Name: HaHa agar
Creator: MediaDive
License: http://creativecommons.org/licenses/by/4.0/

Main solution #

ml

Equipment needed: Autoclave Filter

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Artificial sea water (ASW) 2x	350.00	ml	-	-
1 Add magnetic stir bar.
HEPES (50 mM, pH 7.5)	9.92	g	14.171	59.469
MilleQ water , fill up to	650.00	ml	-	-
2 pH should be between 6.5 and 7.0.
Bacto agar (washed, see below)	18.00	g/l	18	-
3 Direct before autoclavation, mix agar and liquid. Autoclave, then cool down to 60 ℃ (keep at 55 ℃ in a pre-heated water bath).
4 Add sterile from the following stock solutions:
Trace element solution SL-8	1.40	ml	-	-
Selenite-tungstate solution II	0.70	ml	-	-
KH₂PO₄ (50 g/L, autoclaved)	7.00	ml	23.4	171.951
NH₄Cl (50 g/L, autoclaved)	3.50	ml	7.637	142.774
5 Add carbon sources from the following stock solutions:
Glucose (100 g/L, sterile filtered)	3.50	ml	7.72	42.851
Cellobiose (100 g/L, sterile filtered)	3.50	ml	-	-
Yeast extract (BioChemica, 100 g/L, sterile filtered)	3.50	ml	-	-
Casamino acids (Difco, 100 g/L, sterile filtered)	3.50	ml	-	-
Tryptone peptone (Difco, 100 g/L, sterile filtered)	3.50	ml	-	-
6 Adjust pH to pH 7.5 with 1 M HCl or 1 M NaOH (autoclaved).
MgCl₂ x 6 H₂O (500 g/L, autoclaved)	7.90	ml	-	-
MgSO₄ x 7 H₂O (500 g/L, autoclaved)	9.50	ml	-	-
7 Add MilleQ water to a final volume of 700 mL, avoid bubbles (autoclaved).

Artificial sea water (ASW) 2x #

1000

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
NaCl	52.74	g	52.74	902.464
NaHCO₃	1000.00	g	1000	11903.77
CaCl₂ x 2 H₂O	2.94	g	2.94	19.998
KCl	1.44	g	1.44	19.316
KBr	0.20	g	0.2	1.681
H₃BO₃	40.00	mg	0.04	0.647
SrCl2	40.00	mg	0.04	0.252
NaF	6.00	mg	0.006	0.143
Ultra pure water	1000.00	ml	-	-

300

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Bacto agar	12.6	g	42	-
1 Wash the agar: Add 600 mL ultra pure water. Clean inner surface of the bottle with ultra pure water (final volume < 800 mL). Let agar settle. Remove the overlaying water. Repeat washing twice.
2 Note: The final volume of the washed agar should be less than 300 mL.

1000

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Na₂-EDTA	5.2	g	5.2	15.467
FeSO₄ x 7 H₂O	2.0	g	2	7.194
1 Dissolve in 800 ml water, then add:
CoCl₂ x 6 H₂O	190.0	mg	0.19	1.463
MnCl₂ x 2 H₂O	100.0	mg	0.1	-
ZnSO₄ x 7 H₂O	150.0	mg	0.15	0.522
NiCl₂ x 6 H₂O	24.0	mg	0.024	0.185
Na₂MoO₄ x 2 H₂O	36.0	mg	0.036	0.149
H₃BO₃	62.0	mg	0.062	1.003
CuCl₂ x 2 H₂O	17.0	mg	0.017	0.1
Distilled water	fill up to 1000	ml	-	-
2 Adjust pH to 6.0 with 5 M NaOH. Autoclave.

500

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
NaOH	0.1	g	0.2	5
Na₂SeO₃ x 5 H₂O	9.0	mg	0.018	0.068
Na₂WO₄ x 2 H₂O	9.0	mg	0.018	0.055
Distilled water	500.0	ml	-	-

Creator:
Author(s):	Richard L. Hahnke and Jens Harder
Published:	22.07.22
Last modified:	05.09.23
Source:	MediaDive: public
Medium type:	Complex medium
Final pH:	7.5
Equipment needed:	Autoclave, Filter
Reference:	DOI: 10.1016/j.syapm.2013.06.006

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