ATCC medium: 2692 Modified LYI Entamoeba medium
Potassium Phosphate Dibasic ....................... 1.00 g
Potassium Phosphate Monobasic ..................... 0.60 g
Sodium Chloride ................................... 2.00 g
Yeast extract (Sigma Y1625) .......................25.00 g
Liver digest neutralized (Oxoid LP0027) ........... 5.00 g
Glucose ...........................................10.00 g
Cysteine .......................................... 1.00 g
Ascorbic Acid ..................................... 0.20 g
Ferric Ammonium Citrate (brown, 22.8 mg/mL)........ 2.00 ml
1M Sodium Hydroxide (to pH 6.8) ................... 7.50 ml
Water to 830 ml
Dissolve the ingredients in 700.0 ml of distilled water in the order indicated. Adjust pH to 6.8 with 1 N NaOH. Adjust the volume to 830.0 ml with distilled water.
Distribute 83.0 ml aliquots in 100 ml screw-capped bottles and autoclave for 15 minutes. Cool to room temperature. The broth may be stored at -20o C for at least 6 months.
To prepare the complete medium, aseptically add 2.0 ml of Diamond’s Vitamin Solution (see below) and 15 ml of heat inactivated adult bovine serum to 83.0 ml of LYI Broth. Store at 4-8o C in the dark. Use complete medium in 10 days or less.
Diamond’s Vitamin Solution
Solution 1: (DL-6,8-Thioctic acid [DL-alpha-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 (All components from SIGMA):
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 to 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. Store at -20°C.