Reagents 19 



6) Absolute (100 Per Cent) Alcohol. This is a fair killing and 

 fixing agent, it causes but little shrinking of the protoplasm, and is 

 a time-saver if material is to be imbedded in paraffin. The time 

 required for fixing in alcohol is very short. For small fungi, like 

 Eurotium, 1 minute is long enough. Root-tips of the onion, anthers 

 of the lily, and similar objects require 15 to 30 minutes. Larger 

 objects may require an hour. No washing is necessary, but all 

 plant tissues contain water; consequently, if material is to be 

 imbedded in paraffin, the alcohol used for fixing should be poured 

 off and fresh alcohol added before proceeding with the clearing. If 

 material is to be mounted in Venetian turpentine, as is likely to be the 

 case in small filamentous fungi, the transfer to the stain may be 

 made directly from the absolute alcohol. This is only for very small 

 forms, like Aspergillus; neither the fixing nor the rude transfer would 

 be at all satisfactory with a form so large as Saprolegnia. 



Acetic acid is used with alcohols to counteract the tendency to 

 shrink. One of the most widely known of the alcohol combinations is 



c) Carnoy's Fluid. - 



Absolute alcohol 6 parts 



Chloroform 3 parts 



Glacial acetic acid 1 part 



The penetration of the reagent is very rapid. An object like an 

 onion root-tip is doubtless killed in less than a minute, and 10 to 15 

 minutes is long enough for fixing an object of this size. Wash in 

 absolute alcohol, changing frequently, until there is little or no odor 

 of acetic acid. For a root-tip, the entire process of fixing and wash- 

 ing should not require more than an hour. It is better to imbed in 

 paraffin at once, but when this is not convenient the material may be 

 transferred to 85 per cent alcohol, where it may be left until needed. 

 Cyanin and erythrosin, fuchsin and iodine green, and similar com- 

 binations give particularly brilliant staining after this reagent. 



d) Acetic Alcohol. Farmer and Shove recommend for fixing 

 root-tips of Tradescantia virginica a mixture of two parts absolute 

 alcohol and one part of glacial acetic acid. The mixture is allowed 

 to act for 15 to 20 minutes, after which the acid is washed out with 

 absolute alcohol and the material is imbedded as soon as possible. 



