In this section:
Modified Barth's Saline (MBS) Anitbiotics Gentamycin Oxytetracycline Penicillin Streptomycin Sulfate High-salt MBS Amphibian Ringers Modified Frog Ringers (MR) Marc's Modified Ringer's (MMR) Normal Amphibian Medium (NAM) Holtfreter's SolutionModified Barth's Saline (MBS)
Prepare two solutions: 0.1M CaCl2 and 10X MPS salts (Nacl/KCl/MgSO4/HEPES/NaHCO3)
0.1M Cacl2
11.1 g/liter autoclave and store aliquots at -20 deg C or 4 deg C10X MBS salts
880 mM NaCl 10 mM KCl 50 mM HEPES (pH 7.8) 25 mM NaHCO3Adjust final pH to 7.8 with NaOH; autoclave.
Prepare the final MBS solution by missing 100 ml of 10X salt solution with 7 ml of 0.1M CaCl2; adjust the volume up to 1 liter with distilled water. The following are the final concentrations:
88 mM NaCl 1 mM CaCl2 1 mP MgSO4 5 mM HEPES (pH 7.8) 2.5 mM NaHCO3Note: Omit HEPES if MBS is to be used for oocyte maturation.
Anitbiotics
Antibiotics can be added to MBS. The following are the most commonly used antibiotics for embryos and explants.]
Gentamycin (50 ug/ml) Make a 1000x stroke solution, 50 mg/ml, in water, filter sterilize and refrigerate Oxytetracycline (12.5 ug/ml) Make a 1000x stock solution, 12.5 mg/ml, in water, Store frozen. Penicillin (commonly used for oocyte culture at 100 units/ml) Make a 1000x stock stock solution, 100,000 units/ml, in water, Store frozen. Streptomycin Sulfate (Life Technologies) Make a 1000x stock solution, 100 mg/ml, in water. Store frozen.High-salt MBS
0.1 M Cacl2 - 7 ml 10x MBS salts - 100 ml 5 M NaCl - 4 ml water - 888 mlAmphibian Ringers
Amphibian ringers, 5 gal (19 L)
NaCl 125.4 g KCL 2.85g CaCL2 2.85 g NaHCO3 3.8g DI water to 19 LModified Frog Ringers (MR)
0.1 M NaCl 1.8 mM KCl 2.0 mM Cacl2 1.0 MgCl2 5.0 mM HEPES-NaOH (pH 7.6) or 300 mg/liter NaHCO3Modified Ringers
1X Mol. Wt. 10X for 1L 10X* For 10L 10X For 20L 10X NaCl 0.1M 58.44 1 M 58.44 g 584.4 g 1168.8 g KCL 1.8 mM 74.56 18 mM 1.34 g 13.4 g 26.8 g MgCL2 1.0 mM 203.31 10 mM 2.03 g 20.3 g 40.6 g Hepes 5.0 mM 238.31 50 mM 11.92 g 119.2 g 238.4 g- Add 100 ml of 10X to 1 L DI water for 1 L 1X solution
100 ml 1x MR
2.5 ml 4 M NaCl 1 ml 100x MR salts 0.5 ml 1 M Hepes pH 7.6100X MR salts
For 1L 100X:
KCl 13.42 g (180 mM) CaCl2 29.4 g (200 mM) MgCl2 20.33 g (100 mM) Dissolve in dH2O, adjust volume. Store at RT.1/3X Modified Ringers
For 20 L tank:
NaCl 39 g 66.7 ml 100X MR salts 33 ml HEPES 1M pH 7.4Dissolve in dH2O, adjust volume. Store at RT.
1/9X MR + 1% Gentamicin
300ml 1/3 X MR + 600 ml DI water
Add 900ul 1000X gentamicin stock
1/20X MR
100 cc 1/3 X MR 500 ml DI Water
Marc's Modified Ringer's (MMR) (Ubbels et al. 1983)
0.1 M NaCl 2.0 mM KCl 1 mM MgSO4 2 mM CaCl2 5 mM HEPES (pH 7.8) 0.1 mM EDTANote: Most current formulations of MMR omit EDTA and are adjusted to pH 7.4.
Normal Amphibian Medium (NAM) (Slack and Forman 1980)
110 mM NaCl 2 mM KCl 1 mM Ca(NO3)2 1 mM MgSO4 0.1 mM EDTA 1 mM NaHCO3 2 mM sodium phosphate pH 7.4Note: Assemble this solution from autoclaved stocks of 50x phosphate buffer (0.1 M sodium phosphate, pH 7.5) and a 10x stock of remaining salts excluding sodium bicarbonate. A 100x stock of sodium bicarbonate (0.1 M) should be filter-sterilized.
Holtfreter's Solution (Holtfreter 1931)
Prepare a stock of 25 mM NaHCO3 and 10x Holtfreter's salts. Autoclave the salts, and filter-sterilize the NaHCO3.
25 mM NaHCO3
2.1 g/liter10x Holtfreter's salts
600 NaCl 6 KCl 9 mM CaCl2Prepare the final solution by mixing 100 ml of 25 mM NaHCO3 with 100 ml of 10x Holtfreter's salts and adjust the volume of 1 liter with distilled water.
