412
Appendix
*Menadione sodium bisulfite/albumin: 0.002 g menadion sodium bisulfite is homogenized with 0.24 g bovine albumin in 20 ml Shistosoma salt solution, and then diluted
with salt solution to a concentration of 0.1 mg/lOO ml; for use, 1 ml is added to 11
medium.
**Yeast extract/serum: yeast extract is made by combining 4 g Fleishmann's dry yeast
with 16 ml Schistosoma salt solution and 16 ml FBS; the suspension is homogenized
with a Sorvall Omni-Mixer (Sorvall) for 3 min, and then centrifuged at 1,000 g for
10 min; the supernatant is removed, and sterilized by filtration.
***Cholesterollalbumin: a suspension of cholesterol is prepared by adding 0.015 and
0.6 g bovine albumin to 60 ml of a selected reference medium; this is incubated for 1 h
at 56°C with frequent agitation. The suspension is sterilized by filtration, which removes all but the submicroscopic particulate material; three drops of the filtrate are
added to each culture at the time of preparation and again at each change of medium.
Medium 199 (= TC-199 medium) with Hank's salts (Morgan et al., 1950)
The concentrations are given as mg/l,OOO ml culture medium.
NaCl 8,000, KCl 400, MgS0 4 ·7H20 200, Na2HP04·2H20 60, KH 2 P0 4 60, CaCl 2 140,
NaHC0 3 350, glucose 1,000, phenol red 20, L-arginine hydrochloride 70, L-histidine
hydrochloride 20, L-lysine monohydrochloride 70, DL-tryptophan 20, DL-phenylalanine
50, DL-serine 50, DL-methionine 30, DL-threonine 60, DL-leucine 120, DL-isoleucine 40,
DL-valine 50, DL-glutamic acid monohydrate 50, DL-aspartic acid 60, DL-a-alanine 50, Lproline 40, L-hydroxyproline 10, glycine 50, L-glutamine 100, L-cystine 20, L-tyrosine
40, L-cysteine hydrochloride 0.1, sodium acetate 50, adenine 10, guanine hydrochloride 0.3, xanthine 0.3, hypoxanthine 0.3, uracil 0.3, thymine 0.3, sodium a-tocopherol
phosphate 0.Q1, thiamin hydrochloride 0.Q1, pyridoxine hydrochloride 0.025, riboflavin 0.01, pyridoxal hydrochloride 0.025, niacin 0.025, niacinamide 0.025, calcium pantothenate 0.01, i-inositol 0.05, ascorbic acid 0.05, folic acid O.OI,p-aminobenzoic acid
0.05, biotin 0.01, menadione 0.01, glutathione 0.05, vitamin A 0.1, calciferol 0.1, cholesterol 0.2, Tween-80 20, adenosine triphosphate sodium salt 10, adenylic acid 0.2, D-2desoxyribose 0.5, D-ribose 0.5, choline chloride 0.5, and ferric nitrate 0,1.
Medium 199 modified by Heacox et al. (Heacox et al., 1983)
The concentrations are given as mg/l,OOO ml culture medium.
NaCI17,550, CaCI 2 ·2H 2 0 930, KC1510, MgS04· 7H 2 0 10,240, NaHC0 3 340, glucose 1,000,
galactose 500, fucose(D) 250, fucose(L) 25, essential amino acids, non-essential amino
acids and vitamin of Medium 199 as indicated in the instructions for the preparation
of Medium 199, glutamine 100, gentamicin 1.0, penicillin 300,000 IU, Nereis coelomic
fluid ultrafiltrate 0-20 ml, and Nereis vitellin 0 or 500.
Medium C for snail cell culture (Basch and Diconza, 1973)
The concentrations are given as mg/l,OOO ml culture medium.
NaCll,300, KCl 97, MgCI 2 ·6H 2 0 325, Na2HP04 17, CaCI 2 ·2H 2 0 558, NaHC0 3 1,769, Llysine 5.8, L-histidine 2.0, L-arginine 2.4, L-aspartic acid 5.6, L-threonine 6.2, L-serine
12.0, L-glutamic acid 9.4, L-proline 5.8, glycine 4.8, L-alanine 4.8, L-valine 3.2, L-methionine 0.03, L-isoleucine 2.0, L-Ieucine 2.9, L-phenylalanine 1.8, diphosphopyridine
nucleotide 7.0, triphosphopyridine nucleotide 2.0, coenzyme A 2.5, cocarboxylase 2.0,
flavin-adenine dinucleotide 2.0, biotin 1.0, folic acid 1.0, choline chloride 1.0, nicotinamide 1.0, D-calcium pantothenate 1.0, pyridoxal hydrochloride 1.0, thiamine hydro-
Appendix
*Menadione sodium bisulfite/albumin: 0.002 g menadion sodium bisulfite is homogenized with 0.24 g bovine albumin in 20 ml Shistosoma salt solution, and then diluted
with salt solution to a concentration of 0.1 mg/lOO ml; for use, 1 ml is added to 11
medium.
**Yeast extract/serum: yeast extract is made by combining 4 g Fleishmann's dry yeast
with 16 ml Schistosoma salt solution and 16 ml FBS; the suspension is homogenized
with a Sorvall Omni-Mixer (Sorvall) for 3 min, and then centrifuged at 1,000 g for
10 min; the supernatant is removed, and sterilized by filtration.
***Cholesterollalbumin: a suspension of cholesterol is prepared by adding 0.015 and
0.6 g bovine albumin to 60 ml of a selected reference medium; this is incubated for 1 h
at 56°C with frequent agitation. The suspension is sterilized by filtration, which removes all but the submicroscopic particulate material; three drops of the filtrate are
added to each culture at the time of preparation and again at each change of medium.
Medium 199 (= TC-199 medium) with Hank's salts (Morgan et al., 1950)
The concentrations are given as mg/l,OOO ml culture medium.
NaCl 8,000, KCl 400, MgS0 4 ·7H20 200, Na2HP04·2H20 60, KH 2 P0 4 60, CaCl 2 140,
NaHC0 3 350, glucose 1,000, phenol red 20, L-arginine hydrochloride 70, L-histidine
hydrochloride 20, L-lysine monohydrochloride 70, DL-tryptophan 20, DL-phenylalanine
50, DL-serine 50, DL-methionine 30, DL-threonine 60, DL-leucine 120, DL-isoleucine 40,
DL-valine 50, DL-glutamic acid monohydrate 50, DL-aspartic acid 60, DL-a-alanine 50, Lproline 40, L-hydroxyproline 10, glycine 50, L-glutamine 100, L-cystine 20, L-tyrosine
40, L-cysteine hydrochloride 0.1, sodium acetate 50, adenine 10, guanine hydrochloride 0.3, xanthine 0.3, hypoxanthine 0.3, uracil 0.3, thymine 0.3, sodium a-tocopherol
phosphate 0.Q1, thiamin hydrochloride 0.Q1, pyridoxine hydrochloride 0.025, riboflavin 0.01, pyridoxal hydrochloride 0.025, niacin 0.025, niacinamide 0.025, calcium pantothenate 0.01, i-inositol 0.05, ascorbic acid 0.05, folic acid O.OI,p-aminobenzoic acid
0.05, biotin 0.01, menadione 0.01, glutathione 0.05, vitamin A 0.1, calciferol 0.1, cholesterol 0.2, Tween-80 20, adenosine triphosphate sodium salt 10, adenylic acid 0.2, D-2desoxyribose 0.5, D-ribose 0.5, choline chloride 0.5, and ferric nitrate 0,1.
Medium 199 modified by Heacox et al. (Heacox et al., 1983)
The concentrations are given as mg/l,OOO ml culture medium.
NaCI17,550, CaCI 2 ·2H 2 0 930, KC1510, MgS04· 7H 2 0 10,240, NaHC0 3 340, glucose 1,000,
galactose 500, fucose(D) 250, fucose(L) 25, essential amino acids, non-essential amino
acids and vitamin of Medium 199 as indicated in the instructions for the preparation
of Medium 199, glutamine 100, gentamicin 1.0, penicillin 300,000 IU, Nereis coelomic
fluid ultrafiltrate 0-20 ml, and Nereis vitellin 0 or 500.
Medium C for snail cell culture (Basch and Diconza, 1973)
The concentrations are given as mg/l,OOO ml culture medium.
NaCll,300, KCl 97, MgCI 2 ·6H 2 0 325, Na2HP04 17, CaCI 2 ·2H 2 0 558, NaHC0 3 1,769, Llysine 5.8, L-histidine 2.0, L-arginine 2.4, L-aspartic acid 5.6, L-threonine 6.2, L-serine
12.0, L-glutamic acid 9.4, L-proline 5.8, glycine 4.8, L-alanine 4.8, L-valine 3.2, L-methionine 0.03, L-isoleucine 2.0, L-Ieucine 2.9, L-phenylalanine 1.8, diphosphopyridine
nucleotide 7.0, triphosphopyridine nucleotide 2.0, coenzyme A 2.5, cocarboxylase 2.0,
flavin-adenine dinucleotide 2.0, biotin 1.0, folic acid 1.0, choline chloride 1.0, nicotinamide 1.0, D-calcium pantothenate 1.0, pyridoxal hydrochloride 1.0, thiamine hydro-