View source for Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium


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

== Medium components ==
Table 1 MMSM medium composition
{| class="wikitable"
|'''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·3H<sub>2</sub>O (150 g/L)
|2 mL
|-
|Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub>·6H<sub>2</sub>O  (2 g/L)
|1 mL
|-
|Rezasurin (1 g/L)
|0.1 mL
|-
|Trace vitamins
|1 mL
|-
|Trace minerals
|1 mL
|-
|NaHCO<sub>3</sub>
|0.5 g
|-
|Rumen fluid
|3 mL
|-
|NH<sub>4</sub>Cl
|0.1 g
|-
|L-Cysteine hydrochloride·H<sub>2</sub>O
|0.05 g
|-
|Na<sub>2</sub>S·9H<sub>2</sub>O
|0.1 mL
|-
|DTT
|0.05 g
|-
|ddH<sub>2</sub>O
|85 mL
|-
|''Modification of MMSM for growth on  formate''
|
|-
|Add sodium formate
|1.5 g
|-
|Add glycylglycine buffer, 1 M
|20 mL
|-
|ddH<sub>2</sub>O
|65 mL
|}
Table 2 Composition of stock solutions for MMSM medium
{| class="wikitable"
|'''Composition  per 1 L'''
|'''Amount'''
|-
|''Mineral 1''
|
|-
|K<sub>2</sub>HPO<sub>4</sub>
|6 g
|-
|''Mineral 2''
|
|-
|KH<sub>2</sub>PO<sub>4</sub>
|6 g
|-
|(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
|6 g
|-
|NaCl
|12 g
|-
|MgSO<sub>4</sub>·7H<sub>2</sub>O
|2.6 g
|-
|CaCl<sub>2</sub>·2H<sub>2</sub>O
|0.16 g
|-
|''Iron stock solution''
|
|-
|Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub>·6H<sub>2</sub>O
|2 g
|-
|37% HCl
|100 μL
|-
|''Na<sub>2</sub>S·9H<sub>2</sub>O''
|
|-
|2.5 %
|25 g
|-
|0.5 %
|5 g
|-
|''Trace minerals''
|
|-
|Nitriloacetic acid
|1.5 g
|-
|MnSO<sub>4</sub>·H<sub>2</sub>O / MnSO<sub>4</sub>·2H<sub>2</sub>O
|0.45 / 0.5 g
|-
|Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub>·6H<sub>2</sub>O
|0.2 g
|-
|CoCl<sub>2</sub>·6H<sub>2</sub>O
|0.1 g
|-
|ZnSO<sub>4</sub>·7H<sub>2</sub>O
|0.1 g
|-
|CuSO<sub>4</sub>·5H<sub>2</sub>O
|0.01 g
|-
|H<sub>3</sub>BO<sub>3</sub>
|0.01 g
|-
|Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O
|0.01 g
|-
|NiCl<sub>2</sub>·6H<sub>2</sub>O
|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 B<sub>12</sub>
|0.1 mg
|-
|p-aminobenzoic acid
|5 mg
|-
|Lipoic acid
|5 mg
|}
'''Preparation of stock solutions'''.

''Preparation of Mineral 1''. Dissolve 6 g of K<sub>2</sub>HPO<sub>4</sub> 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(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub>·6H<sub>2</sub>O 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 autoclaving. When preparing a filter-sterilized cysteine stock, proceed as follows:

1.    Sparge 20 mL of ddH₂O in an acid-washed serum bottle with N<sub>2</sub>/CO<sub>2</sub> (80:20, v/v) for 1 h. Seal the bottle and transfer it into the anaerobic chamber.

2.    Add 1 g of L-cysteine hydrochloride hydrate in an acid-washed Balch tube, 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 by passage through a 0.22 µm filter. Dispense 5 mL aliquots into sterile 28 mL serum tubes and seal the tubes 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 ddH<sub>2</sub>O 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 ddH<sub>2</sub>O with stirring under room temperature (about 10 min). Then transfer the solution to a serum bottle and sparge with N<sub>2</sub> 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 ddH<sub>2</sub>O. Adjust the pH to 8.0 by adding about 5 g NaOH. Bring the final volume to 1 L with ddH<sub>2</sub>O.

''Preparation of 2.5% and 0.5% sodium sulfide solutions''.

1.   Add 100 mL ddH<sub>2</sub>O to an acid-washed 500 mL round-bottom flask. Add one pellet of NaOH (approximately 0.1 g).

2.   Sparge the solution with N<sub>2</sub> for 90 minutes, then add either 2.5 g or 0.5 g Na<sub>2</sub>S·9H<sub>2</sub>O. 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 N<sub>2</sub> 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.

=== Medium preparation ===
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 N<sub>2</sub>/CO<sub>2</sub> (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 are added. For cultivation of ''Methanobrevibacter'' species, the cultures are repressurized with 207 kPa of H<sub>2</sub>/CO<sub>2</sub> once per day.

For growth on formate, modify the MMSM preparation by adding sodium formate and glycylglycine as listed in Table 1. Replace the H<sub>2</sub>/CO<sub>2</sub> headspace with 103 kPa N<sub>2</sub>/CO<sub>2</sub>.

== Sources ==

* Reference (Used by Metcalf and Whitman Lab)