ATCC Medium: 2695 (Keister's Modified TYI-S-33) old # 1404
Casein Digest…………………..20.00 g
Yeast Extract…………………...10.00 g
Bovine Bile (Sigma B-8381)…...0.75 g
NaCl ……………………………..2.00 g
L-Cysteine HCl………………….2.00 g
Ascorbic Acid…………………...0.20 g
K2HPO4………………………....1.00 g
KH2PO4…………………………0.60 g
Ferric Ammonium Citrate…....22.80 mg
DI Water……………………….880 ml
1. Dissolve the ingredients in 880.0 ml of distilled water in the order indicated.
2. Adjust the pH to 7.0-7.2 with 1N NaOH.
3. Filter sterilize.
4.To prepare the complete medium, aseptically add 20 ml of Diamond’s Vitamins Solution and 100 ml of heat-inactivated adult bovine serum.
5. Mix thoroughly and distribute 13 ml aliquots into 16 x 125 mm screw-capped borosilicate glass test tubes. Store at 4-8°C in the dark. Use within 7-10 d.
NOTE: Serum is heat-inactivated by exposure to 56°C for 30 minutes to inactivate proteins of the complement pathway.
Diamond’s Vitamin Solution
Solution 1: (DL-6,8-Thioctic acid [DL-α-Lipoic acid], 1mg/ml). Dissolve 100 mg of DL-6,8-Thioctic acid (oxidized form, Sigma T1395) in 100 ml of absolute ethanol.
Solution 2: (Vitamin B12, 0.4 mg/ml). Dissolve 40 mg of vitamin B12 (Sigma V2876) in 100 ml distilled water.
Solution 3: (Tween 80, 50% w/v). Dissolve 50 g of Tween 80 (Sigma P1754) in 100 ml absolute ethanol.
Solution 4: alpha-tocopherol phosphate, disodium salt……….0.025 mg
d-biotin……………………………………………….0.025 mg
Calciferol (Vitamin D2)……………………………..0.250 mg
Calcium D-(+)pantothenate………………………..0.025 mg
Choline chloride…………………………………….1.250 mg
Folic acid……………………………………………0.025 mg
i-Inositol……………………………………………..0.125 mg
Menadione (Vitamin K3)…………………………..0.025 mg
Niacin……………………………………………….0.0625 mg
Niacinamide………………………………………..0.0625 mg
p-aminobenzoic acid……………………………….0.125 mg
Pyridoxal HCl……………………………………..0.0625 mg
Pyridoxine HCl……………………………………0.0625 mg
Riboflavin…………………………………………..0.0625 mg
Thiamine HCl……………………………………….0.025 mg
Vitamin A …………………………………………. 0.250 mg
Distilled water 1 L To prepare Diamond’s Vitamin Solution combine the following:
Solution 1………………...0.4 ml Solution 2…………………1.2 ml
Solution 3…………………0.4 ml
Solution 4………………100.0 ml
Sterile distilled water…...18.0 ml
Filter sterilize.