SUGGESTED LABORATORY EXERCISES 421 



chemicals should be dissolved in less than the desired volume of water, 

 and after they are in solution, the medium should be made up to volume 

 by the addition of distilled water. 



Constituents of media. The salts used in making media should be of 

 c.p. grade. These will generally contain enough of the micro essential 

 elements to satisfy the needs of most fungi. Iron, zinc, and manganese 

 should be added routinely to all synthetic media. It is convenient to 

 make up a solution containing the above micro essential elements in such 

 strength that 1 ml. of the master solution will contain 0.1 mg. Fe+ + +, 

 0.1 mg. Zn+ +, and 0.05 mg. Mn+ +. The following amounts of the given 

 salts have been found convenient to use: Fe(N03)3-9H20, 723.5 mg. ; 

 ZnS04-7H20, 439.8 mg.; MnS04-4H20, 203.0 mg. Dissolve these three 

 salts in 600 ml. of distilled water, add sufficient c.p. sulfuric acid to yield 

 a clear solution, and make up the volume to 1 liter with distilled water. 

 Use 2 ml. of this solution per liter of medium. Add the source of carbon 

 at the rate of 10 to 25 g. per liter, depending on the use of the medium. 

 Add the nitrogen source at the rate of 2 g. per liter, or the amount which 

 will give an amount of nitrogen equivalent to that furnished by 2g . of 

 anhydrous asparagine (0.425 g. of N). 



The activated carbon used in Ex. 9 to absorb residual traces of vitamins 

 is a commercial preparation, Norit A (pharmaceutical grade). Use at 

 the rate of 5 g. per liter, or more if necessary. The vitamins used rou- 

 tinely are thiamine and biotin. Inositol and pyridoxine are also used in 

 Ex. 9. These vitamins may be purchased from JMerck and Co., Rahway, 

 N.J., and many other pharmaceutical houses. It is convenient to make 

 up master solutions of these four vitamins. Make the master solutions 

 in 20 per cent alcohol, store in 100-ml. volumetric flasks, and keep in a 

 refrigerator when not in use. It is convenient to make the master solu- 

 tions of thiamine and pyridoxine to contain 100 /xg per ml. (10 mg. per 

 100 ml.). The master solution of inositol contains 5 mg. per ml. Biotin 

 is used at the rate of 5 Mg per liter. Either the contents of an ampule may 

 be made up, or a microbalance used to weigh this vitamin. A master 

 solution containing either 1 or 5 jug per ml. is convenient. 



For a semisolid medium, agar is commonly used at the rate of 20 g. 

 per liter. Agar is not a pure compound, and its use introduces various 

 unsuspected constituents into media. No medium may be said to be of 

 known composition if agar is used, although such media are very useful. 

 Some of the impurities in agar may be removed by leaching with aqueous 

 pyridine solution. Place 1 lb. of agar in a 6-liter flask, add 5 liters of 

 distilled water and 500 ml. of pyridine. Allow to stand 24 hr. Insert 

 a piece of 6-mm. glass tubing of sufficient length to admit air to 

 the flask, tie a piece of cheesecloth over the neck of the flask, invert the 

 flask, and allow the pyridine solution to drain. Wash the agar three 



