62 Botanical Microtechnique 



the parafFin dissolves in 1 or 2 min. With cold slides it is better to 

 allow 5 min. The slide is now in a very dilute solution of paraffin 

 in xylene, which is removed by immersing the slide in anlnclrous 

 alcohol. As outlined in Staining Chart I, progressive transfer to Avater 

 is then made through the indicated grades of ethyl alcohol. Isopropyl 

 alcohol or acetone also may be used in most of the staining charts in 

 this chapter. The slide has been run doiaji to water, and is now ready 

 to be stained in an aqueous dye. Transfers should be made cpiicklv 

 so that the slides do not become dry. The inter\als can be shortened 

 to 30 sec. by moving the slides up and down in the solution with 

 forceps. 



The series of reagents in which slides are deparaffined should be 

 replaced when the 30% alcohol becomes cloudy or when the fluid 

 drains from the slides as if the glass were oily, indicating that paraffin 

 and xylene have been carried down the series until the 70% and 30% 

 cannot hold the xylene-paraffin contaminant in solution. The ad- 

 dition of 10% rj-butyl alcohol to the anh\clrous and 95% grades pro- 

 longs the useful life of the series. 



riie correct staining interval for a given subject must be deter- 

 mined by trial. An experienced worker can make a good guess for 

 a trial slide and make corrections for subsequent slides. One collection 

 of lily ovary killed in Bouin's solution required only 10 min. for a 

 brilliant stain, whereas another collection, fixed in Craf rccpiired 

 1 hr. A collection of lily anther in the microspore stage yielded excel- 

 lent slides with a 30-min. stain. To determine the correct interval, stain 

 three slides of a subject tor three intervals, i.e.. 10. 20, and 40 min., 

 respectively. Mark the slides before staining. The sample slides may be 

 held in distilled water and ])ut into the stain at intervals, or they 

 may be put into the stain simultaneously and removed after the 

 desired intervals. After staining, rinse the slides in two or more 

 changes of distilled water, then rinse in three changes of tap 

 water, or in running tap water for 2-5 min. Note that the 

 color in the tissues changes from purj^le to blue after the trans- 

 fer into tap water. Hematoxylin gives a reddish-purple color Avhen 

 acid and a blue color when alkaline. The latter color is pre- 

 ferred for tlif subjects recommended for this first exercise. If the city 

 water in your ccjnnnunity does not produce the bluish tinge in tissues 

 that have been stained in hemalum, use 0.1% sodium carbonate for 

 the last rinse. This process may be called nlknlizing. 



At this stage, examine the three test slides that were stained as 



