Culture media

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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.