Media Protocols

CTTYE Media (For 1L)

1. Start with 700mL of MiliQ water in a 2L flask with a stir bar. If not making plates, you can mix right in the graduated cylinder.

2. Add 10mL of Tris (pH 8.0), 10 mL of 0.8M MgSO4, 1 mL of 1M KPO4 (pH 7.6).

3. Mix in 10g of casitone (sometimes labeled as Casein Peptone), 2 g of yeast extract.

4. Transfer to graduated cylinder and add water to a final volume of 1L.

5. Transfer back to flask to remove stir bar.

6. Add 15g agar (if making plates). If making broth, transfer to media bottles that are no more than 2/3 full.

7. Label, add autoclave tape, and set aside to be autoclaved.

8. Turn water bath on if making plates so they get returned warm.

TPM Media (For 1L)

1. Start with 700mL of water in a grad cylinder (or 2L flask if plates).

2. Add 10mL of Tris (pH 7.6), 10mL of 0.8M MgSO4, 1 mL of 1M KPO4.

3. Transfer to a graduated cylinder and add water to a final volume of 1L.

4. Add 15g agar (if making plates).

5. Label, add autoclave tape, and set aside to be autoclaved. Cool and pour into plates.

CTTSA for 500mL

1. Start with 300mL of MiliQ water in a 500mL media bottle,

2. Add 5g casitone and a stir bar. Stir on stir plate gently until powder dissolves.

3. Add 5mL of Tris (pH 8.0), 5 mL of 0.8M MgSO4, 0.5 mL of 1M KPO4 (pH 7.6).

4. After casitone dissolves, pour into a graduated cylinder and bring up final volume to 500mL.

5. Pour back into the 500mL bottle to mix, and then split the volume of media between two 500mL bottles so they don't overflow in the autoclave.

6. Add agar to each 250mL aliquot to make 0.7% agar. Important: do not add agar to media before you put it in its final container. Agar only dissolves in the autoclave, so you will not be able to get an even distribution

MMC Media (for 1 L)

1. Autoclave a bottle(s) for your media

2. Start with 700 ml of MiliQ in a 2L flask.

3. Filter sterilize your MOPS buffer! NOTE: YOU CAN'T AUTOCLAVE MOPS

4. Add 10 mL of MOPS (pH 7.6), 5 ml of 0.8 M MgSO4, 4 mL of 1 M CaCl2

5. Transfer to a graduated cylinder and add water to bring the volume to 1 L.

6. Transfer to media bottles that are no more than 2/3 full.

7. Add 15g agar (if making plates).

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