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DSMZ

DSMZ 1267 . FERRIPHASELUS (ES) MEDIUM

Official DSMZ medium: This medium was manually curated by experts from the DSMZ.

Culture medium recipe

Vol.
×

Main sol. 1267 #

ml
Equipment needed: Filter Gassing station Autoclave Hungate tubes
Compound Amount Unit Conc. [g/L] Conc. [mM]
Solution A 901 ml - -
Solution B 1 ml - -
Solution C 100 ml - -
1 Preparation of the bottom layer: Mix 1 volume of solution A (except MES buffer, NaHCO3, and agarose) with 1 volume of solution C and add 1% (w/v) agarose type 1, low EEO. After autoclaving, aseptically fill 1 ml of the suspension in sterile Hungate-type tubes (15 ml total volume). The bottom layer solidifies in approx. 30 min.
2 Preparation of the top layer: Add MES buffer, bicarbonate and low melt agarose to solution A and autoclave. Let the sterile solution cool to 40°C and add 10.00 ml/l of solution B. Sparge solution with sterile CO2 gas until a pH of 6.1 - 6.4 is reached. Then, aseptically pipette aliquots of 5.0 ml over the bottom layer of each tube under 100% N2 gas atmosphere and let medium equilibrate for at least three hours, but not longer than 12 hours.
3 Inoculation: Inoculate the semisolid top layer under a stream of 80% N2 and 20% CO2 gas mixture using a sterile Pasteur pipette that is inserted just above the FeS layer; the pipette tip is drawn upward as the inoculum is dispensed. After inoculation close the tube and add 1.00 ml of sterile air.
4 Note: Some hints on the inoculation of Hungate-tubes under anoxic conditions can be found in the Video tutorial on the „Proper Handling of the Double-Vial Glass Ampoules (anaerobe)" (www.dsmz.de/shop-1/faqs/video-tutorials).

Solution A #

901
Compound Amount Unit Conc. [g/L] Conc. [mM]
NH4Cl 1.00 g 1.11 20.749
MgSO4 x 7 H2O 0.20 g 0.222 0.901
CaCl2 x 2 H2O 0.10 g 0.111 0.755
K2HPO4 0.05 g 0.055 0.319
Modified Wolin's mineral solution 1.00 ml - -
MES
(SIGMA, for top layer) 		1.95 g 2.164 11.085
NaHCO3
(for top layer) 		0.50 g 0.555 6.606
Agarose
(for top layer, low melt) 		1.50 g 1.665 2.64
Distilled water 900.00 ml - -

Solution B #

1
Compound Amount Unit Conc. [g/L] Conc. [mM]
Wolin's vitamin solution (10x) 1 ml - -
1 Sterilize by filtration and store at 4°C in the dark.

Solution C #

100
Compound Amount Unit Conc. [g/L] Conc. [mM]
Ferrous sulfide sludge 100 ml - -

Modified Wolin's mineral solution #

1000
Solution occurs in 166 other media (first defined in Medium 141).
Compound Amount Unit Conc. [g/L] Conc. [mM]
Nitrilotriacetic acid 1.50 g 1.5 7.848
MgSO4 x 7 H2O 3.00 g 3 12.172
MnSO4 x H2O 0.50 g 0.5 2.958
NaCl 1.00 g 1 17.112
FeSO4 x 7 H2O 0.10 g 0.1 0.36
CoSO4 x 7 H2O 0.18 g 0.18 1.161
CaCl2 x 2 H2O 0.10 g 0.1 0.68
ZnSO4 x 7 H2O 0.18 g 0.18 0.626
CuSO4 x 5 H2O 0.01 g 0.01 0.04
AlK(SO4)2 x 12 H2O 0.02 g 0.02 0.042
H3BO3 0.01 g 0.01 0.162
Na2MoO4 x 2 H2O 0.01 g 0.01 0.041
NiCl2 x 6 H2O 0.03 g 0.03 0.231
Na2SeO3 x 5 H2O 0.30 mg 3.0e-4 0.001
Na2WO4 x 2 H2O 0.40 mg 4.0e-4 0.001
Distilled water 1000.00 ml - -
1 First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.

Wolin's vitamin solution (10x) #

1000
Solution occurs in 305 other media (first defined in Medium 120).
Compound Amount Unit Conc. [g/L] Conc. [mM]
Biotin 20 mg 0.02 0.082
Folic acid 20 mg 0.02 0.045
Pyridoxine hydrochloride 100 mg 0.1 0.486
Thiamine HCl 50 mg 0.05 0.148
Riboflavin 50 mg 0.05 0.133
Nicotinic acid 50 mg 0.05 0.406
Calcium D-(+)-pantothenate 50 mg 0.05 0.21
Vitamin B12 1 mg 1.0e-3 7.4e-4
p-Aminobenzoic acid 50 mg 0.05 0.365
(DL)-alpha-Lipoic acid 50 mg 0.05 0.242
Distilled water 1000 ml - -

Ferrous sulfide sludge #

100
Solution occurs in 3 other media (first defined in Medium 1267).
Compound Amount Unit Conc. [g/L] Conc. [mM]
FeSO4 x 7 H2O 15.4 g 154 553.927
Na2S x 9 H2O 12.3 g 123 512.112
Distilled water 100.0 ml - -
1 Heat distilled water to 50°C in a 250 ml beaker with a stir bar present. While rapidly stirring the water, add the ferrous sulfate followed immediately by the sodium sulfide. The formed black FeS sludge is decanted into a glass bottle that can be stoppered. The FeS is allowed to settle for several hours and then the overlying water is decanted and replaced. This procedure is repeated at least five times to wash the FeS. After washing, the pH of the FeS solution should be close to neutrality. The FeS suspension can be kept in closed bottles or tubes under a nitrogen atmosphere for at least three months.

Metadata

Last modified: 18.10.22
Source: DSMZ
Taxonomic range:  Bacteria
Medium type: Defined medium
Final pH: 6.1 - 6.4
Equipment needed: Autoclave, Filter, Gassing station, Hungate tubes
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Gas composition

  • 80 % N2
  • 20 % CO2

Detailed instructions for the cultivation of anaerobes including important warnings can be found in the cultivation instructions .

Associated strains

All strains for this medium: 2

Molecular composition

Compound Conc. [g/L] Conc. [mM]
FeSO4 x 7 H2O 15.369 55.282
Na2S x 9 H2O 12.275 51.109
MES 1.946 9.968
Agarose 1.497 2.374
NH4Cl 0.998 18.658
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Cultivation metadata from 2 strains BacDive

phyla 2 2 strains
oxygen microaerophile 1 strain
temperature mesophilic 2 strains