Method of in vitro cultivation of pathogenic organisms of treponema pallidum and medium therefor

Claim 1

A tissue culture medium suitable for the growth of pathogenic organisms of Treponema Pallidum in the presence of cultured tissue cells wherein one liter of said medium comprises the solid components of: (a) 100 ml of a balanced salt solution selected from modified Earle's balanced salt solution concentrated to between 9.5 and 17 times the standard strength and modified Hank's balanced salt solution concentrated to between 6.5 and 12.6 times standard strength, (b) 20 ml of modified Eagle's MEM amino acid solution without glutamine concentrated to between 35 and 65 times standard strength, and (c) 10 ml of modified Eagle's MEM vitamin solution concentrated to between 100 and 200 times standard strength wherein the modified Earle's balanced salt solution has the following ranges of final standard strength: ______________________________________ mg/l ______________________________________ NaCl 5557.5 - 9945 KCl 380 - 680 NaH.sub.2 PO.sub.4 . H.sub.2 O 133 - 238 MgSO.sub.4 . 7H.sub.2 O 190 - 340 CaCl.sub.2 190 - 340 Glucose 950 - 1700 ______________________________________ the modified Hank's balanced salt solution has the following ranges of final standard strength: ______________________________________ mg/l ______________________________________ CaCl.sub.2 91 - 176.4 KCl 260 - 504 KH.sub.2 PO.sub.4 39 - 75.6 MgCl.sub.2 . 6H.sub.2 O 65 - 126 MgSO.sub.4 . 7H.sub.2 O 65 - 126 NaCl 5200 - 10,080 NaHCO.sub.3 227.5 - 441 Na.sub.2 HPO.sub.4 . 2H.sub.2 O 39 - 75.6 Glucose 650 - 1,260 ______________________________________ the modified Eagle's amino acid solution without glutamine has the following ranges of final standard strength: ______________________________________ mg/l ______________________________________ L-Arginine 73.5 - 136.5 L-Cystine 16.8 - 31.2 L-Histidine 21.7 - 40.3 L-Isoleucine 36.75 - 68.25 L-Leucine 36.68 - 68.12 L-Lysine 40.6 - 75.4 L-Methionine 10.5 - 19.5 L-Phenylalanine 22.4 - 41.6 L-Threonine 33.6 - 62.4 L-Tryptophan 7 - 13 L-Tyrosine 25.2 - 46.8 Valine 32.2 - 59.8 ______________________________________ and the modified Eagle's MEM vitamin solution has the following ranges of final standard strength: ______________________________________ mg/l ______________________________________ Choline Cl 1 - 2 Folic acid 1 - 2 i-Inositol 2 - 4 Nicotinamide 1 - 2 D-Ca pantothenate 1 - 2 Pyridoxal HCl 1 - 2 Riboflavin 0.1 - .2 Thiamine 1.0 - 2 ______________________________________ all the foregoing four solutions being expressed in terms of milligrams per liter (mg/l) and, said medium further comprising: (d) L-Glutamine, 2 - 6.0 mg mols (e) Vitamin B 12 , 0.15 mg to 15 mg, (f) Biotin, 0.1 mg to 1.0 mg, (g) Cobaltous Chloride, 0.05 mg to 0.5 mg, (h) an alkali metal salt of a mono-unsaturated fatty acid of 12 to 22 carbon atoms, 5 mg to 100 mg, (i) an alkali metal salt of a saturated fatty acid of 12 to 22 carbon atoms, 5 to 100 mg, (j) Bovine serum albumin 4 grams to 40 grams, (k) Mannitol, 1 gram to 10 grams, (l) Catalase, 3 times 10 5 units to 1 times 10 6 units, and distilled water to dilute all of said foregoing components up to 1 liter of medium.