Appendix 1 Composition of Salt Solutions and Culture Media (Listed in Alphabetical Order) A Salt Solutions Balanced salt solution (BSSO) for oyster (Tripp et al., 1966) The concentrations are given as g/l,OOO ml solution. NaCI23.50, KCI 0.67, CaCl z 1.10, MgCl z 2.03, MgS04 2.94, NaHC0 3 0.02, K z HP0 4 0.19, glucose 0.50, trehalose 0.50, and phenol red 0.05, made up to 1,000 ml with distilled water. Buffered physiological solution with antibiotics (BPSA; Hansen, 1979) Unless otherwise indicated, the concentrations are given as g/l,OOO ml solution. NaCI 2.2, KCI 0.12, Naz HP04 0.06, glucose 0.8, trehalose 0.8, penicillin 1,000,000 IV, streptomycin 1.0, fungizone 0.0025, and chlortetracycline 0.05, made up to 1,000 ml with distilled water. Carlson's solution (Carlson et at, 1947) The concentrations are given as g/l,OOO ml solution. NaCI7.0, KCI 0.2, CaClz·HzO 0.2, MgClz·6HzO 0.1, NaHz P04 0.2, NaHC0 3 0.12, and glu- cose 8.0, made up to 1,000 ml with distilled water. Chernin's balanced salt solution (BSS; Chernin, 1963) Unless otherwise indicated, the concentrations are given as g/l,OOO ml solution. NaCI 2.80, KCI 0.15, Na2HP04 0.07, MgS0 4·7H20 0.45, CaCI2·2H2 0 0.53, NaHC0 3 0.05, glucose 1.00, trehalose 1.00, penicillin G 100,000 IV, streptomycin sulfate 0.1, and phe- nol red (0.4%) 5 ml, made up to 1,000 ml with distilled water. Chiarandini's saline solution (Chiarandini, 1964) The concentrations are given as g/l,OOO ml solution. NaCI6.45, KCI 0.36, CaCI 2 ·2H z O 0.94, MgS0 4 ·7H z O 0.86, and NaHC0 3 1.54, made up to 1,000 ml with distilled water. Crab saline (Cooke et at, 1989) NaCl 440 mM, KCI 11.3 mM, CaCl z 13.3 mM, MgCl z 26 mM, Na Z S0 4 23 mM, HEPES 10 mM, pH adjusted to 7.4 with NaOH. A mixture of antibiotics (penicillin 100 IV/ml, streptomycin 0.1 mg/ml, and fungizone 0.25 g/ml) is added to the Crab saline. FPl Concentrated complex supplement (Domart-Coulon et al., 1994) Insulin 500 Ilg/ml, hydrocortisone 10- 3 M, epidermal growth factor 250 ng/ml, plate- let-derived growth factor 100 ng/ml, catalase 500 Ilg/ml, Ex-Cyte VLE (Pentex-Miles) 1 %, egg yolk extract 10%, prostaglandin F z 10- 7 M.
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Appendix 1
Composition of Salt Solutions and Culture Media (Listed in Alphabetical Order)
A Salt Solutions
Balanced salt solution (BSSO) for oyster (Tripp et al., 1966) The concentrations are given as g/l,OOO ml solution. NaCI23.50, KCI 0.67, CaClz 1.10, MgClz 2.03, MgS04 2.94, NaHC03 0.02, KzHP04 0.19, glucose 0.50, trehalose 0.50, and phenol red 0.05, made up to 1,000 ml with distilled water.
Buffered physiological solution with antibiotics (BPSA; Hansen, 1979) Unless otherwise indicated, the concentrations are given as g/l,OOO ml solution. NaCI 2.2, KCI 0.12, NazHP04 0.06, glucose 0.8, trehalose 0.8, penicillin 1,000,000 IV, streptomycin 1.0, fungizone 0.0025, and chlortetracycline 0.05, made up to 1,000 ml with distilled water.
Carlson's solution (Carlson et at, 1947) The concentrations are given as g/l,OOO ml solution. NaCI7.0, KCI 0.2, CaClz·HzO 0.2, MgClz·6HzO 0.1, NaHzP04 0.2, NaHC03 0.12, and glucose 8.0, made up to 1,000 ml with distilled water.
Chernin's balanced salt solution (BSS; Chernin, 1963) Unless otherwise indicated, the concentrations are given as g/l,OOO ml solution. NaCI 2.80, KCI 0.15, Na2HP04 0.07, MgS04·7H20 0.45, CaCI2·2H20 0.53, NaHC03 0.05, glucose 1.00, trehalose 1.00, penicillin G 100,000 IV, streptomycin sulfate 0.1, and phenol red (0.4%) 5 ml, made up to 1,000 ml with distilled water.
Chiarandini's saline solution (Chiarandini, 1964) The concentrations are given as g/l,OOO ml solution. NaCI6.45, KCI 0.36, CaCI2·2HzO 0.94, MgS04·7HzO 0.86, and NaHC03 1.54, made up to 1,000 ml with distilled water.
Crab saline (Cooke et at, 1989) NaCl 440 mM, KCI 11.3 mM, CaClz 13.3 mM, MgClz 26 mM, NaZS04 23 mM, HEPES 10 mM, pH adjusted to 7.4 with NaOH. A mixture of antibiotics (penicillin 100 IV/ml, streptomycin 0.1 mg/ml, and fungizone 0.25 g/ml) is added to the Crab saline.
Gey's salt solution (Gey, 1949) The concentrations are given as gll,OOO ml solution. NaCI 8.0, KCI 0.38, CaCl2 0.13, MgClz-6H20 0.21, Na2HP04·12H20 0.3, KH2P04 0.025, NaHC03 1.0, and glucose 2.2, made up to 1,000 ml with distilled water.
Hydra culture solution (Loomis and Lenhoff, 1956) Chemically defined solution: CaCl2 1O-3M, NaHC03 1O-3M, Convenient solution: NaHC03 20 g, disodium ethylendiaminetetraacetic acid 10 g/ tap water 1,000 ml.
Insect Ringer's solutions (Yamasaki and Narahashi, 1963)
Isotonic buffer for Echinococcus (IBE; Dieckmann and Frank, 1988) NaCl121.00 mM, KCI 5.36 mM, MgS04 0.77 mM, MgCl2 0.93 mM, Na2HP04 0.34 mM, KH2P04 0.44 mM, glucose 5.56 mM, ethyleneglycol-bis-(~-aminoethylether) N,N'tetra acetic acid 0.20 mM, N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid 10.00 mM, pH 7.40.
Laverdure's physiological solution (Laverdure, 1971) The concentrations are given as g/I,OOO ml solution. CaCl2 0.6, MgCI2·6H20 1.0, MgS04·7H20 5.5, NaH2P04 1.0, KCI5.5, trehalose or glucose 1.6, made up to 1,000 ml with distilled water. The pH is adjusted to 7.0 with 5.5 % NaHC03•
Leech Ringer's solution (Nicholls and Kuffler, 1965) NaCl115 mM, KCl4 mM, CaCl2 1.8 mM, Tris maleate buffered with NaOH 10 mM, and glucose 11 mM.
Composition of Salt Solutions and Culture Media 403
Locke's salt solution (Locke, 1895) The concentrations are given as gll,OOO ml solution. NaCI 9.0, KCI 0.42, CaCl2 0.24, and NaHC03 0.2, glucose 1.0 made up to 1,000 ml with distilled water.
Modified Alsever's anti-coagulant solution (Lebel et al., 1996) 115 mM glucose, 27 mM sodium citrate, 11.5 mM EDTA, and 3S2 mM NaCI.
Molluscan balanced salt solution (MMBSS; Machii and Wada, 1989) The concentrations are given as gll,OOO ml solution. NaCI26.22, KCI1.0S, MgS04 3.1S, MgCl2 2.20, CaCl2 1.12, NaHC03 0.30, NaH2P04 0.044, and glucose 0.30, made up to 1,000 ml with distilled water.
Necco's physiological solution (Burnett et al., 1968) The concentrations are given as gll,OOO ml solution. NaCI 25.S00, KCI 0.644, CaCl2 0.644, MgCI2·6H20 3.0S0, KH2P04 0.020, Na2HP04 O.OSO, NaHC03 0.200, Na2S04 0.920, sodium pyruvate 1.500, sodium fumarate 1.050, Lglutamine 1.090, and phenol red 0.020. The pH of the solution is adjusted to 7.5. The antibiotics used in the solution are kanamycin 0.300 gIl, streptomycin 0.100 gIl, and penicillin 0.100 gIl.
Nereis balanced salt solution (NBSS; Heacox et al., 1983) To prepare NBSS, add 6.39 g NaCl, 1.00 g NaHC03, 0.34 g KCI, and 0.169 g CaCI2·2H20 to 742.2 ml natural seawater. Dilute the solution with distilled water to attain the proper osmolarity (920 mOsmollkg). Adjust the pH to between 7.5 and 7.7.
Octopus physiological solution (Necco and Martin, 1963) The concentrations are given as g/l,OOO ml solution. NaCl25.S, KCI 0.644, CaCl2 0.644, MgCI2·6H20 3.0S, KH2P04 0.02, Na2HP04 O.OS, NaHC03
0.2, Na2S04 0.92, sodium pyruvate 1.5, sodium fumarate 1.05, L-glutamine 1.09, and phenol red 0.01, made up to 1,000 ml with distilled water (pH 7.5).
Planarian salt solution (Murray, 1928) The concentrations are given as gll,OOO ml solution. NaCI3.0, CaCI2·6H20 4.0, KCI 0.1, KN03 0.1, MgCl2 1.0 g, MgS04 0.2, NaHC03 0.1, and CaHP04·2Hp 0.1, made up to 1,000 ml with distilled water.
Rinaldini's salt solution (Rinaldini, 1959) The concentrations are given as gll,OOO ml solution. NaCIS.O, KCI 0.20, NaH2P04 0.044, NaHC03 1.0, glucose 1.0, and sodium citrate anhydrous O.SS, made up to 1,000 ml with distilled water.
Ringer's salt solution (Ringer, 1886) The concentrations are given as gil ,000 ml solution. NaCI 7.5, CaCl2 0.125, KCI 0.1, and NaHC03 1.0, made up to 1,000 ml with distilled water.
404 Appendix
SA solution (Furuta and Shim ozawa, 1983) Solution A: NaCI 2,486 mg, KCI 496 mg, CaClz 6 mg, and MgClz 152 mg, made up to 500 ml with distilled water. Solution B: fungizone 5 mg, kanamycin 1,000 mg, and streptomycin 1,000 mg, made up to 40 ml with distilled water. Ready-to-use solution: Solution A 50 ml, solution B 2 ml, 0.1 N NaOH 2 ml, phenol red 0.2 mg, and distilled water 44 ml.
Schistosoma salt solution (Weller and Wheeldon, 1982) The concentrations are given as gll,OOO ml solution. CaClz·2HzO 0040, KCI1.0, MgS04·7HzO 0040, NaC13.0, NaHC03 2.5, and NaHzP04 0.01, made up to 1,000 ml with distilled water.
Snail physiological saline solution (Ivic et aI., 1995) NaCI 100 mM, KCI 4 mM, MgClz 3 mM, CaClz 7 mM.
Tyrode's salt solution (Tyrode, 1910) The concentrations are given as gll,OOO ml solution. NaC18.0, KCI 0.2, CaClz 0.2, NaHC03 1.0, NaHzP04·HzO 0.05, MgClz·6HzO 0.1, glucose 1.0 made up to 1,000 ml with distilled water.
van Harreveld's saline (van Harreveld, 1936) The concentrations are given as gll,OOO ml solution. NaCl 12, CaClz 1.5, KCI 004, MgClz 0.25, and NaHC03 0.2, made up to 1,000 ml with distilled water.
WS washing solution (Rinkevich and Rabinovitz, 1993) MgS04·7HzO 12.06 g (49 mM), CaClz 1.25 g (11.3 mM), NaCl 28.64 g (490 mM), solution of 20 mM HEPES (sodium salt N-2-hydroxyethylpiperazine-N' -2-ethenesulphonic acid) 20 ml, and distilled water to 1,000 ml. The pH of the solution is adjusted to 7.3 with 1 N NaOH. The osmolarity is 1,200 mOsmol/kg. The solution should be sterilized by filtration. The sterilized solution can be stored at 4°C until use.
B Culture Media
Barnacle organ culture medium IV (Fyhn and Costlow, 1975) NaCl466 mM, KCl8 mM, CaClz 20 mM, MgClz 12 mM, NaHC03 10 mM, glucose 0.1 %, bovine embryo extract (Difco Laboratories) 1 %, and TC-Yeastolate (Difco Laboratories) 0.1%.
Basch and DiConza's medium C (Basch and DiConza, 1973) The concentrations are given as mgll,OOO ml medium. NaCl1,300, KCI 97, MgClz·6HzO 325, NazHP04 17, CaClz·2HzO 558, NaHC03 1,769, Llysine 5.8, L-histidine 2.0, L-arginine 204, L-aspartic acid 5.6, L-threonine 6.2, L-serine 12.0, L-glutamic acid 904, L-proline 5.8, glycine 4.8, L-alanine 4.8, L-valine 3.2, L-me-
Composition of Salt Solutions and Culture Media 405
thionine 0.03, L-isoleucine 2.0, L-Ieucine 2.9, L-phenylalanine 1.B, 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, o-calcium pantothenate 1.0, pyridoxine hydrochloride 1.0, thiamine hydrochloride 1.0, riboflavin 0.1, inositol1.B, cyanocobalamin 10.0, nicotinic acid 2.0,paminobenzoic acid 2.0, pyridoxine hydrochloride 2.0, uridine 2.0, deoxyadenosine 10.0, deoxycytidine 10.0, deoxyguanosine 10.0, thymidine 10.0, cytosine 1.0, glucuronolactone 1.B, glutathione 10.0, phenol red 20.0, and glucose 100.0. The pH of the solution should be adjusted to 7.2-7.4. The osmolarity of the solution is 155 mOsmol/kg. The medium is supplemented with 10% heat-inactivated FBS and 10% whole egg ultrafiltrate. Sometimes hemolymph from adult Biomphalaria glabrata is added at a concentration of 5% after inactivation at 60°C for 10 min. An antibiotic mixture (penicillin G 100 IV, streptomycin sulfate 100 !lg, gentamicin sulfate 10 !lg/ml) is added to the medium.
Botryllid cell culture medium (BCCM; Rinkevich and Rabinovitz, 1993) The medium basically consists of Dulbecco's modified Eagle's medium x5 (DMEM) and a salt solution, the osmolarity of which is adjusted to that of botryllid ascidian hemolymph. The salt solution is made up of two major components at a 2x concentration. Component A: NaCI13.67 g, KCI 0.412 g, and CaClz·2HzO 0.721 g, made up to 125 ml with distilled water. Component B: MgS04·7HzO 5.57 g and MgClz·6HzO 3.05 g, made up to 125 ml with distilled water. Both components are mixed (1 : 1), and the solution is sterilized by filtration through a membrane filter with a 0.2 !lm pore size. Both stock solutions can be stored at 4°C. The ready-to-use BCCM should be prepared before use. Each 100 ml BCCM is made by mixing 20 ml DMEM (x5), 50 ml salt solution, 26 ml distilled water, 2 ml L-glutamine solution (200 mM),2 ml HEPES buffer (1 M,pH 7.0) and 1 ml antibiotic mixture (penicillin sodium salt 104 IU, streptomycin 10 mg, and nystatin 1,250 IU). The medium is sterilized by filtration through a membrane filter with a 0.2 !lm pore size, and can be stored at 4°C for up to 3 weeks. Heat-inaysate (5%) 10.0 ml, and yeast extract (10%) 5.0 ml.
Cecil's medium modified by Heacox et al. for Nereis cell culture (Heacox et al., 1983) Distilled water 147 ml, NaCI 6,390 mg, NaHC03 1,000 mg, KCI 340 mg, CaClz·2HzO 169 mg,glucose 1,000 mg,galactose 500 mg,fucose(o) 250 mg,fucose(L) 250 mg,minimal essential medium (MEM) essential amino acids (lOOx) 20.0 ml, MEM non-essential amino acids (100x) IB.O ml, MEM vitamin (100x) 3.3 ml, glutamine 200 mg, gentamicin 1.0 mg, penicillin 300,000 IV, Nereis coelomic fluid ultrafiltrate 0-20 ml, horse serum 0-100 ml, hens' whole egg ultrafiltrate 0-100 ml, and Nereis vitellin 0 or 500 mg.
Cp medium (Miltenburger et aI., 1984) The concentrations are given as mgll,OOO ml medium. KCI 2,870.0, CaCI2·2H20* 1,320.0, MgCI2·6H20* 2,280.0, MgS04·7H20* 2,780.0, NaH2P04·2H20* 1,140.0, NaHC03 350.0, L-arginine hydrochloride 350.0, L-aspartic acid 175.0, L-asparagine 175.0, L-alanine 112.5, ~-alanine 100.0, L-cystine 11.0, L-glutamic acid 300.0, L-glutamine 300.0, glycine 320.0, L-histidine hydrochloride 1,250.0, L-isoleucine 25.0, L-Ieucine 37.5, L-Iysine hydrochloride 312.5, L-methionine 25.0g, L-proline 175.0, L-phenylalanine 75.0, DL-serine 550.0, L-threonine 87.5, L-tryptophan 50.0, L-tyrosine 25.0, L-valine 50.0, p-aminobenzoic acid 0.010, biotin 0.005, D-calcium pantothenate 0.010, choline chloride 0.100, folic acid 0.010, i-inositol 0.010, nicotinic acid 0.010, pyridoxine hydrochloride 0.010, riboflavin 0.010, thiamine hydrochloride 0.010, glucose 1,000.0, and tryptose 2,600.0, made up to 1,000 ml with distilled water. The medium is supplemented with 10% FBS. The pH is adjusted to 6.4 with 1 N KOH.
Crab integument medium I and II (Ballard et aI., 1993) Stock solutions: Modified medium 199 (MI99). Dissolve 10,900 mg Medium 199 (Sigma) in 900 ml deionized-distilled water (DDW).Add 5,960 mg HEPES,200 mg MgCI2·6H20, 15,000 mg NaCI, and 200 mg L-proline and adjust the pH to 7.5 with 1 N NaOH or 1 N HCI. Make the solution up to a final volume of 1,000 ml with DDW. Sterilize with a 0.22-l1m filter and store at 4°C. Antibiotic cocktails (HCA). In 900 ml DDW, dissolve 100 mg Benomyl (Dragon Chemical), 50 mg gentamicin sulfate, 25 mg nystatin, 100 mg penicillin G. When in solution, add 1.65 ml penicillin-streptomycin solution (Sigma), 0.5 ml gentamicin sulfate solution (Sigma), and 0.5 ml fungizone (Gibco).Adjust the pH to 7.5 with 1 N NaOH or 1 N Hel. Make the solution up to a final volume of 1,000 ml with DDW. Sterilize through a 0.22-l1m filter and store in 7.65 ml aliquots at -20°C (thaw before use in final medium). Antibiotic cocktails (LCA). Sterilize the penicillin-streptomycin solution (Sigma), gentamicin sulfate solution (Sigma), and fungizone (Gibco) by passage through a 0.22-l1m filter. Into separate sterile tubes, aliquot 1.65 ml penicillin-streptomycin, 0.5 ml gentamicin sulfate and 0.5 ml fungizone. Store the aliquots at -20°C (thaw before use in final medium). Complete medium I: mix 95 ml M119 and 5 ml FBS. Take 7.65 ml this mixture and add a 7.65 ml HCA aliquot. Sterilize the mixture by passage through a 0.22-l1m filter and store the solution at 4°C (warm to room temperature before use). Complete medium II: mix 95 ml M119 and 5 ml FBS. Take 2.65 ml this mixture and add one aliquot of LCA. Sterilize the solution by passage through a 0.22-l1m filter and store at 4°C (warm to room temperature before use).
Crayfish integument medium (Daig and Spindler, 1979) Mix 2 parts of van Harrevelt saline with 1 part horse serum; osmolarity 349 mOsmoll kg, pH 7.6.
CSM-2F medium (Mitsuhashi, 1967) Unless otherwise indicated, the concentrations are given as mg/1,000 ml medium. NaH2P04·Hp 500, MgCI2·6H20 1,200, MgS04·7H20 1,600, KCl1,200, CaCI2·2H20 400, glucose 800, fructose 800, lactalbumin hydrolysate 5,200, Bacto-peptone (Difco Laboratories) 5,200, choline chloride 400, TC-Yeas to late 2,000, TC-199 medium 200 ml, FBS
Composition of Salt Solutions and Culture Media 407
200 ml, and dihydrostreptomycin sulfate 100, made up to 1,000 ml with distilled water. The pH of the medium is adjusted to 6.2 with KOH.
Durchon and Schaller's medium for Annelida (Durchon and Schaller, 1963) Mix the following: 7 drops of 1 % agar in sterilized natural seawater containing 0.1 % glucose, 3 drops of sterilized seawater containing 0.1% glucose, 1 drop of 50% egg albumin in sterilized seawater containing 0.1 % glucose, 1 drop of horse serum (Difco Laboratories), 1 drop of 50% chicken embryo extract in sterilized seawater containing 0.1 % glucose, 100 IV of penicillin G, and 0.009 mg of streptomycin.
Gottschewski's medium (Gottschewski, 1960) The concentrations are given as mg/l,OOO ml medium. L-Arginine 170, L-cysteine 40, L-cystine 3, glycine 12, L-histidine 196, L-Ieucine 200, Llysine 140, o-methionine 50, L-phenylalanine 200, L-threonine 120, L-tryptophan 20, glucose 1,600, mannose 1,600, fructose 1,600, choline 5, folic acid 0.5, inositol 1.0, nicotinic acid 5.0, pantothenate 1.0, thiamine 0.5, riboflavin 0.5, pyridoxine 1.0, DPN 10, TPN 5, adenine deoxyribonucleoside 0.5, guanine deoxyribonucleoside 0.5, uridine-5-triphosphate O.5,deoxycytidine diphosphate 0.5,deoxy-5-methylcytidilic acid 0.5, thymidine diphosphate 0.5, ATP 5.0, RNA 50, DNA 50, NaCI 5,000, sodium acetate 50, NazHP04·5HzO 10,000, KC1300, CaClz·2HzO 20, MgClz·6HzO 300, MgS04·7HzO 300, glutathione 500, peptone 1,000, glycylglycine 1,000, casein hydrolysate 5,000, and yeast extract 15. The pH of the medium is adjusted to 7.0-7.2 with 1 N NaOH and 1 N HCl.
Grace's medium (GMA; Grace, 1962) The concentrations are given as mg/l,OOO ml medium. NaHzP04·2HzO 1,140, NaHC03 350, KCI2,240, MgClz·6HzO 2,280, MgS04·7HzO 2,780, CaClz l,OOO, L-a-alanine 225, ~-alanine 200, L-arginine hydrochloride 700, L-asparagine 350, L-aspartic acid 350, L-cystine hydrochloride 25, L-glutamic acid 600, L-glutamine 600, glycine 650, L-histidine 2,500, L-isoleucine 50, L-Ieucine 75, L-Iysine hydrochloride 625, L-methionine 50, L-phenylalanine 150, L-proline 350, oL-serine 1,100, L-threonine 175, L-tryptophan 100, L-tyrosine 50, L-valine 100, sucrose 26,680, glucose 700, fructose 400, malic acid 670, a-ketoglutaric acid 370, succinic acid 60, fumaric acid 55, thiamine hydrochloride 0.02, riboflavin 0.02, calcium pantothenate 0.02, pyridoxine hydrochloride 0.02, p-aminobenzoic acid 0.02, folic acid 0.02, niacin 0.02, isoinositol 0.02, biotin 0.01, choline chloride 0.2, penicillin G sodium salt 30, and streptomycin sulfate 100, made up to 1,000 ml with distilled water. The pH is adjusted to 6.5 with KOH. The medium is used by supplementing it with 3% heat-inactivated hemolymph of Lepidoptera or 10% FBS.
408 Appendix
Haliotis discus culture medium (Naganuma et aI., 1996) Leibovitz's L-15 medium (Gibco) with the following components added: NaCI 1.5% (w/v) final concentration (originally 0.85%), NaHC03 2 mM final concentration, antibiotic-antimycotic solution (lOOx; Gibco; amphotericin B 25 mg/ml, ampicillin 5 mgt ml, and streptomycin sulfate 10 mg/ml), lipid mixture (lOOx; Sigma), and FBS (lOx; GIBCO). The final pH of the culture medium should be 7.8.
Hansen's M-182 medium (Hansen, 1976) The concentrations are given as mg/l,OOO ml culture medium. NaCI2,800, KC1150, CaCl2 530, MgS04·7H20 450, Na2HP04 70, NaHC03 50, L-alanine 6.0, L-arginine 4.2, L-asparagine 2.0, L-aspartic acid 6.0, L-cysteine 6.0, L-cystine 2.4, Lglutamic acid 10.0, L-glutamine 20.0, glycine 6.0, L-histidine 4.0, L-isoleucine 5.2, Lleucine 5.2, L-Iysine 5.8, L-methionine 1.5, L-phenylalanine 3.5, L-proline 6.0, L-serine 12.0, L-threonine 4.8, L-tryptophan 0.8, L-tyrosine 3.6, L-valine 4.7, galactose 2,000, glucose 860, trehalose 860, biotin 0.038, calcium folinate 0.038, o-calcium pantothenate 0.075, cyanocobalamin 0.038, folic acid 0.075, N-acetylglucosamine 0.150, niacin 0.075, niacinamide 0.075, pantethine 0.038, p-aminobenzoic acid 0.075, pyridoxal hydrochloride 0.038, pyridoxamine hydrochloride 0.038, pyridoxine hydrochloride 0.075, riboflavin 0.075, thiamine hydrochloride 0.075, thioctic acid 0.038, Bacto-peptone (Difco Laboratories) 4,500, and FBS 130 ml. The pH of the medium should be 7.0-7.2 and the osmolarity 185 mOsmollkg.
Composition of Salt Solutions and Culture Media 409
440, and FBS 130 m!. The pH of the medium should be 7.0-7.2 and the osmolarity 155 mOsmollkg.
Haliotis rufescens culture medium (Naganuma et al., 1994) Leibovitz's L-15 medium (Gibco) with the following components added: NaCI3% (w/ v) final concentration (originally 0.85%), L-glutamine 2 mM final concentration, NaHC03 2 mM final concentration, antibiotic solution (100x; amphotericin B 25 mgt ml, ampicillin 5 mg/ml, penicillin G 10,000 IU/ml, and streptomycin sulfate 10 mg/ml), lipid mixture (1,OOOx; Sigma), yeast extract 200 mg/ml (100x; Sigma), calf serum (10-20x). The final pH of the solution should be 7.6 and final osmolarity 1,080 mOsmollkg.
Heacox et al?s Medium G (Heacox et aI., 1983) Natural seawater (sterile) 742 ml, distilled water 218 ml, NaCI 6,390 mg, NaHC03
Honeybee medium (Kaatz et al., 1985) The concentrations are given as mg/l,OOO ml culture medium. NaCI 1,580, KCI 1,790, CaClz·2HzO 1,029, MgClz·6HzO 406, MgS0 4·7HzO 493, NaHzP04·HzO 690, NaHC03 336, L-a-alanine 300, L-arginine 500, L-asparagine 200, Laspartic acid 100, L-cysteine 50, L-glutamic acid 250, L-glutamine 1,000, glycine 200, Lhistidine 200, L-isoleucine 100, L-Iysine 550, L-methionine 50, L-phenylalanine 100, Lproline 3,300, DL-serine 200, L-threonine 150, L-tryptophan 50, L-tyrosine 100, L-valine 150, glucose 4,000, fructose 2,500, sucrose 55,370, PIPES 7,560, bovine serum albumin 50,000, penicillin G ISO, streptomycin 250. The pH is adjusted to 6.70 with 1 N KOH and 1 N HC!.
Hydra growth medium (Yu-Ying et aI., 1963) Eagle's medium modified by Swin and Parker (1958) 10 m1,horse serum 100 ml,penicillin 500 IU, Fumidil B 200 mg, NaCI 5.8 mg, MgS04·7HzO 100 mg, NaHC03 168 mg, KCI 7.5 mg, CaClz III mg, dextrose 1,000 mg, phenol red 10 mg, streptomycin 12.5 mg, triple-distilled water 800 ml, and 0.15 M phosphate buffer, to maintain the pH of the medium at 6.8, made up to 1,000 ml with distilled water.
IPL-41 medium (Weiss et aI., 1981) The concentrations are given as mg/l,OOO ml culture medium. NaHzP04·HzO 1,160, NaHC03 350, KCl1,200, CaClz 500, MgS04·7HzO 1,880, B-alanine 300, L-arginine hydrochloride 800, L-asparagine 1,300, L-aspartic acid 1,300, L-cystine 100, L-glutamic acid 1,500, L-glutamine 1,000, glycine 200, L-histidine 200, hydroXY-Lproline 800, L-isoleucine 750, L-leucine 250, L-Iysine hydrochloride 700, L-methionine 1,000, L-proline 500, L-phenylalanine 1,000, oL-serine 400, L-threonine 200, L-tryptophan 100, L-tyrosine 250, L-valine 500, sucrose 16,500, glucose 2,500, maltose 1,000, malic acid 53.6, a-ketoglutaric acid 29.6, succinic acid 4.8, fumaric acid 4.4, thiamine hydrochloride 0.08, riboflavin 0.08, calcium pantothenate 0.008, pyridoxine hydrochloride O.4,p-aminobenzoic acid 0.32, folic acid 0.08, niacin 0.16, inositol 0.4, biotin 0.16, cyanocobalamin 0.24, choline chloride 20, ZnClz 0.04, MnCl2·4H20 0.02, CuCl2·2HzO 0.2, (NH4)Mo70Z4·4H20 0.04, CoClz·6HzO 0.05, and FeS04·7H20 0.55. The osmolarity of the medium should be 360-375 mOsmollkg. FBS 10% may be added to the medium.
410 Appendix
K-6 medium (Horikawa, 1959) The concentrations are given as mg/l,OOO ml culture medium. NaCl 7,000, KC1200, CaCI2·2H20 20, MgCI2·6H20 100, NaHC03 50, NaH2P04·2H20 200, glucose 800, casein hydrolysate 50,000, tryptophan 5,000, cystine 1,000, cysteine 260, oL-kynurenine 4 g, oL·3-hydroxykynurenine 2 g, ribonucleic acid 500, thiamine 0.05, riboflavin 0.05, pyridoxine hydrochloride 0.125, niacinamide 0.125, calcium pantothenate 0.05, ascorbic acid 250, biotin 0.05, folic acid 0.05, choline chloride 2.5, inosite 0.25,p-aminobenzoic acid 0.25, vitamin A 0.5, vitamin B12 0.5, sodium acetate SO, glutathione 10, glutamic acid 100, diphosphopyridine nucleotide (DPN) 1.0. The pH of the medium should be adjusted to 7.2 with 1 N KOH.
Kuroda and Tamura's medium for Drosophila melanotic tumors (Kuroda and Tamura, 1955) The concentrations are given as mg/1,OOO ml culture medium. NaCI 7,000, KC1200, CaCI2·2H20 20, MgCI2·6H20100, NaHC03 SO, NaH2P04·2Hp 200, glucose 800, casein hydrolysate 50,000, tryptophan 1,000, cystine 1,000, cysteine 260, ribonucleic acid 500, thiamine 0.05, riboflavin 0,05, pyridoxine hydrochloride 0.125, niacinamide 0.125, calcium pantothenate 0.05, ascorbic acid 250, biotin 0.05, folic acid 0.05, choline chloride 2.5, inosite 0.25, p-aminobenzoic acid 0.25, cyanocobalamin 0.5, vitamin A 0.5, sodium acetate SO, glutathione 10, glutamic acid 100, and DPN 1.0.
L-15B (Munderloh and Kurtti, 1989) Unless otherwise indicated, the concentrations are given as mg/1 ,000 ml culture medium. Leibovitz's L-15 medium powder (Gibco) 1 package 03.88g), L-aspartic acid 299, Lglutamic acid 500, L-glutamine 292, L-proline 300, a-ketoglutaric acid 299, o-glucose 2,239, mineral stock* 1 ml, vitamin stock** 1 ml. The pH of the medium is adjusted to 6.4-6.6 with 1 N NaOH. *Mineral stock (x103) contains stock solutions A, B, and C. The components of stock solution A (xl0S) are: CoCI·6H20 20 mg, CuS04·5H20 20 mg, MnS04·H20 160 mg, ZnS04·7H20 200 mg, and distilled water 100 ml. The components of stock solution B (xlOS) are NaMo04·2H20 20 mg and distilled water 100 ml. The components of stock solution C (xlOS) are Na2Se03 20 mg and distilled water 100 ml. The complete mineral stock (xl03) are: reduced glutathione 1,000 mg, ascorbic acid 1,000 mg, FeS04·7H20 50 mg, stock solution A 1 ml, stock solution B 1 ml, stock solution C 1 ml, and distilled water to 100 ml. **Vitamin stock (xl03): p-aminobenzoic acid 100 mg, cyanocobalamin 50 mg, o-biotin 10 mg, and distilled water 100 ml.
Composition of Salt Solutions and Culture Media 411
Laverdure's medium (Laverdure, 1971) Laverdure's medium is made by mixing 1 part of horse serum (Difco Laboratories; dissolve the content of one ampoule in 5 ml bidistilled water), 1 part chicken embryo (11 days old) extract (Difco Laboratories; dissolve the contents of one ampoule in 8 ml Laverdure's physiological solution), 1 part Laverdure's physiological solution containing 2% peptone (Merck, Darmstadt, Germany), and 2 parts Laverdure's physiological solution containing 1 % agar. Penicillin (20,000 IU) and streptomycin (0.5 mg) are added to 20 ml medium. The pH of the medium is adjusted to 6.84-7.1 with 5.5% NaHC03•
Leloup's medium 1 (Leloup, 1964) The concentrations are given as mg/l,OOO ml culture medium. NaCI6,500, KC1250, MgS04·7H20 350, CaCl2 300, trehalose 800, glucose 700, malic acid 500, a-ketoglutaric acid 250, succinic acid 50, fumaric acid 50, yeast extract 1,000, and lactalbumin hydrolysate 10,000. The pH of the medium is adjusted to 7.0-7.1 with 1 N NaOH.
Limulus organ culture medium (Bayer and Barlow, 1978) The concentrations are given as mg/l,OOO ml culture medium. NaCI 16,100, KCI 276, CaCI2·2H20 1,184, MgCI2·6H20 1,423, MgS04·7H20 5,669, medium 199 with Hanks salts (lOx; Gibco) 100 ml, horse serum 100 ml (Gibco), glucose 2,703, L-glutamine 292, retinol* (2 gil, Sigma) 10 ml, tocopherol* (200 mg/l, Sigma) 10 ml,penicillin-streptomycin (10,000 U/ml) 20 ml,HEPES (50 mM,Sigma) 10 ml,and TES (N-tris (Hydroxymethyl) methyl-2-aminoethane-sulfonic acid; 2-( (2Hydroxy-l, I-bis (hydroxymethyl)-ethyl) amino) ethansulfonic acid; 50 mM, Sigma) 10 ml, made up to 1,000 ml with distilled water. The pH should be adjusted to 7.3. * Add as an alcoholic solution.
Medium A-12 (Tripp et aI., 1966) Mix 5% casamino acid 1.0 ml, 10% yeast extract 10 ml, 5% lactalbumin hydrolysate 10 ml, calf serum 10 mI, and BSSO (see Salt Solutions) 969 ml. The pH of the medium should be 7.2-7.4.
*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 modified by Heacox et al. (Heacox et al., 1983) The concentrations are given as mg/l,OOO ml culture medium. NaCI17,550, CaCI2·2H20 930, KC1510, MgS04· 7H20 10,240, NaHC03 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, MgCI2·6H20 325, Na2HP04 17, CaCI2·2H20 558, NaHC03 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-
Composition of Salt Solutions and Culture Media 413
chloride 1.0, riboflavin 0.1, inositol 1.8, vitamin B12 10.0, nicotinic acid 2.0, p-aminobenzoic acid 2.0, pyridoxine hydrochloride 2.0, uridine 2.0, deoxyadenosine 10.0, deoxycytidine 10.0, deoxyguanosine 10, thymidine 10.0, cytosine 1.0, glucuronolactone 1.8, glutathione 10.0, phenol red 20.0, and glucose 100.0, made up to 1,000 ml with distilled water. The pH of the medium should be adjusted to 7.2-7.4 with 1 N KOH and 1 N HCl.
Medium X for Drosophila imaginal discs (Davis and Shearn, 1977) The concentrations are given as mg/1,000 ml culture medium. MgS04·7H20 1,230, MgCI2·6H20 3,192, KH2P04 200, K2HP04 256, KC1446, KHC03 300, potassium citrate 312, potassium fumarate 800, potassium a-ketoglutarate 870, potassium acetate 750, sodium citrate 3,122, sodium 1,4-piperazine diethanesulfonate (PIPES) 3,604, calcium succinate monohydrate 518, lactalbumin hydrolysate 9,000, L-proline 1,000, L-tryptophan 100, ~-alanine 50, L-cysteine 100, L-serine 250, y-aminobutyric acid 70, Bacto-peptone 5,000, trehalose 4,000, glucose 4,400, L-a-phosphatidylcholine, dimyristoyl1.0, L-a-phosphatidylcholine, distearoyl1.0, cholesterol 2.6, TC-Yeastolate 2,000, vitamin A (trans-retinol) 0.1, vitamin E (o-a-tocopherol) 0.1, sodium a-glycerophosphate 1,620, calcium phosphorylcholine 1,290, phosphorylethanolamine 70, BSA fraction V 10,000, polyvinylpyrrolidone (MW = 10,000) 250, polyvinylpyrrolidone (MW = 40,000) 1,000, and polyvinylpyrrolidone (MW = 360,000) 9,000. The pH of the medium should be adjusted to 6.8 with 1 N KOH and 1 N HCl. This medium can be used following supplementation with Altosid (a juvenile hormone analog; Zoecon) at a concentration of 31.0 ng/lOO ml medium and bovine insulin at 0.04 units/ 1 00 ml medium. Furthermore, 100 ml of this medium is conditioned either by preincubation with the fat bodies of five larvae or by co culturing of the discs with fat bodies.
MGM-434, MGM-443, MGM-448, MGM-450 and MGM-464 MGM-434 (chemically defined medium) The concentrations are given as mg/1,OOO ml medium. NaH2P04·2H20 958, NaHC03 292,KCl1,875,CaCI2 833, MgCI2·6H20 1,917, MgS04·7H20 2,333, glucose 3,333, fructose 417, sucrose 22,080, malic acid, 558, a-ketoglutaric acid 308, succinic acid 50, fumaric acid 46, L-a-alanine 263, ~-alanine 167, L-arginine hydrochloride 583, L-asparagine 293, L-aspartic acid 293, L-cystine 2l, L-glutamic acid 500, L-glutamine 500, glycine 542, L-histidine 2,083, L-isoleucine 42, L-Ieucine 63, Llysine hydrochloride 521, L-methionine 42, L-phenylalanine 125, L-proline 292, oL-serine 917, L-threonine 146, L-tryptophan 83, L-tyrosine 42, L-valine 83, thiamine hydrochloride 0.16, riboflavin 0.16, pyridoxine hydrochloride 0.16, niacin 0.16, calcium pantothenate 0.16, biotin 0.08, folic acid 0.16, isoinositol 0.16,p-aminobenzoic acid 0.16, choline chloride 1.6, polyvinylpyrrolidone K-90 500. The pH of the medium should be adjusted to 6.5 with concentrated KOH. MGM-443 (Mitsuhashi, 1980) This is MGM-434 medium modified by the addition of 10% FBS. The pH of the medium is adjusted to 6.3 with concentrated KOH solution. MGM-448 (Mitsuhashi, 1984) This is MGM-443 medium fortified by the addition of inosine 200 mg/l, cytochrome c 100 mg/I, fetuin 20 mg/l, and bovine plasma albumin fraction V 10,000 mg/l, FBS 100 mll
414 Appendix
1, or Antheraea pernyi hemolymph 30 mlll. The pH of the medium is adjusted to 6.3 with concentrated KOH solution. MGM-450 (Mitsuhashi and Inoue, 1988) This medium is prepared by adding 3,000 mg tryptose phosphate broth to 1,000 ml of MGM-448 medium. The pH is adjusted to 6.3 with concentrated KOH solution. This medium can also be used by adding FBS at 10%, or FBS 5% and Antheraea pernyi hemolymph 3%. MGM-464 (Mitsuhashi, 2001) This medium is prepared by adding 3,000 mg tryptose phosphate broth, 3,000 mg TCYeastolate (Difco), and 3,000 mg lactalbumin hydrolysate to 1,000 ml of MGM-448 medium. The pH of the medium is adjusted to 6.3 with concentrated KOH solution. This medium is used by adding FBS at 20%.
MM medium (Mitsuhashi and Maramorosch, 1964) The concentrations are given as mg/l,OOO ml culture medium. NaCl 7,000, KCl 200, CaC12·2H20 200, MgC12·6H20 100, NaH2P04 200, NaHC03 120, glucose 4,000, lactalbumin hydrolysate 6,500, TC-Yeastolate 5,000, and FBS 0-200 ml, made up to 1,000 ml with distilled water. The pH is adjusted to 6.3 with KOH (the pH of the medium is already around 6.3 without any adjustment). The medium can be sterilized before the addition of FBS by autoclaving at 121°C for 15 min. The medium can be used as a serum-free medium (= MM-SF medium).
Nudibranch culture medium (Tamse et aI., 1995) This is Leibovitz's L-15 medium supplemented with 444 mM NaCl , 10.0 mM KCl, 28.0 mM MgC12·6H20, 27.0 mM MgS04·7H20, 2.0 mM NaHC03, 35.0 mM D-glucose, 15.0 mM HEPES, 11.0 mM CaC12·2H20, penicillin 50 lUI ml, and streptomycin 50 Ilg1 ml. The pH of the medium should be 7.4-7.5.
Ogura's medium (Ogura, 1989) The concentrations are given as mg/l,OOO ml culture medium. NaCl 7,900, KCl 290, CaClz·2H20 200, MgS04·7H20 150, Na2HP04·12Hp 380, KH2P04
80, NaHC03 1,200, glucose 1,500, lactalbumin hydrolysate 500, TC-Yeastolate 250, chicken embryo extract 2.5 ml, nematode hemolymph * 5 ml, penicillin 105 IV, and streptomycin 100, made up to 1,000 ml with distilled water. *Nematode hemolymph is prepared from the female adults of Toxocara canis.
Oncomelania culture medium (Iwanaga et aI., 1985) Vnless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium. NaC12,800, KCl150, Na2 HP04 70, MgS04·7H20 450, CaC12·2H20 530, NaHC03 50, galactose 5,000, glucose 5,000, trehalose 1,000, a-ketoglutaric acid 1,200, fumaric acid 800, malic acid 800, succinic acid 800, L-lysine hydrochloride 208, L-histidine 100, Larginine hydrochloride 144, L-aspartic acid 532, L-threonine 288, L-serine 360, L-glutamic acid 516, L-proline 248, glycine 388, L-alanine 352, L-valine 104, L-methionine 88, Lisoleucine 116, L-leucine 292, L-tyrosine 128, L-phenylalanine 156, Eagle's MBE vitamin (xl00; Gibco) 40 ml, and phenol red 5, made up to 1,000 ml with distilled water. Vse the medium by adding heat-treated horse serum at a concentration of 10%. The pH of the medium should be 7.0-7.2.
Composition of Salt Solutions and Culture Media 415
Planarian cell culture medium (Betchaku, 1967) Unless otherwise indicated, the concentrations are given as mgll,OOO ml culture medium. L-Arginine hydrochloride 5.6, L-cystine 2.0, L-histidine 1.1, L-isoleucine 8.7, L-Ieucine 4.7, L-Iysine 6.1, L-methionine 2.7, L-phenylalanine 2.8, L-threonine 4.0, L-tryptophan 0.7, L-tyrosine 6.0, L-valine 3.9, L-glutamine 48.9, 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, sodium pyruvate 150.0, glucose 300.0, calf serum 3 ml, NaCll,750.0, KCI25.0, CaCl2 50.0, NaHC03 100.0, neomycin 100.0. The pH of the medium should be adjusted to 7.2-7.4 with 1 N KOH and 1 N HCl.
Rasmont medium M (Rasmont, 1961) The concentrations are given as milliequivalent II. Ca++ 2.00, Mg++ 1.00, Na+ 1.00, K+ 0.05, Cl~ 2.05, S04 1.00, Si03~~ 0.50, HC03~ 0.50.
RML-375 medium (Moulton, 1978) AI: 1 : 2 mixture of Eagle's MEM, Hank's BSS and Leibovitz's L-15 medium is supplemented with 20% FBS (heat-inactivated for 1 h at 56°C), 0.1% bovine albumin, 10% tryptose phosphate broth stock solution, made by dissolving 109 tryptose phosphate broth in 100 ml distilled water, and 50 glml gentamicin.
cium pantothenate 0.24, made up to 1,000 ml with distilled water. The pH of the medium should be adjusted to 6.7 with 1 N KOH and 1 N HCl.
Sengel's medium (Sengel, 1960) Salt solution (Holtfreter's solution, 1931): NaC13,500 mg, KCI SO mg, CaCl22H20 100 mg, and NaHC03 200 mg, made up to 1,000 ml with distilled water. Culture medium: 5 parts of 1 % agar in the above salt solution, 4 parts of 0.1 % glucose in the above salt solution, and 3 parts chicken embryo (9 days old) extract diluted to 50% with the above salt solution.
SH-7 medium (Furuta and Shimozawa, 1983) Unless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium. NaCI 2,483.0, KCI 496.0, CaCl2 6.0, MgCl2 152.0, NaHC03 1,769.0, glucose 50.0, trehalose 50.0, L-a-alanine 41.2, L-arginine hydrochloride 6.1, L-asparagine monohydrate 14.5, L-aspartic acid 3.1, L-cystine 3.3, L-glutamic acid 3.7, L-glutamine 3.7, glycine 3.0, L-histidine dihydrochloride 4.1, L-isoleucine 3.3, L-Ieucine 2.3, L-Iysine hydrochloride 5.8, L-phenylalanine 5.7, L-serine 11.8, L-threonine 27.2, L-tyrosine 2.8, L-valine 4.9, thymidine 0.35, uridine 22.4, hypoxanthine 2.0, tricine 2,750.0, putrescine 0.15, phenol red 2.0, D-biotin 0.007, choline chloride 7.0, calcium pantothenate 0.25, folic acid 0.5, inositol 9 .0, pyridoxal hydrochloride 0.03, riboflavin 0.02, thiamine hydrochloride 0.15, cyanocobalamin 0.5, nicotinamide 0.02, linoleic acid 0.04, a-lipoic acid 0.1, FBS (dialyzed and inactivated) 100.0 ml, and chicken embryo extract 40.0 ml, made up to 1,000 ml with distilled water.
Shields and Sang's Medium (Shields and Sang, 1970) The concentrations are given as mg/l,OOO ml culture medium. MgS04·7H20 5,130, CaCI2·6H20 1,740, KCI3,130, NaC1860, NaH2P04·2H20 880, KHC03
180, monosodium malate·2H20 950, monosodium a-ketoglutarate 420, disodium fumarate 80, disodium succinate·6H20 140, monosodium L-glutamate 2,460, L-aspartic acid ISO, L-threonine 500, L-serine 350, L-asparagine 300, L-glutamine 600, L-proline 400, glycine 500, L-a-alanine 1,650, L-valine 420, L-methionine 120, L-isoleucine 270, Lleucine 400, L-tyrosine 260, L-phenylalanine 240, ~-alanine 100, L-histidine 550, L-tryptophan 100, L-arginine 500, L-Iysine 680, L-cystine 200, L-cysteine 800, glutathione 5, glucose 4,600, and TC-Yeastolate (Difco Laboratories) 2,000. The pH of the medium is adjusted to 6.9 with 1 % NaOH. Use the medium by adding 10% FBS.
Shields and Sang's medium modified by Poels (1972) The concentrations are given as mg/l,OOO ml culture medium. NaCI 860, NaH2P04·2H20 880, KCI 3,130, KHC03 180, CaCl2·2H20 1,160, MgS04·7H20 5,130, monosodium malate·2H20 950, monosodium a-ketoglutarate 420, disodium succinate·6H20 140, disodium fumarate 80, monosodium pyruvate 20, monosodium Lglutamate 760, L-aspartic acid 90, L-threonine 150, L-serine 175, L-asparagine 150, Lglutamine 300, L-proline 400, glycine 175, L-a-alanine 750, L-valine 90, L-methionine 150, L-isoleucine SO, L-Ieucine 90, L-tyrosine 30, L-phenylalanine 30, ~-alanine 120, Lhistidine 190, L-tryptophan 100, L-arginine 210, L-Iysine 70, L-cystine 10, L-cysteine 60, TC-Yeastolate 2,000, glucose 300, trehalose 800, and FBS 100 ml. The pH of the medium is adjusted to 6.9 with 1 N NaOH.
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, NaHzP04·2HzO 8S0, KCl 3,130, KHC03 ISO, CaClz·2HzO 1,160, MgS04·7HzO 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, CaClz 53.6, MgS04·7HzO 90.1, MnClz·4HzO 0.3, NaHC03 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-
418 Appendix
nine 12 mg, DL-valine 14 mg, glycine 2.5 mg, L-tyrosine 1 mg, L-hydroxyproline 2.5 mg, DL-serine 12.5 mg, taurine 5 mg, ornithine dihydrochloride 5 mg, and L-asparagine 5 mg. Adjust the pH to 7.5 with 1 N KOH and 1 N HC!. To prepare medium C, dissolve 0.25 mg glutathione into solution C.
Wyatt's medium (Wyatt, 1956) The concentrations are given as mg/l,OOO ml culture medium. NaH2P04 1,100, KCl 2,980, MgCl2·6H20 3,040, MgS04·7H20 3,700, CaCl2 810, glucose 700, fructose 400, sucrose 400, malic acid 670, a-ketoglutaric acid 370, succinic acid 60, fumaric acid 55, DL-a-alanine 450, ~-alanine 200, L-arginine 700, L-asparagine 350, Laspartic acid 350, cysteine 80, L-cystine 25, L-glutamic acid 600, L-glutamine 600, glycine 650, L-histidine 2,500, DL-isoleucine 100, DL-leucine 150, DL-lysine 1,250, DL-methionine 100, L-phenylalanine 150, L-proline 350, DL-serine 1,100, DL-threonine 350, Ltryptophan 100, L-tyrosine 50, and DL-valine 200. The pH of the medium is adjusted to 6.35 with 1 N KOH and 1 N HC!. Use the medium by adding heat-treated Bombyx mori hemolymlph.
ZH 1% medium (Wyss, 1977) Unless otherwise indicated, the concentrations are given as mg/l,OOO ml culture medium. CaCl2·2H20 890, KCl 2,000, MgCl2·6H20 1,000, MgS04·7H20 3,700, NaCl 2,050, NaH2P04·2H20 470, CuS04·5HzO 0.01, FeS04·7Hp 0.6, MnS04·7H20 0.3, ZnS04·7H20 0.6, L-arginine 170, L-asparagine l30, L-cysteine hydrochloride 310, L-glutamine 700, Lglutamic acid 290, glycine 750, L-histidine 150, L-isoleucine l30, L-leucine l30, L-lysine 180, L-methionine 150, L-phenylalanine 160, L-proline llO, L-serine 520, L-threonine 120, L-tryptophan 100, L-tyrosine 90, L-valine 120, glucose 2,000, malic acid 670, oxaloacetic acid 250, sodium acetate·3H20 25, succinic acid 60, biotin 0.01, calcium pantothenate 0.02, choline chloride 0.2, folic acid 0.02, m-inositol 0.02, niacin 0.02, niacinamide 0.02, pyridoxine hydrochloride 0.02, pyridoxal hydrochloride 0.02, riboflavin 0.02, thiamine hydrochloride 0.02,p-aminobenzoic acid 0.02, cyanocobalamin 0.1, phenol red 10, high molecular weight fraction of yeast extract* 1,500 (equivalent to original yeast extract), and heat-inactivated horse serum 10 m!. The pH is adjusted to 6.6 with NaOH. *The fractionation of yeast extract was made using Sephadex G25 (Sigma) chromatography, and fractions 23-46 are used (Wyss, 1977).
Composition of Salt Solutions and Culture Media 419
2,000, MgS04·7H20 1,230, NaCI3,200, NaH2P04·H20 420, CuS04 0.001, FeS04·7H20 1.0, MnS04·7H20 1.0, ZnS04·7H20 1.0, biotin 0.05, calcium pantothenate 5.0, choline chloride 50.0, folic acid 0.05, m-inositoI50.0, niacinamide 0.5, pyridoxal hydrochloride 5.0, riboflavin 0.01, thiamine hydrochloride 5.0, cyanocobalamin 0.05, cholesterol 0.01, linoleic acid 0.01, and phenol red 10.0, made up to 1,000 ml with distilled water. The pH of the medium is adjusted to 6.6 with 1 N NaOH. The final osmolarity of the medium is 250 mOsmollkg.
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Appendix 2
List of Reported Continuous Invertebrate Cell Lines
How many invertebrate cell lines exist is uncertain, because there are many cell lines that have not been reported properly, and some of the reported cell lines have been lost already. There are also some cell lines that are known to be mislabeled or cross-con-taminated. The following list contains only properly reported cell lines; cell lines that no longer exist or those that have been ascertained as mislabeled are marked with asterisks.
Blattella germanica Embryo Whole UM-BGE-1, -2, Kurtti and Brooks, -4, and-5 1977
B. germanica Embryo Whole Landureau, 1966 Blattidae Gromphadorhina Larva Dorsal Quiot et ai., 1985
laevigata vessel
Periplaneta Embryo Whole EPa Landureau, 1968 americana
p. americana Larva Hemocyte HPa-33 Landureau and -34 and Grellet, 1975
P. americana Adult Fat body APa Philippe, 1982 Coleoptera Cerambycidae Xylotrechus Larva Fat body XP-1 Iwabuchi, 1999 pyrrhoderus Chrysomelidae Diabrotica Embryo Whole IPLB-DU182A, Lynn and
undecimpunctata IPLB-DU182E Stoppleworth, 1984
Leptinotarsa Embryo Whole ZIZ-LD-1 Dubendorfer and decemlineata Liebig, 1992
L. decemlineata Embryo Whole IPLB-CPB2 Lynn, 1995 Culcurionidae Anthonomus grandis Embryo Whole AGE,AGF Barcenas et ai., 1989 A. grandis Embryo Whole BRL-AG-1,-2, Stiles et ai., 1992
-3A, -3C, and -4
422 Appendix
Species Stage Tissue Designation Report
Scarabaeidae
Antitragus parvulus Embryo Whole APE-1 Fernon et aI., 1996
A. parvulus Larva Hemocyte APHL-1 Fernon et aI., 1996
Hydrozoa Millepora dichotoma Colony Fragment NIO-MD-l Frank et aI., 1994
Anthozoa
Clathrarcia Colony Fragment NIO-CR-l Frank et aI., 1994 rubinoides
Plexaura A Colony Fragment NIO-PC Frank et aI., 1994
Stylophora pistillata Colony Fragment NIO-SPP-l Frank et aI., 1994 planula larva
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Appendix 3
Commercially Available Culture Medium Products and List of Suppliers
Agfa: Geneva, Switzerland American Ins.: Silver Spring, MD, USA American National Can Co.: Menasha, WI, USA American Type Culture Collection: Rockville, MD, USA Amicon Corp.: Danvers, MA, USA Aquarium Systems: Eastlake, OH, USA Arthur H. Thomas: Philadelphia, PA, USA Becton-Dickinson Labware: Lincoln Park, N], USA Bel-Art: Pequannock, N], USA Belleo Glass: Vineland, N], USA Biochrom: Berlin, Germany Bio-Rad: Richmond, CA, USA
436 Appendix
Boehringer-Mannheim Biochemicals: Indianapolis, IN, USA Brickman Instruments: Logan, UT, USA BTX, San Diego, CA, USA Calbiochem: La Jolla, CA, USA Clorox: Oakland, CA, USA Collaborative Research: Lexington, MA, USA Corning Medical, Corning Glass Works: Medfield, MA, USA Difco Laboratories: Detroit, MI, USA Dragon Chemical: Roanoke, VA, USA Drummond Sci, Co.: Broomall, PA, USA Falcon: Oxnard, CA, USA Flow Laboratories: Irvine, Scotland, UK FMC Bioproducts: Cambridge, MA, USA Fuji Photo Film: Tokyo, Japan Geisler Pet Products: New York, NY, USA Gentra Systems: Minneapolis, MN, USA Gibco-BRL: Grand Island, NY, USA Godo Shusei: Tokyo, Japan Greiner: Nuertingen, Germany Hampton Research: Laguna Niguel, CA, USA Hawaiian Marine Imports: Houston, TX, USA HEM Research: Rockville, MD. USA Humco-Sheffield: Norwich, NY, USA Ikemoto Rika: Tokyo, Japan Instant Ocean Aquarium System: Menton, OH, USA Jamarin Laboratory: Osaka, Japan KC Biological, Inc.: Lenexa, KS, USA Kontes Glass Co.: Vine land, NJ, USA Lab-Crest Scientific, Warminster, PA, USA LKB Instuments: Rockville, MD, USA Marco Specialty Steel, Inc.: Houston, TX, USA Marinus: Long Beach, CA, USA Merck: Darmstadt, Germany Microbiological Associates: Bethesda, MD, USA Millipore: Bedford, MA, USA Mitsubishi, Tokyo, Japan Nalgene (Nalge): Rochester, NY, USA New Brunswick Scientific: Edison, NJ, USA Nihon Nosan Kogyo, Yokohama, Japan Nikon, Tokyo, Japan Nipponseiyaku: Tokyo, Japan Nisseiken: Tokyo, Japan Nunc: Roskilde, Denmark Nutritional Biochemical: Cleveland, OH, USA Osi: Maurepas, France Oxoid: London, UK Pentex-Miles, Kankakee, IL. USA
Commercially Available Culture Medium Products and List of Suppliers 437
Perkin Elmer: Norwalk, CT, USA Pharmacia Fine Chemicals: Uppsala, Sweden Polaroid Corporation: Cambridge, MA,USA Razel Scientific Instruments, Inc., Stamford, CT, USA Riken Cell Bank: Ibaraki, Japan Robinson Laboratory: San Francisco, CA, USA Roche Chemicals: Nutley, NJ, USA Rohm and Haas: Philadelphia, PA, USA Roth: Karlsruhe, Germany Sartorius: G6ttingen, Germany Shin Etsu Chemical: Tokyo, Japan Sigma Chemical: St. Louis, MO, USA Societe Langlois: Niot, France Soden Chemicals: Toronto, Ontario, Canada Sovall: Manchester, UK Takara: Kyoto, Japan Tetko Inc.: Elmsford, NY, USA Toa Gosei Co. Ltd.: Tokyo, Japan VWR Scientific (Division ofUnivar): Columbus, OH, USA Verax Corporation: Lebanon, NH, USA Vestal Laboratories: St. Louis, MO, USA West Agro, Inc.: Kansascity, MO, USA Winthrop Laboratories: Surbiton, England; New York, NY, USA Woods End, Inc.: Weston, CT, USA Zoecon: Palo Alto, CA, USA
Balanus amphitrite 139, 271, 275 barnacle organ culture medium IV 404 Basch and DiConza's medium C 404 bees 101 benzalkonium chloride 15 Biomphalaria glabrata 163 Blaberus craniifer 262
gorgonian tissue (Hydra) 301 Gottschewski's medium 407 Grace 20 Grace's medium 27, 407 graduated pipettes 11 granular hemocytes 60 grid micrometer 349 growth factors 20 gut (Lepidoptera) 51
H
Haliotis discus 183 Haliotis discus culture medium 408 Haliotis rufescens 183 Haliotis rufescens culture medium 409 Haliotis tuberculata 179 Hansen's M-182 medium Hansen's M-260 medium Hansen's S-301 medium Heacox et al.'s Medium G heart
- Lepidoptera 57
408 408
408 409
- Mollusca (oysters) 175 - Mollusca (snails) 173
Heliothis virescens 52, 54 Helix aspersa 191 Helix pomatia 186 hemagglutinating virus of Japan (HVJ)