Robert L. Metzenberg, Department of Chemistry and Biochemistry, University of California, Los Angeles CA 90095
Fungal Genetics Newsletter 51:19-20
This medium was designed to circumvent some problems that arise in the use of Medium N (Vogel 1964 Am. Naturalist 98:435-446). These are, among others, the presence of high levels of citrate, a chelator which leaves the concentration of calcium and trace elements uncertain; the use of ammonium nitrate, which leaves the actual source of nitrogen ambiguous; the use of MgSO4, which does not allow the experimenter to vary the concentration of magnesium and sulfur independently; the high activity coefficient for the pKa values of citrate, which makes the pH unnecessarily sensitive to ionic strength; the use of sucrose, which leaves uncertain the nature and relative amounts of the hexose(s) being used at any particular moment; the need to use chloroform as a preservative, which results in the gradual depletion of the aqueous phase of complexes of trace elements. Molybdate ion is excluded from the trace elements used for Solution 1 because, in concentrated stock solutions, it forms water-insoluble complexes with phosphate plus ammonium ion; instead, it is included in Solution 2, where its presence is benign. Finally, concentrations are expressed in moles rather than in grams, which eases the experimenter's task of thinking in terms of stoichiometry and biomass yield. Bird Medium is not meant to supplant Vogel Medium for routine auxanography, stock-keeping, searches for mutants, or growth of Neurospora for preparing DNA, mitochondria, etc. However, it should be seriously considered for critical applications such as preparation of samples for microarrays and for analysis of subtle phenotypes of new mutants. Bird Medium supports rapid germination of conidiospores and rapid growth of mycelium in good yield, with apparently normal morphology. It appears at least equal to, or better than, Vogel Medium in this regard. It should be noted, however, that mycelial pads harvested from Bird Medium have a subtly different texture from those grown on Vogel Medium, being somehow more slippery to the touch. It is not evident that more slippery is less "normal" for Neurospora, nor more so, than less slippery.
Solution 1
| INGREDIENT | CONCENTRATION AT 1X | |
| g/liter | mM | |
| water | 45 ml | |
| MES(Sigma M-8250) | 4.85 g | 22.75 |
| K2HPO4 | 1.74 g | 10 |
| NH4Cl | 1.34 g | 25 |
| K2SO4 | 0.174 g | 1. |
| NaCl | 0.058 g | 1. |
| trace elements without molybdate, 10,000x * | 0.1 ml. | |
Solution 2
| INGREDIENT | CONCENTRATION AT 1X | |
| g/liter | mM | |
| water | 38 ml. | |
| MgCl2.6H2O | 0.203 g | 1. |
| CaCl2.2H2O | 0.074 g | 0.5 |
| glucose | 18 g | 100 |
| biotin, 10,000x solution * | 0.1 ml. | |
| sodium molybdate, 10,000x * | 0.1 ml. | |
Make up MgCl2. 6H2O, CaCl2. 2H2O, biotin, molybdate solution, and glucose in 38 ml. of warm water, which will produce a volume of 50 ml. ("Solution 2; 20X final strength"). Store at room temperature over a few ml. of chloroform.
Obviously, it will usually be convenient to make up these two solutions on at least ten times the above scale.
Solutions 1 and 2 can be autoclaved separately in their concentrated form, if desired, and diluted into sterile water, or each can be diluted to 2X, autoclaved, and then combined. The pH of the diluted medium, about 5.8, equals that of Vogel medium, and should not be adjusted. Solution 1 can instead be made up to 20 ml. of 50X instead of 50 ml. of 20X by dissolving the ingredients in 15 ml. of warm water rather than in 45 ml. In practice, however, there are likely to be fewer calculational errors in medium-making if both stock solutions are kept as 20X stock solutions for dilution to 2X, rather than as 50X and 20X. The minor savings in space for Solution 1 are probably not be worth the increased risk of error.
* BIOTIN, 10,000X
| INGREDIENT | CONCENTRATION AT 10,000X | |
| mg/100 ml | mM | |
| water | 100 ml | |
| biotin | 6.1 | 0.25 |
* TRACE ELEMENTS WITHOUT MOLYBDATE, 10,000X
| INGREDIENT | CONCENTRATION AT 10,000X | |
| mg/100 ml | mM | |
| water | 95 ml | |
| citric acid. H2O | 4200 | 20 |
| ZnSO4.7H2O | 5750 | 20 |
| Fe(NH4)2(SO4)2.6H2O | 980 | 2.5 |
| CuSO4.5H2O | 250 | 1.0 |
| H3BO3 | 62 | 1.0 |
| MnSO4. H2O | 33.3 | 0.2 |
* SODIUM MOLYBDATE, 10,000X
| INGREDIENT | CONCENTRATION AT 10,000X | |
| mg/100 ml | mM | |
| water | 100 ml | |
| Na2MoO4.2H2O | 48.3 | 0.2 |
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