Composition of Salt Solutions and Culture Media
417
Shields and Sang's medium modified by Sinha and Lakhotia (1980)
Unless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium.
NaCl 860, NaH z P0 4 ·2H z O 8S0, KCl 3,130, KHC0 3 ISO, CaCl z ·2H z O 1,160, MgS0 4 ·7H z O
5,130, malic acid 950, a-ketoglutaric acid 420, fumaric acid SO, succinic acid 140, pyruvic acid 20 Ill, glucose 300, trehalose SOO, lactalbumin hydrolysate S,120, Bacto-peptone 5,000, yeast extract paste 2,000, streptopenicillin 1,000, insulin 0.4 units. The pH
of the medium is adjusted to 7.0 with I N KOH and 1 N HCl. For maintaining the
salivary glands of Drosophila, preconditioning of the medium or co culture with fat
bodies provides better results.
Snail organ culture medium (Wattez, 1975)
Stock solution 1: Agar 1% (Difco Laboratories) in Gey's solution mixed with the same
amount of peptone (Difco Laboratories)
Stock solution 2: mix 63 parts TC-199, S5 parts of 0.3% sodium bicarbonate, S50 parts
Hank's solution, and 2 parts yeast extract.
Mix 7 drops of stock solution 1 and 6 drops of stock solution 2, supplement this mixture with penicillin G 100 IU and polymyxin B 25 IU. The pH of the medium is adjusted to 7.45 with sodium bicarbonate.
TC-199 (see Medium 199).
TC-199MK (Mcintosh et al., 1973)
This medium consists of a mixture of equal parts of TC-199 with Hanks' salts and
Melnick's monkey kidney medium "N', both without sodium bicarbonate. To this, 10%
inactivated FBS is added. Antibiotics are incorporated at final concentrations of 50 IU /
ml penicillin and 50 Ilg/ml streptomycin. 1 ml/lOO ml medium of 200 mM glutamine
is added to the final product. The pH of the medium is 6.9 without adjustment. The
osmolarity is 266 mOsmollkg.
TTP medium (Azumaya and Teshirogi's medium) for planarians (Teshirogi, 1998)
Unless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium.
NaCll,S59.S, KCl 26.4, CaCl z 53.6, MgS04·7H z O 90.1, MnCl z ·4H z O 0.3, NaHC0 3 SOO.O,
L-aspartic acid I.S, L-threonine 6.2, L-serine 6.0, L-glutamic acid 7.7, L-glutamine 4S.9,
glycine 1.3, L-alanine 5.3, L-cystine 2.0, L-valine 3.1, L-methionine 2.0, L-isoleucine 1.5,
L-leucine I.S, L-tyrosine 1.2, L-phenylalanine 2.S, L-lysine 7.7, L-histidine 4.1, L-tryptophan 0.7, choline chloride 0.3, nicotinamide 0.3, calcium-o-pantothenate 0.3, pyridoxal hydrochloride 0.3, riboflavin 0.03, thiamine hydrochloride 0.3, i-inositol 0.3, biotin 0.3, folic acid 0.3, glucose 300.0, trehalose 50.0, tricine 2.5, phenol red 2.0, and FBS
(heated at 56°C for 30 min) 10.0 ml. The pH of the medium is adjusted to 7.4-7.6 with
1 N KOH and 1 N HCl. The osmolarity of the medium is approximately SO mOsmollkg.
Wolff et al?s medium C (Wolff et al., 1953)
First, prepare solution C by dissolving following the chemicals in 25 ml Tyrode's solution (see "Salt Solutions" in Appendix I): L-lysine hydrochloride 15.1 mg, L-arginine
7.7 mg, DL-methionine 2.6 mg, oL-histidine hydrochloride 3.1 mg, L-glutamic acid
14.1 mg, DL-aspartic acid 5.9 mg, L-proline 5.1 mg, L-cysteine hydrochloride 1.5 mg, Ltryptophan 5 mg, OL-phenylalanine 7 mg, L-leucine 9 mg, OL-isoleucine 10 mg, OL-threo-
417
Shields and Sang's medium modified by Sinha and Lakhotia (1980)
Unless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium.
NaCl 860, NaH z P0 4 ·2H z O 8S0, KCl 3,130, KHC0 3 ISO, CaCl z ·2H z O 1,160, MgS0 4 ·7H z O
5,130, malic acid 950, a-ketoglutaric acid 420, fumaric acid SO, succinic acid 140, pyruvic acid 20 Ill, glucose 300, trehalose SOO, lactalbumin hydrolysate S,120, Bacto-peptone 5,000, yeast extract paste 2,000, streptopenicillin 1,000, insulin 0.4 units. The pH
of the medium is adjusted to 7.0 with I N KOH and 1 N HCl. For maintaining the
salivary glands of Drosophila, preconditioning of the medium or co culture with fat
bodies provides better results.
Snail organ culture medium (Wattez, 1975)
Stock solution 1: Agar 1% (Difco Laboratories) in Gey's solution mixed with the same
amount of peptone (Difco Laboratories)
Stock solution 2: mix 63 parts TC-199, S5 parts of 0.3% sodium bicarbonate, S50 parts
Hank's solution, and 2 parts yeast extract.
Mix 7 drops of stock solution 1 and 6 drops of stock solution 2, supplement this mixture with penicillin G 100 IU and polymyxin B 25 IU. The pH of the medium is adjusted to 7.45 with sodium bicarbonate.
TC-199 (see Medium 199).
TC-199MK (Mcintosh et al., 1973)
This medium consists of a mixture of equal parts of TC-199 with Hanks' salts and
Melnick's monkey kidney medium "N', both without sodium bicarbonate. To this, 10%
inactivated FBS is added. Antibiotics are incorporated at final concentrations of 50 IU /
ml penicillin and 50 Ilg/ml streptomycin. 1 ml/lOO ml medium of 200 mM glutamine
is added to the final product. The pH of the medium is 6.9 without adjustment. The
osmolarity is 266 mOsmollkg.
TTP medium (Azumaya and Teshirogi's medium) for planarians (Teshirogi, 1998)
Unless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium.
NaCll,S59.S, KCl 26.4, CaCl z 53.6, MgS04·7H z O 90.1, MnCl z ·4H z O 0.3, NaHC0 3 SOO.O,
L-aspartic acid I.S, L-threonine 6.2, L-serine 6.0, L-glutamic acid 7.7, L-glutamine 4S.9,
glycine 1.3, L-alanine 5.3, L-cystine 2.0, L-valine 3.1, L-methionine 2.0, L-isoleucine 1.5,
L-leucine I.S, L-tyrosine 1.2, L-phenylalanine 2.S, L-lysine 7.7, L-histidine 4.1, L-tryptophan 0.7, choline chloride 0.3, nicotinamide 0.3, calcium-o-pantothenate 0.3, pyridoxal hydrochloride 0.3, riboflavin 0.03, thiamine hydrochloride 0.3, i-inositol 0.3, biotin 0.3, folic acid 0.3, glucose 300.0, trehalose 50.0, tricine 2.5, phenol red 2.0, and FBS
(heated at 56°C for 30 min) 10.0 ml. The pH of the medium is adjusted to 7.4-7.6 with
1 N KOH and 1 N HCl. The osmolarity of the medium is approximately SO mOsmollkg.
Wolff et al?s medium C (Wolff et al., 1953)
First, prepare solution C by dissolving following the chemicals in 25 ml Tyrode's solution (see "Salt Solutions" in Appendix I): L-lysine hydrochloride 15.1 mg, L-arginine
7.7 mg, DL-methionine 2.6 mg, oL-histidine hydrochloride 3.1 mg, L-glutamic acid
14.1 mg, DL-aspartic acid 5.9 mg, L-proline 5.1 mg, L-cysteine hydrochloride 1.5 mg, Ltryptophan 5 mg, OL-phenylalanine 7 mg, L-leucine 9 mg, OL-isoleucine 10 mg, OL-threo-