TITLE: Ney&#39;s Spring Media Preparation 
AUTHORS: Annette Rowe, Leah  Trutschel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is for protocol for making Ney's Spring high pH media and plates. These consist of a basal minimal media with differing electron donors and acceptors depending on what organism is targeted. </div><div class = "text-block">This includes instructions for:</div><div class = "text-block">sulfur/acetate plates & media</div><div class = "text-block">Methanol plates & media</div><div class = "text-block">Sulfate/acetate plates & media</div></div>

[STEPS]
?. [Ney's Spring Basal Salts]
These are the basal salts used in all medias. If making liquid media, add salts to 700mL milli Q water. If making plates, then start with  400 mL Milli Q (additional components will then be added to bring up to 1L later)ComponentMW (g/mol)Amount added (g)Concentration (mM)NaCl58.4411.7200Na2SO4142.040.2842NH4Cl53.490.53510MgCl2 * 6H2O203.30.1020.5
ComponentMW (g/mol)Amount added (g)Concentration (mM)NaCl58.4411.7200Na2SO4142.040.2842NH4Cl53.490.53510MgCl2 * 6H2O203.30.1020.5
?. [Components added from Stock]
These are all prepared as stock solutions first, then are added to the media after the basal salts. This should bring the liquid media up to ~1L, and media to be used for plates to ~700 mL. After adding these, check the pH. Media should be between pH 10-11. Aim for closer to 10.5 for plates, while liquid media can be closer to 11. For plates, prepare a second solution of 300 mL milli Q water with 15g NOBLE agar. Regular agar burns incredibly easy at high pH and won't be sufficient for this media. Add a stir bar and autoclave separately from the salt solutionComponentMW (g/mol)Amount of Stock preparedAmount added to stock (g)Concentration (mM)amount added to mediaFinal conc.K2HPO4174.1810X (500mL)0.87110100 mL1 mMNa2CO3105.9910X (500 mL)531000100 mL100 mMCAPS (N-cyclohexyl-3-aminopropanesulfonic acid)221.3210X (500 mL)11100100 mL10 mMCH3COONa (sodium acetate)136.08100X (200 mL)5.4420010 mL2 mMNaOH39.99100X (200 mL)450010 mL5 mM
ComponentMW (g/mol)Amount of Stock preparedAmount added to stock (g)Concentration (mM)amount added to mediaFinal conc.K2HPO4174.1810X (500mL)0.87110100 mL1 mMNa2CO3105.9910X (500 mL)531000100 mL100 mMCAPS (N-cyclohexyl-3-aminopropanesulfonic acid)221.3210X (500 mL)11100100 mL10 mMCH3COONa (sodium acetate)136.08100X (200 mL)5.4420010 mL2 mMNaOH39.99100X (200 mL)450010 mL5 mM
?. [Components added from Stock]
Additions after autoclaving:Once media has cooled you may also add vitamins and minerals (1mL of each from a 10,000X conc. Recipes for these can be found here:  ).Note for plates:*It is very important to let the media cool almost entirely before any more additions! Plates especially will burn and turn black almost immediately if your media is still hot when you combine the 700mL salt solution and 300 mL agar solution. Wait until just warm and then very slowly pour the basal salt solution into the agar solution, stirring the entire time. Note for anaerobic media:*While liquid media is still hot and fresh out of the autoclave, immediately begin purging with N2 gas. Purge until cool, then you may add vitamins, minerals, and other components.
?. [Electron donor & Acceptor variants]
***Check first to see if whether these need to be added before or after autoclaving!!Methylotroph/Methanogen enrichmentComponentMW (g/mol)StockAmount added for stock (g)ConcentrationAmount added to mediaMeOH32.04Pure MeOH-2.5 mM2.5 mLNa2S * 9H2O (sodium Sulfide)240.18100 mM2.41 mM10 mLSulfate Reducers EnrichmentComponentMW (g/mol)Amount addedConcentration (mM)NaSO4 (Sodium sulfate)142.041.42 g10 CH3COONa (sodium acetate)136.080.82 g 10Sulfur reducers/oxidizers EnrichmentComponentMW (g/mol)Amount addedConcentration (mM)Polysulfide-8.8 mL20mMCH3COONa (sodium acetate)136.080.41g5mM
ComponentMW (g/mol)StockAmount added for stock (g)ConcentrationAmount added to mediaMeOH32.04Pure MeOH-2.5 mM2.5 mLNa2S * 9H2O (sodium Sulfide)240.18100 mM2.41 mM10 mL
ComponentMW (g/mol)Amount addedConcentration (mM)NaSO4 (Sodium sulfate)142.041.42 g10 CH3COONa (sodium acetate)136.080.82 g 10
ComponentMW (g/mol)Amount addedConcentration (mM)Polysulfide-8.8 mL20mMCH3COONa (sodium acetate)136.080.41g5mM