Preparation of the CAS Blue agar

Protocol

    - This protocol is based on using MM9 minimal media
    - Not all microbes grow on MM9
    - Any media base can be used, given that that media is buffered and can be maintained at pH 6.8
        - This is very important for when mixing CAS solution with base media
    - Media known to work with CAS Blue Dye:
        - LB agar
        - Oatmeal agar
        - TSA agar
        - Potato dextrose agar
        - There are cerainly more.....

Blue Dye:

A. Solution I: - Dissolve 0.06g CAS in 50ml DI-H~2~O B. Solution 2: - Dissolve 0.0027g FeCl~3~ * 6H~2~O in 10ml of 10mM HCl C. Solution 3: - Dissolve 0.073g of HDTMA in 40ml DI-H~2~O D. Combine solutions: - Mix solution 1 with 9 ml of solution 2. - Then mix with solution 3. - Solution should now be blue in color. - Filter sterilize and store in plastic bottle in the dark. - Alternatively, the mixed solution can be autoclaved if appropriate filters are not available.

Growth solution:

A. Minimal Media 9 (MM9) salt stock solution - The minimal media can be changed to an isolate specific media type if needed. - In 500ml DI-H~2~O, dissolve: - 15g KH2PO4 - 25g NaCl - 50g NH4Cl - Filter sterilize - Store at room temperature B. Carbon source - For glucose, sucrose, mannitol, and other standard carbon sources. - In 100ml DI-H~2~O, dissolve: - 20g carbon source - Filter sterilize - Store at room temperature C. Casamino acid solution: - Dissolve 3g casamino acids in 27ml of DI-H~2~O - Extract with 3% 8-hydrozyquinolin in chloroform to remove any trace iron - Filter sterilize - Store at room temperature

CAS agar preparation:

A. Add 100ml of MM9 salt solution to 750ml DI-H~2~O B. Dissolve 32.24g PIPES buffer - NOTE: PIPES will not dissolve below pH 5. - start with pH 6, the pH will drop as PIPES dissolves - Slowly bring pH up to 6.8 - DO NOT EXCEED pH 6.8 - When adding Blue Dye in later steps, the solution will turn green and you will have to start over C. Add 15g agar D. Autoclave at 121^o^C and 15 psi for 20 min E. Cool to ~50^o^C F. Add 30ml of sterile casamino acids solution G. Add 10ml of carbon stock solution H. Slowly add 100ml of Blue Dye - pipette Blue Dye solution down the glass wall of the flask with media - Add while stirring just enough to mix solutions. - if the pH is not correct this is where the solution will turn green - This media can be the worst sometimes. I. Once mixed pour into plates