General Culturing Tips

General Culturing Tips

3. General Tips for Isolation, Purification and Growth of Methanotrophs

In this section, we describe general methodologies for the culturing of extant methanotrophs and isolation and purification of novel methanotrophic cultures.


3.1.  General Methanotrophic Growth Media

3.1.1. Nitrate Mineral Salts (NMS). By far the most common growth medium used in methanotrophy is “Nitrate Mineral Salts” (NMS) medium first described by Whittenbury, et al. (1970).  The recipe is:

Ingredient Amount (per liter)
MgSO4•7H2O 1.0 g
KNO3 1.0 g
CaCl2•H2O 0.2 g
3.8% (w/v) solution Fe-EDTA 0.1 ml
0.1% (w/v) NaMo•4H2O 0.5 ml
Trace element solution (recipe below) 1.0 ml
Bacto-agar (if making plates) 15 g

Add all above components to 700 mls water and dissolve.  Then add additional water to 1 liter.  Autoclave medium and then add:

  • 10 ml/liter of autoclaved phosphate stock (recipe below)
  • 10ml/liter of filter-sterilized vitamin stock (recipe below)


Trace element solution (1X)

Ingredient Amount (per liter)
FeSO4•7H2O 500 mg
ZnSO4•7H2O 400 mg
MnCl2•7H2O 20 mg
CoCl2•6H2O 50 mg
NiCl2•6H2O 10 mg
H3BO3 (boric acid) 15 mg
EDTA 250 mg

Add above components to 700 mls water and dissolve.  Then add additional water to 1 liter.  NOTE:  This solution need not be sterilized as it is added to the base components of NMS medium prior to autoclaving.  Stock solutions of 1X or 10X can be prepared and stored at 4°C.


Phosphate stock solution (1X)

Ingredient Amount (per liter)
KH2PO4 26 g
Na2HPO4•7(H2O) 62 g

Add above components to 700 mls water and dissolve.  Then add additional water to 1 liter.  Autoclave and store at room temperature.  NOTE:  pH should be 6.8


Vitamin stock (1X)

Ingredient Amount (per liter)
Biotin 2.0 mg
Folic acid 2.0 mg
Thiamine HCl 5.0 mg
Ca pantothenate 5.0 mg
Vitamin B12 0.1 mg
Riboflavin 5.0 mg
Nicotiamide 5.0 mg

Add above components to 700 mls water and dissolve.  Then add additional water to 1 liter and filter sterilize using a 0.2 µm filter.  NOTE:  It is easier to make 10X stock solutions and then dilute to1X prior to use.  Store at 4°C.


Add after autoclaving (per liter)

Ingredient Amount (per liter)
CuCl2.2H2O 1.4 mg (for 10 µM)

Key tips:

  • It is preferable to use the purest water source available for growth of methanotrophs, e.g., deionized-distilled water, BUT on occasion methanotrophic growth can be poor in such water.  If this is the case, deionized water can be used for medium preparation.
  • It is often of interest to vary the copper concentration as it is well-known that copper has a strong effect on methanotrophic activity, e.g., the canonical “copper-switch” (Semrau et al., 2013; Stanley et al., 1983).  Copper is typically added after autoclaving the base components using a stock solution of filter-sterilized CuCl2.  It is common to vary the copper concentration from 0 (no additional copper) to 20 µM, although in some experiments as much as 100 µM has been added.  The amount noted above (10 µM) usually allows good growth. NOTE:  It is IMPORTANT that copper be added AFTER autoclaving via a filter-sterilized 10 mM stock solution to reduce the probability of forming copper precipitates.
  • The Fe-EDTA and NaMO4•4(H2O)4 solutions are typically made at 1X and stored at 4°C
  • It is important that the vitamin and Fe-EDTA solutions not be exposed to light to prevent photodegradation (wrapping stock bottles in aluminum foil is sufficient).
  • Various labs have different variations of NMS medium, with some omitting vitamins to decrease risk of contamination.  Vitamins often stimulate growth of methanotrophs. Also, some labs add the components of the phosphate buffer directly to the base NMS recipe and then autoclave (i.e., the addition of 0.26 g KH2PO4 and 0.62 Na2HPO4•7(H2O) in conjunction with Fe-EDTA, KNO3, etc. to achieve the final desired phosphate concentration).  This protocol is not recommended, as it is more likely that precipitates will form after autoclaving, which may complicate the maintenance of methanotrophic cultures.


3.1.2.  Ammonium Mineral Salts Medium (AMS). Another common medium used for methanotrophic growth is “Ammonium Mineral Salts” medium (AMS) in which 0.5 g NH4Cl is substituted for the 1 g of KNO3 (Whittenbury et al., 1970).  This medium is less common in practice than NMS, as some methanotrophs do not grow well with ammonium as a nitrogen source.  The rest of the recipe and steps of preparation are identical to NMS medium.


3.1.3.  Ammonium Nitrate Mineral Salts Medium (ANMS). This is another variation on NMS medium, in which both ammonium (0.25 g NH4Cl) and nitrate (0.25 g KNO3) are added, with the rest of the medium preparation identical to that for NMS as described above.


3.1.4.  Alternative media for growth of mesophilic methanotrophs. Many alternative growth media have been designed for methanotrophs.  One such example is BTZ, a growth medium (Sharpe et al., 2007) designed for Methylomonas species (a genus within the ?-Proteobacteria) although one could conceivably use this for any mesophilic methanotroph:


Recipe for BTZ Growth Medium

Ammonium Liquid Medium (BTZ)**

Component Conc.(mM) Amount (per liter)
NH4Cl 10 537 mg
KH2PO4 3.67 500 mg
Na2SO4 3.52 500 mg
MgCl2 x 6H2O 0.98 200 mg
CaCl2 x 2H2O 0.68 100 mg
1 M HEPES (pH 7.0) 50 mL
Solution 1 10 mL


**Dissolve in 900 mL H2O.  Adjust to pH=7.0, and add H2O to give a final volume of 1 L. For agar plates: Add 15 g of agarose in 1 L of medium, autoclave, cool liquid solution to 50°C, mix, and pour plates.

Solution 1*

Component Conc.(mM) Amount (per liter)
Nitrilotriacetic acid 66.90 12.80 g
CuCl2 x 2H2O 0.15 25.4 mg
FeCl2 x 4H2O 1.50 300 mg
MnCl2 x 4H2O 0.50 100 mg
CoCl2 x 6H2O 1.31 312 mg
ZnCl2 0.73 100 mg
H3BO3 0.16 10 mg
Na2MoO4 x 2H2O 0.04 10 mg
NiCl2 x 6H2O 0.77 184 mg

Mix the amounts designated above in 900 mL of H2O, adjust to pH 7.0, and add H2O to a final volume of 1L.  Keep refrigerated at 4°C.


3.2.  Media for Growth of “Extreme” Methanotrophs

As noted above, the physiological diversity of methanotrophs is quite broad, they can be isolated from many intriguing locations, including acidic bogs and hot springs, mud volcanoes, hypersaline lakes, etc.  These strains require different growth conditions, and specific media have been developed for these strains.

  • Growth of acidophilic/thermoacidophilic methanotrophs as described by Dedysh, et al. (1998)and used by Pol, et al. (2007); see also Semrau et al. (2008).
  • Growth of psychrophilic methanotrophs as described by Bowman, et al. (1997)
  • Growth of halophilic methanotrophs as described by Heyer, et al (2005), Horz, et al. (2002), and of haloalkaliphilic methanotrophs, as described by Kaluzhnaya et al., (2001).


3.3  Growth on Methane

Growth of methanotrophs can be challenging due to the necessity of having gas-tight systems in which methane can be easily provided to promote growth.


Typically for growth of methanotrophs on plates, a simple gassing system is used to provide methane.  In these systems, vessels initially designed for maintenance of anaerobic cultures (e.g., Oxoid jars) are used.  Plates are streaked and placed in the jar under ambient atmospheric conditions.  A vacuum is then applied to create a partial vacuum in the gas-tight vessel (typically -15 psig).  High-purity methane (99.999%) is then added back to create an internal vessel pressure of 0 psig.  This protocol creates a methane:air ratio of ~1:2.  For batch cultures of methanotrophs, a similar set-up can be created in which cultures are cultivated in Erlenmeyer flasks using rubber stoppers with connectors to allow for the removal and addition of gasses, or in serum vials with crimp-sealed stoppers.  In the latter case, the appropriate volume of methane is added by syringe.


In order to cultivate methanotrophs at larger scale (e.g., > 1 liter) or in continuous culture, it is necessary to continuously add methane and oxygen (in the form of sterile air or oxygen) and vent off-gasses to the outside for safety reasons. It is recommended that these systems include a dissolved oxygen probe to monitor oxygen levels in solution.  NOTE: METHANE/AIR MIXTURES ABOVE 5% METHANE ARE EXPLOSIVE.  It is best to use non-explosive mixtures in continuous culture, and consult an Environmental Health & Safety expert before initiating such experiments.


NOTE:  It is important that high-purity methane be used.  Low purity methane can contain trace amounts of contaminants that can inhibit methanotrophic growth.  Natural gas is inhibitory to most methanotrophs, especially if they were not isolated with natural gas as the methane source, and normally is not used as a methane source.


Maintaining pure cultures of methanotrophs requires vigilance and constant testing, because most strains grow better in mixed culture.  It is wise to be skeptical of any culture that seems to be growing particularly well. On plates, it is common for contaminants to grow under methanotroph colonies, and only after the methanotroph cells have lysed (often after weeks of incubation) is it possible to see thin films of contaminants growing out from the colonies. It is recommended that cultures routinely be checked under the microscope for low levels of contamination (at the few % level), and routinely streaked onto a minimal medium with methanol and also onto a rich medium, such as nutrient broth, to test for the presence of contaminants.  LB is not recommended as a test medium, since the higher salt may inhibit heterotrophic contaminants.  See the isolation and purification section below for more tips on testing cultures for purity.


It is important to maintain good freezer stocks and use them regularly as a source of pure culture.  Glycerol is inhibitory to many methanotrophs.  A protocol that is successful for most strains is to freeze in 10% DMSO (0.9 ml of a culture with OD600 0.5-1 plus 100 µl DMSO, stored at -80C).  It is advisable to check the cultures for freezer stocks carefully for purity and discard any that are suspect.


3.4  Isolation and purification of novel methanotrophs

We would like to stress that the isolation procedures described below have been most commonly used for the isolation and purification of mesophilic methanotrophs, i.e., those that grow best at pH values between 6-8, with Toptimal of ~ 30°C, and an optimal salt concentration of ~0.5% (w/v).  The physiological diversity of methanotrophs, however, is quite broad, including thermoacidophilic, psychrophilic, haloalkaliphilic strains, etc. [see (Semrau et al., 2010) for a recent summary of methanotrophic diversity].  If one is interested in examining such methanotrophs, although the general isolation procedure described below can be followed, the appropriate selective conditions should be utilized and the appropriate growth medium should be chosen as described above.


It should be noted that the isolation and purification of novel methanotrophs from environmental samples requires significant effort, and attention to detail is critical to ensure success.  Although the procedure outlined below may appear to be simple to use to achieve pure methanotrophic cultures, it is alarmingly easy to obtain mixed cultures of methanotrophs and heterotrophs, and one must be very thorough to ensure culture purity. A number of suggested assays are described, with additional references provided for further information.


3.4.1. General Procedure for Isolation of Methanotrophs. Environmental samples (e.g., pore water, sediment, etc.) are usually first enriched in liquid culture with methane as the sole carbon and energy source (typically using a CH4/air mixture of 1:4) and the appropriate environmental conditions (e.g., temperature, pH, salt concentration, etc.) with a simple growth medium (e.g. NMS). If microaerophiles are desired, air should be kept under 10%. After 2-3 passages in liquid culture, 50-100 µl is spread on plates and incubated under the same CH4/air mixture.  Alternatively, samples can be plated directly onto plates, although the growth of oligotrophs can complicate identification of true methanotrophs.  A control plate incubated without methane is a good guide in this case. Once colonies are apparent, single colonies should be selected and continuously re-streaked onto fresh selective methanotrophic medium and on rich medium (e.g., nutrient agar) until no growth on the rich medium is observed.  Culture purity should then be confirmed using phase contrast and electron microscopy, as well as extracting DNA from methane-grown cultures with the16S rRNA gene amplified and cloned.  Scores of clones should then be sequenced to verify culture purity.  One should then also verify the existence of genes encoding for sMMO and pMMO polypeptides using specific PCR primer sets.  Many bacteria are present in nature that grow on very low amounts of organic compounds (oligotrophs), and it is always important to test growth in the absence of added methane, to ensure that the bacteria isolated show methane-dependent growth.


It should be noted here that some methanotrophs are facultative, i.e., some strains can utilize multi-carbon compounds for growth (Semrau et al., 2011).  If it is suspected that novel isolates are facultative, it is necessary to be much more thorough to ensure culture purity. Specifically, the ability of methanotrophic isolates to grow on various multi-carbon compounds should first be determined.  It must be stressed that to date no methanotrophs have been shown to grow on sugars or rich media, but some can grow on short-chain organic acids and ethanol (Semrau et al., 2011). If facultative methanotrophy is suspected, culture purity should be verified by performing the following assays in addition to those described above: (1) whole-cell hybridization with genus/species specific probes; (2) 16S rRNA gene library sequence analysis of scores of clones grown under all substrates added separately; (3) dilution-extinction experiments using both methane and multi-carbon compounds as the sole carbon source, and; (4) quantification of MMO gene(s) when grown on multi-carbon compounds.  For a very thorough and detailed description of the suite of assays one should perform to verify the existence of facultative methanotrophs, the reader is directed to Dedysh and Dunfield (2011).