28 CULTURE METHODS AND TECHNIQUE 



method requires more of the personal attention of the operator, but 

 it insures successful solution of the agar. The medium may be 

 then cleared in the usual manner. 



In making nutrient agar, a chief difficulty for the beginner has 

 been with relation to nitration, which is necessary in order that a 

 clear product may be obtained in which the development of micro- 

 colonies may be carefully followed. If the agar is thoroughly dis- 

 solved, it filters with comparative ease ; whereas, if partly dissolved, 

 filtration is next to impossible. In any case a grooved or ridged 

 filter, good filter paper wet with hot water immediately before using, 

 and well-dissolved agar direct from the pan, steamer, or autoclave 

 are the requirements. Nevertheless, in case of difficulty, the filter 

 stand with funnel and flask may be placed in the sterilizer or auto- 

 clave to be kept thoroughly hot during the process. Again, a side- 

 neck filter flask may be used so that connection with a filter pump 

 attached to the tap may be secured. In the latter case porcelain 

 supports and cotton may be substituted for filter paper. 



After filtration the agar may be poured into flasks or test tubes 

 (usually about 8 cc. per tube, when used for isolation cultures), 

 subsequently sterilized and stored. 



A synthetic liquid medium may be used as a nutrient base with 

 agar. The standard salt solution previously mentioned and many 

 others are serviceable ; however, since agar is a medium the com- 

 position of which is complex, it is often too " impure " as to known 

 qualities for certain physiological studies. Long washing is of value, 

 but this may not remove all materials furnishing food substances. 

 A few drops of hydrochloric acid in the water will also materially 

 improve the purity of the agar, but it may injure or entirely destroy 

 the solidifying properties. In most instances it is best to substitute 

 for the agar Winogradsky's silicate jelly, or resort to cultures on 

 tightly folded bars of filter paper or some other pure substance. 

 Glycerin agar is particularly serviceable in culturing slow-growing 

 fungi. It is made by the addition of 5 per cent glycerin to the 

 prepared medium. 



A stiff agar, made by using from twenty to thirty grams of agar 

 for each liter of solution, is desirable when cultures are to be trans- 

 ported. It is also valuable, employed in large flask cultures, in 

 order to obtain the fruiting stages of many fungi. 



