Media recipes

Most of the following recipes can be found in Shimkets et al. 2006

Table of Contents

1. Liquid media
   1.1. CTT
   1.2. TPM
   1.3. CY

2. Solid media
   2.1 CTT Hard and Soft Agar (CTT-HA and CTT-SA)
   2.2 TPM Hard and Soft Agar (TPM-HA and TPM-SA)
   2.3 CY-Agar
   2.4 CY^min-Agar
   2.5 VY/2
   2.6 Black-Agar
   2.7 WAT and WCX Agar

3. Soil extraction media

Liquid media

CTT

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Ingredient	Final [ ]	per L	note
Tris-HCl (pH 8.0)	10 mM	10 ml 1M stock
MgSO4	8 mM	10 ml 0.8 M stock
Bacto Casitone	10 mg/ml	10 g

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2). Autoclave and cool down to 50 °C before adding KH₂PO₄-K₂HPO₄;1 mM; 1 ml of 1M stock.

TPM

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Ingredient	Final [ ]	per L	note
Tris-HCl (pH 8.0)	10 mM	10 ml 1M stock
MgSO4	8 mM	10 ml 0.8 M stock

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2). Autoclave and cool down to 50 °C before adding KH₂PO₄-K₂HPO₄;1 mM; 1 ml of 1M stock.

CY

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Ingredient	Final [ ]	per L	note
Bacto Casitone	0.3%	3 g
CaCl2•2H2O	0.1%	1 g
Yeast Extract	0.1%	1 g

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2).

Solid media

CTT Hard and Soft Agar (CTT-HA and CTT-SA)

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Ingredient	Final [ ]	per L	note
Tris-HCl (pH 8.0)	10 mM	10 ml 1M stock
MgSO4	8 mM	10 ml 0.8 M stock
Bacto Casitone	10 mg/ml	10 g
Bacto Agar	1.5%	15 g	for soft, 0.5% add 5 g

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2). Autoclave and cool down to 50 °C before adding KH₂PO₄-K₂HPO₄;1 mM; 1 ml of 1M stock.

NOTE: the evolutionary experiment MyxoEE-3 was conducted on Select Agar instead of Bacto Agar! While the two Agar type lead to different quantitative phenotypic outcomes with myxo, they are relatively similar in the qualitative output.

TPM Hard and Soft Agar (TPM-HA and TPM-SA)

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Ingredient	Final [ ]	per L	note
Tris-HCl (pH 8.0)	10 mM	10 ml 1M stock
MgSO4	8 mM	10 ml 0.8 M stock
Bacto Agar	1.5%	15 g	for soft, 0.5% add 5 g

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2). Autoclave and cool down to 50 °C before adding KH₂PO₄-K₂HPO₄;1 mM; 1 ml of 1M stock.

CY-Agar

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Ingredient	Final [ ]	per L	note
Bacto Casitone	0.3%	3 g
CaCl2•2H2O	0.1%	1 g
Yeast Extract	0.1%	1 g
Bacto Agar	1.5%	15 g	for soft, 0.5% add 5 g

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2).

CY^min-Agar

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Ingredient	Final [ ]	per L	note
Bacto Casitone	0.1%	1 g
CaCl2•2H2O	0.1%	1 g
Yeast Extract	0.1%	1 g
Bacto Agar	1.5%	15 g	for soft, 0.5% add 5 g

Bring to volume with deionized water. Adjust pH 7.4 (myxobacteria grow well also at pH 7.2).

VY/2

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Ingredient	Final [ ]	per L	note
Baker’s yeast (commercial)	0.5%	5 g	this can be prepared as a stock solution (10 - 20)
CaCl2•2H2O	0.13 %	1.3 g
Bacto Agar	1.5%	15 g
Cynocobalamin	0.5 mg/L	-	Do NOT autoclave add later when needed

Bring to volume with deionized water. Adjust pH 7.2.
NOTE: B12 should be filtered with 0.2 µm filters before is added to the media.
Check literature if the cultured myxobacteria need B12. For example, Cystobacteriaceae can be grown without.

VY/4

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Ingredient	Final [ ]	per L	note
Baker’s yeast (commercial)	0.25%	5 g	this can be prepared as a stock solution (10 - 20)
CaCl2•2H2O	0.13 %	1.3 g
Bacto Agar	1.5%	15 g
Cynocobalamin	0.5 mg/L	-	Do NOT autoclave add later when needed

Bring to volume with deionized water. Adjust pH 7.2.
Note: first time seen in Zhang et al. 2023. Used to grow Archangium sp. AC19.

Black-Agar

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Prepare media using the standard protocol (e.g., CTT-HA)

Ingredient	Final [ ]	per L	note
Tris-HCl (pH 8.0)	10 mM	10 ml 1M stock
MgSO4	8 mM	10 ml 0.8 M stock
Bacto Casitone	10 mg/ml	10 g
Bacto Agar	1.5%	15 g	for soft, 0.5% add 5 g
Black Mars	2% of the total agar	0.3 g	!work under chemical hood! The fine pouder can be easily inhaled!

WAT and WCX Agar

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Ingredient	Final [ ]	per L	note
CaCl2•2H2O	0.1%	1 g
Agar	1.5%	15 g	vary amount to increase or decrease stiffness
HEPES	20 mM		The addition of HEPES helps also to pH the solution
Cyclohexamide	25 mg/mL		Add after autoclaving. Can use directly the filtered prepared solution stored at -20 °C

Soil Extraction Media

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