1. Combine and dissolve the following reagents:
500 ml ddH2O, 20.0 g casein digest,
10.0 g yeast extract, 10.0 g glucose, 2.0 g
NaCl, 0.6 g KH2PO4, 1.3 g K2HPO4 . 3H2O,
2.0 g L-cysteine HCl monohydrate (i.e.
Sigma-Aldrich C7880), 0.2 g L-ascorbic
acid, and 0.012 g ferric ammonium citrate.
Note: if biosate (2:1 casein digest to yeast
extract) is preferred, use 30.0 g per 1 liter in
place of the casein digest and yeast extract.
2. pH the solution with 5 M NaOH to 7.1, and
then add ddH2O quantity suffi cient (QS) to
890 ml volume. Filter sterilize with 0.22 M
fi lter unit (i.e. Millipore Steritop, GP Millipore
Express® PLUS Membrane). Do not
autoclave any components of modifi ed TYIS-
33 medium.
3. After fi ltration, add 10 ml of sterile 100
bovine bile and 100 ml of heat-inactivated
bovine serum.