Sulfur Limitation Carboy

• Autoclave carboy
• Make 5L stock 10X salts for sulfur limitation:

• Fill 6L carboy/flask with approx. 4L MilliQ H₂O
• Add with constant stirring:

• CaCl₂-2H₂O                            5g
• NaCl                                        5g
• MgCl₂-6 H₂O                          20.6g
• NH₄Cl                                     202.5g
• KH₂PO₄ (monobasic)             50g

o      Bring up to 5L with MilliQ H₂O

o      Aliquot into 2L media bottles

o      Autoclave

To prepare 1 10L carboy:

• Add the following to 1L of MilliQ H₂O in a 1L flask and mix with magnetic stirrer until glucose is completely dissolved:

• 1000x vitamins                        10mL
• 1000x metals                           10mL
• 2 g/L (NH₄)₂SO₄                     15mL
• Dextrose 50g

• Add contents of flask to separate carboy with approx. 7L of MilliQ H₂O, rinse flask into carboy
• Measure out 1L of 10X sulfur limited salts and add to carboy, rinse grad. cylinder into carboy
• Bring up to 10 L with MilliQ H₂O
• Mix for 5 minutes with magnetic stirrer

• Filter sterilize into autoclaved carboy

Dunham, Maitreya. http://genomics.princeton.edu/dunham/MDchemostat.pdf
Modified, S. Silverman 6/2006