36 CULTURE METHODS AND TECHNIQUE 



Frequently, it will be found in practice that in making cultures 

 of many parasitic fungi so few spores will be available that they 

 may be inserted directly into tube No. I, and it will be necessary 

 to pour a few drops of the agar from No. I to No. 2. In fact, 

 No. 2 will usually give a very good isolation ; and then No. 3 may 

 be used as a No. 2, to which is added a drop of 50 per cent lactic 

 acid. Fig. 8 shows an isolation series of Glomerclla rnfomacnlans, 

 the bitter rot fungus. 



Elimination of bacteria in isolating fungi. The use of lactic acid 

 in culture media is an important aid in eliminating certain trouble- 

 some bacteria. In general it may be well to prepare some tubes 

 with lactic acid in about the amount above indicated, practically .5 

 per cent, so that all tubes used in the isolation series may be thus 

 acidulated without danger of contamination. Acidulated media are 

 especially valuable when separation cultures must be made by using 

 hyphae from a mixed culture, or from any other source which permits 

 extraneous organisms to come in. In such cases the mycelium 

 should be washed as carefully as possible in distilled water, and then 

 on being placed in the tubes of liquefied agar, the tubes should be 

 vigorously shaken before the contents are poured into the Petri 

 dishes. If, however, there is nothing to indicate the relations of 

 a fungus to acidity, one isolation series should be made with 

 neutral, or very slightly acidulated agar. 



Colony counting. In bacteriological work, and sometimes in 

 purely mycological work, it is desirable to make accurate count of 

 the number of spores or cells which may have been present in the 

 material from which the culture is made. Under such circumstances 

 a leveling table must be employed in making the poured plates or 

 Petri dish isolation cultures. Plates of glass or other devices, such 

 as cardboard charts, especially calibrated for counting colonies will 

 also be necessary. 



Study of the isolation colony. In the study and transference 

 of the fungi which may appear in isolation cultures, there is a 

 rough method which may be pursued, and there is a careful method 

 which must be followed if one is to be sure that the life history of 

 a particular fungus has been accurately traced. In the first place, 

 one may wait until the colonies have appeared, and perhaps until 

 growth has been considerably advanced. Then, if isolation is 



