Culture media
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 Solution
Modified 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 C
10X MBS salts
- 880 mM NaCl
- 10 mM KCl
- 50 mM HEPES (pH 7.8)
- 25 mM NaHCO3
Adjust 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 NaHCO3
Note: 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 ml
Amphibian 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 L
Modified 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 NaHCO3
Modified 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 7.6
100X 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 7.4
Dissolve 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 EDTA
Note: 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 7.4
Note: 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/liter
10x Holtfreter's salts
- 600 NaCl
- 6 KCl
- 9 mM CaCl2
Prepare 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.