424 PHYSIOLOGY OF THE FUNGI 



stock culture should be to another stock-culture tube, and the old tube 

 should be kept until the new culture begins to grow free from contamina- 

 tion. Other methods of storing stock cultures of fungi are described by 

 Greene and Fred (1934), Thom and Raper (1945), Fennell et al. (1950), 

 and Buell and Weston (1947). 



Methods of inoculation. It is customary to use a bit of mycelium from 

 a growing culture to inoculate fresh media. For ordinary uses this is 

 satisfactory, if only a few cultures are to inoculated at a time and no 

 special precautions need be taken. Some fungi produce a tough mat of 

 mycelium difficult to cut with a needle. Often this can be overcome by 

 growing the mycelium for inoculum on an agar medium quite low in 

 sugar. A small cork borer may be used for cutting out uniform disks of 

 mycelium and agar from Petri dishes. 



Spores alone may be transferred by a dry needle, or they may be 

 suspended in water and inoculated by use of a loop or a sterile pipette 

 with a cotton plug at the upper end. The use of a pipette fitted w^ith a 

 small rubber bulb greatly decreases the inoculation time when many 

 cultures of the same fungus are made. It is preferable to use spores as 

 inoculum in studies of vitamins or micro elements, where none of the 

 previous medium should be added. 



Nonsporulating mycelium may be fragmented by placing it with about 

 50 ml. water in a sterile Waring Blendor jar for about 30 sec. Either 

 agar or liquid medium may be used if the addition of the medium is of no 

 consequence. In vitamin studies the mycelium may be grown in liquid 

 medium and, w^hen ready for use, w'ashed in sterile distilled water and 

 fragmented in the Blendor. Either a loop or a pipette may be used to 

 dispense the mycelial suspension. 



Methods of obtainmg single-spore cultures. In certain physiological 

 studies it is desirable to use single-spore cultures. These may be obtained 

 by a number of different methods. A review of the literature on these 

 techniques has been given by Hildebrand (1938). Other modified tech- 

 niques are described by Georg (1947) and Thom and Raper (1945). 

 Still another modification may be worthy of brief mention. In this 

 laboratory we have used a specially prepared small sewing needle as a 

 tool for picking out single germinated spores. The eye of the needle is 

 rounded and the thick metal portion filed down, making a rather thin 

 edge for cutting agar. The pointed end is fastened in a convenient 

 holder, and the needle bent in such a way that, when held over a Petri 

 dish, the eye portion will be parallel with the surface of the medium so 

 that it can be pushed straight down into the agar. An isolated, germi- 

 nated spore is located on a dilution plate by use of a microscope. The 

 needle is then held in place under the objective so that the spore is visible 

 through the eye of the needle. The eye is pressed down around the spore 



