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.