Forsburg Lab Recipes: Fission Yeast Media

Also see recipes for drugs and DNA damaging agents. These are our standard media; other pombe groups may have different preferences. Fission yeast can be grown on S. cerevisiae media (YPD and SD). However, the cells are not as happy on peptone (the P in YPD), and our experiments suggest that standard yeast nitrogen base (SD) contains enough thiamine to repress the commonly used nmt promoter. But for general culturing purposes, S. cerevisiae media will work fine.

Prepared media can be obtained as a powdered homogenate from several biotech firms. While these can be more expensive than making it yourself, they are time-saving, and this may be more practical especially if your use is occassional. You can also grind your own medium to make an equivalent powder. Note that all media should be sterilized by autoclaving (15 or 20min max to avoid caramelizing the sugar), although liquid media can be filter sterilized.

YES: Yeast Extract with supplements

amt	component	final conc
5 g/l	yeast extract	0.5% w/v
30 g/l	glucose	3.0% w/v
supplements: 225 mg/l adenine, histidine, leucine, uracil and lysine hydrochloride.
Solid media is made by adding 2% Difco Bacto Agar

Variations

Yeast extract + phloxin B (YEP - Checking ploidy)
YES + 5 mg/l phloxin B (Sigma No. P 4030). Add when the medium has cooled below 60*C from a 5g/l stock solution in sterile distilled water.

YSO
As YES, but at 2g/l cas-amino acids to final concentration of 0.2%. (No adenine).

EMM: Edinburgh minimal medium

amt	component	final conc
3 g/l	potassium hydrogen phthallate	14.7mM
2.2 g/l	Na2HPO4	15.5 mM
5 g/l	NH4Cl	93.5 mM
20 g/l	glucose	2% w/v
20 ml/l	salts
1 ml/l	vitamins
0.1 ml/l	minerals
Solid media is made by adding 2% Difco Bacto Agar

PMG: Pombe Glutamate medium

amt	component	final conc
3 g/l	potassium hydrogen phthallate	14.7mM
2.2 g/l	Na2HPO4	15.5 mM
3.75 g/l	L-glutamic acid, monosodium salt (Sigma G-5889)
20 g/l	glucose	2% w/v
20 ml/l	salts
1 ml/l	vitamins
0.1 ml/l	minerals
Solid media is made by adding 2% Difco Bacto Agar PMG is recommended because of more even growth of Ura+ and Ura- strains, and is also compatible with G418 selection. Not to be confused with EMM(low)Glut, below, which uses a limiting amount of glutamate (nitrogen) to induce sporulation.

Variations

Minimal supplemented with amino acids etc
EMM + 225 mg/l supplements (ade, leu, his, lys, ura...) as required. We make stock solutions at 7.5mg/ml (3.75 for uracil) and autoclave.

Minimal low adenine (used to see red color associated with ade6 mutations)
Reduce adenine to 10 mg / l. (Concentrations from 7.5mg/l to 30 mg/l can be used) .

Minimal low glucose (Used for Yeast transformations)
As EMM but 0.5% (w/v) glucose instead of 2% (w/v) .

Minimal sorbitol (EMMS)
As EMM. Add 1.2M sorbitol.

Minimal (low) glutamate (EMMGlut, Used for sporulating diploids in liquid)
As EMM. Replace NH4Cl with 1 g/l sodium glutamate (5.91 mM). Use 40mg/l supplements. Note PMG has higher amount of glutamate.

Minimal free phosphate (EMMP - Used for 32P-Phosphate labelling)
As EMM. Remove Na2HPO4 and replace potassium hydrogen phthallate with 2g/l Na-acetate.3H2O (14.6 mM). The medium is adjusted to pH 5.5.

Minimal + thiamine (to repress nmt promoter)
add 15uM thiamine (5ug/ml; from filter sterilized 2000x stock at 10mg/ml)

For intermediate repression (described in this reference): 0.05uM thiamine (0.016ug/ml).

Why buffer with phthallate? Paul Nurse says this choice was made to reduce clumping, which was a particular problem with cdc mutants .

Stock Solutions

filter sterilize and store at 4*C

50x Salt stock

amt	component	final conc
52.5 g/l	MgCl2.6H20	0.26 M
0.735 g/l	CaCl2.2H20	4.99 mM
50 g/l	KCl	0.67 M
2 g/l	Na2SO4	14.l mM

1000x Vitamin stock

amt	component	final conc
1 g/l	pantothenic acid	4.20 mM
10 g/l	nicotinic acid	8l.2 mM
10 g/l	inositol	55.5 mM
10 mg/l	biotin	40.8 uM

10,000x Mineral stock

amt	component	final conc
5 g/l	boric acid	80.9 mM
4 g/l	MnSO4	23.7 mM
4 g/l	ZnSO4.7H2O	13.9 mM
2 g/l	FeCl2.6H2O	7.40 mM
0.4 g/l	molybdic acid	2.47 mM
1 g/l	KI	6.02 mM
0.4 g/l	CuSO4.5H2O	1.60 mM
10 g/l	citric acid	47.6 mM

MB media

This is a very stringent minimal medium that is used for lithium acetate transformation protocol
amt	Compound
0.5 g/l	KH₂PO₄
0.36 g/l	K-acetate
0.5 g/l	MgSO₄.7H₂O
0.1 g/l	NaCl
0.1 g/l	CaCl₂.2H₂O
5 g/l	(NH₄)₂SO₄
500 ug/l	H₃BO₄
40 ug/l	CuSO₄.5H₂O
100 ug/l	KI
200 ug/l	FeCl₃.6H₂O
400 ug/l	MnSO₄.H₂O
200 ug/l	Na₂MoO₄.2H₂O
400 ug/l	ZnSO₄.7H₂O
5g/l	glucose
10ug/l	biotin
1mg/l	calcium pantothenate
10mg/l	nicotinic acid
10mg/l	myo-inositol
150 mg/l 150 mg/l	uracil for ura4- strains or leucine for leu1- strains.
Filter sterilize

Mating/Sporulation media

Autoclave as usual

ME: Malt extract

amt	component	final conc
30 g/l	Bacto-malt extract	3% (w/v)
Supplements added as for YES except lysine. Adjust to pH 5.5 with NaOH.
Solid media is made by adding 2% Difco Bacto Agar

SPAS mating media

amt	component	final conc
10 g/l	glucose	1% (w/v)
1 g/l	KH2PO4	7.3mM
1 ml	1000x vitimins	(w/v)
supplements: 45 mg/l adenine, histidine, leucine, uracil and lysine hydrochloride (1/5 normal).
Solid media is made by adding 3% Difco Bacto Agar

Growth on nonfermantable carbon sources
Recently (20 Nov 07) a discussion on the pombelist mailing list addressed growth on nonfermentable carbon sources. S. pombe grows very poorly on non-fermentable carbon sources. People report various results, which I summarize here: Strains will grow better if ura4+ You need to include replacements for citric acid cycle intermediates. Kurt Runge quotes, "fats burn in the flame of carbohydrates", but done without glycosis. and recommends the old Gutz method of 0.37% mono sodium glutamate
Suggested recipes
1% yeast extract, 0.5% casaminoacids, 3% glycerol, 3% ethanol, 40 microg/ml adenine. (Nathalie Bonnefoy) use galactose as non-fermentable carbon source, in 1% yeast extract, 1% bactopeptone, 0.1% glucose, 2% galactose, 20 microg/ml adenine (the peptone here comes from an old cerevisiae recipe, it could be eliminated). But you can also try galactose without the 0.1% glucose (they will be slower to start growing), or you can reduce the glucose to 0.05%.(Nathalie Bonnefoy). Charlie Hoffman notes that sucrose, maltose, or raffinose may work. 0.67% Yeast nitrogenbase with 150 mg/l supplements for auxotrophies plus 3% glycerol + 0.37% mono sodium glutamate (from a 7.4% MSG filter-sterilized stock). After 2 weeks, cells reach about 4e7 cells/ml by hemocytometer counts, where as 3% glucose gives 6 e7 in similar medium without MSG. (Kurt Runge) Yeast Extract -dextrose + 3% glycerol as a growth assay for S. pombe. Wild type colonies in 3-4 days. (Nate Blewitt,) liquid 0.5%YE + glycerol (2%? 3%?) without any glucose. (Peter Fantes) EMM doesn't work. Thanks to Peter Fantes, Charlie Hoffman, Nate Blewitt, Nathalie Bonnefoy , and Kurt Runge for responses quoted here.