Modified Methanobrevibacter smithii and Methanosphaera stadtmanae medium (MMSM) - Revision history

Nanashao: /* Sources */

2026-10-28T18:42:21Z

Sources

← Older revision		Revision as of 18:42, 28 October 2026
Line 232:		Line 232:
	*External Links		*External Links
	<references />		<references />
	* ~~Reference (~~Used by Metcalf and Whitman Lab)		* Used by Metcalf and Whitman Lab

Nanashao: /* Medium preparation */

2026-10-28T18:41:59Z

Medium preparation

← Older revision		Revision as of 18:41, 28 October 2026
Line 220:		Line 220:

	== Medium preparation ==		== 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.		'''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> (80:20, v/v) 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>.		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>.

	'''Preparation of MMSM solid medium.''' ~~This protocol prepares~~ 1.5% agar ~~medium~~ in 70 mL serum bottles for plating ''Methanobrevibacter'' strains ~~and is~~ modified from Long et al. <ref>Long F, Wang L, Lupa B, Whitman WB. A Flexible System for Cultivation of ''Methanococcus'' and Other Formate-Utilizing Methanogens. Archaea. 2017 Nov 19;2017:7046026. doi: [https://onlinelibrary.wiley.com/doi/10.1155/2017/7046026 10.1155/2017/7046026].</ref>		'''Preparation of MMSM solid medium.''' MMSM solid medium containing 1.5% agar (w/v) was prepared in 70 mL serum bottles for plating ''Methanobrevibacter'' strains using a protocol modified from Long et al. <ref>Long F, Wang L, Lupa B, Whitman WB. A Flexible System for Cultivation of ''Methanococcus'' and Other Formate-Utilizing Methanogens. Archaea. 2017 Nov 19;2017:7046026. doi: [https://onlinelibrary.wiley.com/doi/10.1155/2017/7046026 10.1155/2017/7046026].</ref>. Before medium preparation, 0.15 g of agar was added to each serum bottle. The remaining medium components were prepared as described above for MMSM liquid medium. The liquid medium and agar-containing bottles were transferred into an anaerobic chamber, and 10 mL of medium was dispensed into each bottle. The bottles were sealed with blue rubber stoppers (Bellco Glass, Inc.), and secured with aluminum crimp seals. The sealed bottles were removed from the chamber, pressurized to 103 kPa of H<sub>2</sub>/CO<sub>2</sub> (80:20, v/v), and autoclave at 121 °C for 20 min.

			To obtain individual colonies of ''Methanobrevibacter'', cultures were serially diluted in MMSM broth. Undiluted and diluted culture were plated by transferring 1 mL of each suspension onto agar bottle surface using a 1 mL syringe, with each bottle resting on its side. The bottles were gently swirled to distribute the suspension evenly across the agar surface, then left on their sides overnight (~20 h), with the agar surface level and horizontal, to allow the suspension to be fully absorbed. The inoculated bottles were subsequently incubated upright outside of the anaerobic chamber and repressurized to 207 kPa with H₂/CO₂ (80:20, v/v) every 2 days. Colonies appeared after ~7 days of incubation.

	== Sources ==		== Sources ==

Nanashao at 18:13, 28 October 2026

2026-10-28T18:13:07Z

← Older revision		Revision as of 18:13, 28 October 2026
Line 224:		Line 224:
	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>.		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>.

	'''Preparation ~~of 100 mL~~ of MMSM solid 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 indicate~~		'''Preparation of MMSM solid medium.''' This protocol prepares 1.5% agar medium in 70 mL serum bottles for plating ''Methanobrevibacter'' strains and is modified from Long et al. <ref>Long F, Wang L, Lupa B, Whitman WB. A Flexible System for Cultivation of ''Methanococcus'' and Other Formate-Utilizing Methanogens. Archaea. 2017 Nov 19;2017:7046026. doi: [https://onlinelibrary.wiley.com/doi/10.1155/2017/7046026 10.1155/2017/7046026].</ref>

	== Sources ==		== Sources ==

			*External Links
			<references />
	* Reference (Used by Metcalf and Whitman Lab)		* Reference (Used by Metcalf and Whitman Lab)

Nanashao at 17:40, 28 October 2026

2026-10-28T17:40:50Z

← Older revision		Revision as of 17:40, 28 October 2026
Line 182:		Line 182:
	\|5 mg		\|5 mg
	\|}		\|}
	~~'''~~Preparation of stock solutions~~'''.~~		== 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 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.

Line 220:		Line 219:
	5. Store the sodium sulfide tubes inside the anaerobic chamber. They remain stable for up to two months. Discard if a precipitate forms.		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 ===		== 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.		'''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.

Nanashao at 17:39, 28 October 2026

2026-10-28T17:39:13Z

← Older revision		Revision as of 17:39, 28 October 2026
Line 221:		Line 221:

	=== Medium preparation ===		=== 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.		'''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>.		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>.

			'''Preparation of 100 mL of MMSM solid 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 indicate

	== Sources ==		== Sources ==

	* Reference (Used by Metcalf and Whitman Lab)		* Reference (Used by Metcalf and Whitman Lab)

Nanashao at 16:43, 18 October 2026

2026-10-18T16:43:20Z

Show changes

Nanashao: Blanked the page

2026-10-18T16:41:21Z

Blanked the page

Show changes

Nanashao: Created page with " == 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 |- |Try..."

2026-10-18T16:38:59Z

Created page with " == 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 |- |Try..."

New page

== 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·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
{| class="wikitable"
|'''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 autoclaving. 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 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 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.

=== 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 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 are added. For cultivation of ''Methanobrevibacter'' species, the cultures are 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)