74 Botanical Microtechnique 



bottle. Tapping the specimen bottle gently against the sink aids in 

 the loosening of air bubbles within or on the specimen. Highly 

 buoyant materials should be placed into a tall vial of the killing fluid 

 and held below the surface by means of a plug of cheesecloth. A 

 screw-topped wide-mouthed bottle is necessary for evacuating large 

 objects (Fig. 3.1 C). W'hen most of the pieces remain submerged 

 after the suction is released, push any floating pieces under the 

 surface with a matchstick, and most of them will then sink. Remo\e 

 and discard all pieces that do not sink after pumping and submersion. 

 Materials from which it is difficult to evacuate air do not become 

 infiltrated readily and should be pimiped again when nearing the 

 end of the dehydrating series, and again when in the final change 

 of paraffin solvent, before any paraffin has been added. Connect a 

 second safety bottle between the regular safety bottle and the speci- 

 men. The possible entry of water vapor into the specimen bottle 

 when the pump is shut oft is prevented by having a deep layer of 

 calciinn chloride and a layer of cotton in the second safety bottle. 

 The ingenious and precisely controllable vacuum apparatus of Witt- 

 lake (1942) may be used for the killing, as well as the subsequent 

 operations of embedding. 



Killing and Fixing of Tissues 



Killing solutions may be grouped into types on the basis of the 

 ingredients used. Some formulas are stable and may be kept on hand 

 ready for inmnediate use. Other formulas must be made up innnedi- 

 ately before use. The forjnulas given on the following pages have 

 been comj)uted so that they can be made up from the above stock 

 solutions by volumetric measurements, llic system of letters and 

 numbers used in this manual to designate killing fluids is explained 

 later in this chapter. 



The length of time necessary to bring about killing and harden- 

 ing of material varies greatly and is determined by the character of 

 the fluid used, the bulk of llie indixidual pieces, and the resistance 

 of materials to jKuetraiion by reagents. Fluids of the anhydrous type, 

 such as Carnoy's absolute alcohol-glacial acetic acid fornuda. pene- 

 trate small objects almost instantaneously, and killing and hardening 

 are a matter of miniues. The chrome-acetic fluids penetrate slowlv 

 into the interior of a piece of tissue, and have poor hardening action. 

 Re(f)nnnendations coiucrning the duration in killing fluids are given 

 in the description of the various fonnulas. Washing of tissues, which 



