42 CULTURE METHODS AND TECHNIQUE 



of the fungous hyphae within other plant cells or tissues is desired ; 

 while an entirely different method may be essential if the investi- 

 gation is to concern itself with more minute cytological details. 



Even when material is to be used for immediate casual study it 

 is often necessary to kill and fix it on account of the greater ease, 

 with which the subsequent operation of staining may be performed. 

 In the examination of hyaline filamentous fungi it is unnecessary 

 to use any but the simplest methods of fixing on the slide. It has 

 been found desirable to treat such hyphae for a minute or two with 

 a few drops of a 3 per cent solution of acetic acid. This treatment 

 will also generally dispel bubbles of air. The acid should be well 

 washed out with water before using basic stains. In the same way 

 a 3 per cent solution of potassium hydrate or a weak solution of 

 chloral hydrate will often give good results, the former, particu- 

 larly, if the material has suffered any drying out and needs restor- 

 ing by the swelling process to which the hydrate is adapted. 



Alcohol. The fixing qualities of alcohol are well known. When 

 employed alone it is usually recommended to use either very low 

 or very high grades of this agent, and it is serviceable only for 

 gross work. Of the lower grades, 15 to 25 per cent are generally 

 used, for at this concentration little harm will result from the effects 

 of diffusion currents. When the higher grades are used, those 

 from 96 per cent to absolute alcohol are preferable, in order to 

 effect rapid penetration and fixing. Where the weaker grades are 

 employed first, the process is also essentially one of dehydration. 

 The size and consistency of the material will determine the length 

 of time that the object should be left in the lower grades. It is 

 usually left in each lower grade from two to four hours and in each 

 higher grade from four to twelve hours. If one begins with 1 5 per 

 cent alcohol, the material should subsequently be passed through 

 30, 50, and 70 per cent, and for safety in hardening 85 per cent, 

 and finally 95 per cent may also be used. Material that is to be 

 kept for any length of time should, however, be stored in from 65 

 to 75 per cent alcohol, since the higher grades are more apt to ren- 

 der it brittle. If material is fixed in from 96 per cent to absolute 

 alcohol, it may remain at this concentration for from twenty-four to 

 thirty-six hours, and then, if storage is desired, it should be passed 

 back to the weaker grade mentioned. 



