-50g of KOH pellets (as few as possible)

Set the EDTA solution aside and prepare the metal stocks.

Prepare the following stocks and mix together. DO NOT ADD EDTA YET!

Chemical	Supplier and Cat#	Amount	MQ-H₂O	Notes
ZnSO₄•7 H₂O		22 g	100 mL
H₃BO₃		11.4 g	200 mL	Carefully heat in a microwave to dissolve
MnCl₂•4 H₂O		5.06 g	50 mL
CoCl₂•6 H₂O		1.61 g	50 mL
CuSO₄•5 H₂o		1.57 g	50 mL
(NH₄)₆Mo₇O₂₄•4 H₂O		1.10 g	50 mL
Mix all these solutions in the order listed
FeSO₄•7 H₂O		4.99 g	50 mL	Dissolve in water immediately before use. FeSO₄•7 H₂O will oxidize over time, so if the powder is not light green purchase a new stock

Bring this solution to a boil under a Bunsen burner for about 10 minutes
Heat EDTA in a microwave to warm it up and slowly add it to the mixture.
Cool to ~70°C
Check the pH with a pH paper, should be ~4
Use pH paper to bring the pH to ~6.7 with 20% KOH (typically 20-30 mL), perform this step slowly. Do NOT use NaOH.
Cool to room temperature and bring the volume to ~950 mL MQ-H₂O
At this point the solution should be a greenish color.
Bubble the solution with air for about 1 week to oxidize. When oxidation is complete, it should a a deep rich purple color with no signs of precipitate present. If a precipitate is present either one of the steps above was not followed correctly, or an expired or low quality reagent was used to prepare the solution. The usual culprit is and old or oxidized bottle of FeSO₄•7 H₂O. Note in many classical protocols, including the one listed at the Chlamydomonas stock center, a precipitate is formed when the Hutner’s trace is made. However, this is an improved version of the original and no precipitate should be formed. This improved version of the original is more consistent between batches since unknown quantities of trace metals are not lost due to precipitation.
Following oxidation, bring the volume to 1 L with MQ-H2O, aliquot in 50 mL tubes and freeze at -20°C. Stocks stored at -20°C do not generally go bad. Working stocks can be stored at 4°C for several months.

Special thanks to Matt Zones in the lab of Jim Umen at the Danforth Plant Science Center for troubleshooting the improved Hutner’s trace element method.

HSM Stocks

Sueoka, N. (1960) Proc. Natl. Acad. Sci. USA 46, 83-91

The traditional way of making HSM is to use 2X Beijerink’s in place of the separate NH₄Cl and Ca/Mg stock solutions. However, of you laboratory routinely does mating of Chlamydomonas it is more convenient to separate these salt stocks to that N-free HSM can be more easily prepared. The Chlamydomonas Stock Center has a HSM recipe based off of Beijerink’s salt stocks.

NH4Cl Stock Solution (1L)

Chemical	Amount
NH₄Cl	125 g

Bring the volume to 1 L with MQ-H₂O
Autoclave

Ca/Mg Stock Solution (1 L)

Note, a high grade of CaCl₂ needs to be used or this solution will precipitate. Generally, CaCl₂>98% is sufficient, but if this solution precipitates, the CaCl₂•2 H₂O is the likely culprit.

Chemical	Amount
MgSO₄•7 H₂O	5 g
CaCl₂•2 H₂O	2.5 g

Bring the volume to 1 L with MQ-H₂O
Autoclave

HSM-PO4 Stock Solution

Chemical	Amount
K2HPO4	180 g
KH2PO4	90 g

Bring the volume to 1 L with MQ-H₂O
Autoclave

HSM (1 L)

TAP media is expensive to prepare due to the cost of Tris. As an alternative to TAP, HSM can be supplemented with acetate (HSMA, see the overview for more information). HSMA is prepared by adding NaAcetate to 200 mg/L. A 1000X stock of NaAcetate is described for Volvox media.

Stock	Volume
MQ-H₂O	983 mL
NH4Cl Stock	4 mL
Ca/Mg Stock	4 mL
PO4 Stock	8 mL
Hutner’s Trace	1 mL

N-free HSM (1 L)

Stock	Volume
MQ-H₂O	983 mL
Ca/Mg Stock	4 mL
PO4 Stock	8 mL
Hutner’s Trace	1 mL

TAP Stocks

2M Tris (1L)

Chemical	Amount
Tris base	242.28 g

Bring the volume to 1 L with MQ-H₂O
Autoclave

2M Tris pH 7.4 for TP (1L)

Chemical	Amount
Tris base	242.28 g

Dissolve in about 700 mL of MQ-H₂O
Add concentrated HCl to bring the pH to 7.4, ensure that the pH is stable for at least 15-20 min. You will use several hundred mL of HCl in this step. You can start adding HCl before all of the Tris base is dissovled, but just make sure that the pH is stable at 7.4 before continuing.
Bring the volume to 1 L with MQ-H₂O
Autoclave

2X Beijerink’s Solution (1 L)

Chemical	Amount
NH4Cl	7.5 g
MgSO₄•7 H₂O	2.0 g
CaCl₂•2 H₂O	1.0 g

Bring the volume to 1 L with MQ-H₂O
Autoclave

1M K₂HPO₄

Chemical	Amount
K₂HPO₄	174.18 g

Bring the volume to 1 L with MQ-H₂O
Autoclave

1M KH₂PO₄

Chemical	Amount
KH₂PO₄	136.09 g g

Bring the volume to 1 L with MQ-H₂O
Autoclave

1M TAP-PO4 (100 ml) pH 7.0

Stock Chemical	Volume
1M K₂HPO₄	61.5 mL
1M KH₂PO₄	38.5 mL

Autoclave
Verify pH with pH paper

TAP Media

Stock	Volume
MQ-H₂O	937 mL
2M Tris	10 mL
2X Beijerink’s	50 mL
1M TAP-PO4 Stock	1 mL
Hutner’s Trace	1 mL
Acetic Acid (Glacial)	1 mL

pH 7.4 (Check with pH paper to be sure)

TP Media

Stock	Volume
MQ-H₂O	937 mL
2M Tris pH 7.4	10 mL
2X Beijerink’s	50 mL
1M TAP-PO4 Stock	1 mL
Hutner’s Trace	1 mL

pH 7.4, if needed, adjust with dilute HCl NOT acetic acid (Check with pH paper to be sure)