MMSM

Description

Modified Methanobrevibacter smithii and Methanospheara stadtmanae medium (MMSM) is a rich, undefined medium commonly used for the anaerobic cultivation of Methanobrevibacter species.

Recipe

Table 1 MMSM medium composition

Composition per 100 mL	Amount
Modified Rich medium for Methanobrevibacter (MMSM)
Mineral 1	3.3 mL
Mineral 2	3.3 mL
Yeast extract	0.2 g
Tryptone	0.2 g
Na Acetate·3H2O (150 g/L)	2 mL
Fe(NH4)2(SO4)2·6H2O (2 g/L)	1 mL
Rezasurin (1 g/L)	0.1 mL
Trace vitamins	1 mL
Trace minerals	1 mL
NaHCO3	0.5 g
Rumen fluid	3 mL
NH4Cl	0.1 g
L-Cysteine hydrochloride·H2O	0.05 g
Na2S·9H2O	0.1 mL
DTT	0.05 g
ddH2O	85 mL
Modification of MMSM for growth on formate
Add sodium formate	1.5 g
Add glycylglycine buffer, 1 M	20 mL
ddH2O	65 mL

Table 2 Composition of stock solutions for MMSM medium

Composition per 1 L	Amount
Mineral 1
K2HPO4	6 g
Mineral 2
KH2PO4	6 g
(NH4)2SO4	6 g
NaCl	12 g
MgSO4·7H2O	2.6 g
CaCl2·2H2O	0.16 g
Iron stock solution
Fe(NH4)2(SO4)2·6H2O	2 g
37% HCl	100 μL
Na2S·9H2O
2.5 %	25 g
0.5 %	5 g
Trace minerals
Nitriloacetic acid	1.5 g
MnSO4·H2O / MnSO4·2H2O	0.45 / 0.5 g
Fe(NH4)2(SO4)2·6H2O	0.2 g
CoCl2·6H2O	0.1 g
ZnSO4·7H2O	0.1 g
CuSO4·5H2O	0.01 g
H3BO3	0.01 g
Na2MoO4·2H2O	0.01 g
NiCl2·6H2O	0.025 g
Trace vitamins
Biotin	2 mg
Folic acid	2 mg
Pyridoxine hydrochloride	10 mg
Thiamine hydrochloride	5 mg
Riboflavin	5 mg
Nicotinic acid	5 mg
DL-Pantolactone	5 mg
Vitamin B12	0.1 mg
p-aminobenzoic acid	5 mg
Lipoic acid	5 mg

Preparation of stock solutions.

Preparation of Mineral 1. Dissolve 6 g of K₂HPO₄ in 1 liter of glass-distilled water. Use 3.3 mL per 100 mL of medium.

Preparation of Mineral 2. Use 3.3 mL per 100 mL of medium (see Table 2 for details).

Preparation of Iron stock solution. Add 0.2 g of Fe(NH₄)₂(SO₄)₂·6H₂O into a small screw top bottle. Add 100 μL of concentrated HCl, then bring the volume to 100 mL with glass-distilled water.

Preparation of L-cysteine hydrochloride hydrate solution. Use 0.05 g per 100 mL of medium. L-cysteine hydrochloride hydrate can be added directly to the medium before autoclave. When preparing a filter-sterilized cysteine stock, proceed as follows:

1.    Sparge 20 mL of ddH₂O in an acid-washed serum bottle with N₂/CO₂ (80:20, v/v) for 1 h. Seal the bottle transfer it into the anaerobic chamber.

2.    Add 1 g of L-cysteine hydrochloride hydrate in an acid-washed Balch tube and transfer the tube into the chamber and leave it overnight.

3.    Add 20 mL anaerobic water to the tube containing cysteine and mix well.

4.    Filter-sterilize the cysteine solution through a 0.22 µm filter. Dispense 5 mL aliquots into sterile 28 mL serum tubes and seal with sterile blue stoppers and aluminum seals.

5.   For medium preparation, add 0.05 mL of the cysteine stock solution to each 5 mL of medium prior to inoculation.

Preparation of Trace mineral solution. Use 1 mL per 100 mL of medium (see Table 2 for details). Nitriloacetic acid was first dissolved in ~800 mL of ddH₂O, and the pH was adjusted to 6.5 with KOH. Add minerals in order, allowing each one to dissolve before adding the next mineral. At the end, bring the final volume to 1 L and adjust pH to 7.0.

Preparation of Trace vitamin mix solution. Use 0.05 mL per 5 mL of medium (see Table 2 for details). Dissolve the 10 ingredients listed in Table 2 in 1 liter of ddH₂O with stirring under room temperature (about 10 min). Then transfer the solution to a serum bottle and sparge with N₂ for 1 h. Inside the anaerobic chamber, the solution is filter-sterilized by passage through a 0.22 µm filter. Aliquots of 20 mL are transferred to sterile serum bottles and stored inside the chamber.

Preparation of Glycylglycine solution. Use 20 mL per 100 mL of formate medium (see Table 1 for details). Dissolve 132 g glycylglycine with 800 mL ddH₂O. Adjust the pH to 8.0 by adding about 5 g NaOH. Bring the final volume to 1 L with ddH₂O.

Preparation of 2.5% and 0.5% sodium sulfide solutions.

1.   Add 100 mL ddH₂O to an acid-washed 500 mL round-bottom flask. Add one pellet of NaOH (approximately 0.1 g).

2.   Sparge the solution with N₂ for 90 minutes, then add either 2.5 g or 0.5 g Na₂S·9H₂O. Loosely stopper the flask and continue sparging for an additional 10 min. Transfer the flask to the anaerobic chamber and dispense the solution into 5 mL aliquots in 28 mL aluminum-sealed tubes.

3.   Remove the tubes from chamber and pressurize with N₂ to 103 kPa.

4.   Autoclave the tubes using gravity cycle 121 °C, 20 min, D: 5 min.

5.   Store the sodium sulfide tubes inside the anaerobic chamber. They remain stable for up to two months. Discard if a precipitate forms.

Directions

Preparation of 100 mL of MMSM liquid medium. Prior to medium preparation, glassware was cleaned with 5% HCl overnight and dried at 150 °C for five hours to ensure that it was free of organic matter before use. MMSM liquid medium was prepared by combining the components indicated in Table 1 within an acid-washed 500 mL round-bottom flask. Sparge the medium with N₂/CO₂ (80:20, v/v) for 1 hour. Add 0.05 g DL-Dithiothreitol (DTT) and 0.05 g L-cysteine hydrochloride hydrate to reduce the medium and continue sparging for 0.5 hour. Tightly stopper and transfer the flask into the anaerobic chamber. Dispense the medium into Balch tubes or serum bottles. Seal the tubes with 20 mM blue stoppers (Bellco Glass, Inc.) and secure each stopper with an aluminum seal. Remove the tubes from the chamber and sterilize the tubes by autoclaving at 121 °C for 20 min. Prior to inoculation, 0.05 mL of sterile vitamin mix and 0.1 mL of sterile 0.5% sodium sulfide was added. For cultivation of Methanobrevibacter species, the cultures were repressurized with 207 kPa of H₂/CO₂ once per day.

For growth on formate, modify the MMSM preparation by adding sodium formate and glycylglycine as listed in Table 1. Replace the H₂/CO₂ headspace with 103 kPa N₂/CO₂.

Sources

• Reference (Used by Metcalf and Whitman Lab)