246 PRINCIPLES OF SOIL MICROBIOLOGY 



examining the inverted dish under the microscope, using the low power 

 and marking with a colored pencil the spot where an individual spore 

 has germinated. If such a growth is found, it is transferred with a 

 small amount of substrate, by means of a sterile platinum loop, 

 to a new dish or sterile agar slant. Povah sprayed a spore dilution 

 upon a poured plate by means of capillary pipettes, then proceeded as 

 before. Within twenty-four hours, after the spore was removed, 

 transfers were made from the edge of the growth to a fresh tube; the 

 possibility of contamination through a neighboring spore delayed in 

 germination was thus avoided. 



The following procedure may also be followed : 



A small amount of the spore material is well shaken in 50 to 100 cc. of sterile 

 water in an Erlenmeyer flask; a sterile platinum loop is then dipped into this 

 suspension and carefully streaked out 3 or 4 times over the solidified agar in a 

 Petri dish. The spores drop heavily at first, then singly, so that the third or 

 fourth streak will have only single spores separated from one another. The plate 

 is incubated for 36 to 48 hours, then examined in an inverted condition with the 

 low power of the microscope. The streaks make easier the location of the spores. 

 Where single spores have been dropped, they can be readily recognized, marked, 

 and transferred with small blocks of agar into fresh dishes with agar or 

 slants. By transferring again, within 24 hours, from the edge of the colony, and 

 repeating the whole process when spore development takes place, single spore 

 cultures can be assured. Of course, when the Barber 43 pipette can be employed, 

 there is greater certainty of obtaining single spore cultures. This is, however, 

 not so necessary, in the case of fungi, as in the case of bacteria. 49 



Blakeslee 50 devised a procedure for the isolation of two strains of 

 Mucorineae, plus and minus strains. This consisted in teasing out an 

 immature zygospore and placing it in a nutrient medium favorable 

 for growth. In some cases, growth occurred from both suspensors in 

 sufficient amount so that they could both be transferred to a fresh culture. 



CLASSIFICATION OF FUNGI, WITH SPECIAL REFERENCE TO THOSE 

 OCCURRING IN THE SOIL 



EUMYCETES. Vegetative tissues deprived of chlorophyll, unicellular or 

 multicellular, with a typical apical growth and formation of ramified my- 

 celium. Reproduction sexual and asexual. No locomotion in developed 

 cells. 



48 Barber, 1907 (p. 55). 



49 Roberts, J. W. A method of isolating single spores. Phytopathol., 13: 

 558-560. 1923. 



60 Blakeslee, A. F. Sexual reproduction in the Mucorineae. Proc. Amer. 

 Acad. Arts. Sci., 40: 205-315. 1904. 



