70 Botanical Microtechnique 



xylene. If red color is still evident in cellulose walls and cytoplasm, 

 carry the slide backward through the series to fast green, double the 

 previous interval in fast green, run upward again to carbol-xylene 

 and examine. This process can be repeated until the desired color 

 contrast between chromatin, lignified walls, cellulose and cytoplasm is 

 obtained. 



If the red color is too pale when the slide is examined out of 

 carbol-xylene, transfer to the de-waxing xylene and proceed as with 

 a new slide. The green will be removed in the down series. If the 

 green is too intense at the carbol-xylene stage, back downward to 

 70% alcohol, in which the green is removed rapidly. Try 10 seconds 

 and carry up to carbol-xylene again and examine. 



Several stains can be substituted for fast green in Chart IV. The 

 most commonly used other green stains are light green and malachite 

 green. Several excellent blue counterstains arc cotton blue, methylene 

 blue, gentian violet (crystal violet) , and aniline blue. Any of these 

 green or blue counterstains can be used in solution in 95% alcohol, 

 in the sequence shown in the chart, or they may be dissolved in 50% 

 alcohol or in clove oil and introduced at the appropriate place in 

 the series. The above safranin-grcen or safranin-hlue combinations 



STAINING CHART IV 



Safranin-Fast Green 

 Pre-Staining Operations and Intervals as in Chart I 



Aqueous resin and 



safranin cover glass 

 1-12 In. I 



i wlvuc J II 



water, change a 



until colorless 



i 

 30% 



alcohol 



i 

 50% 



xylene II 



wlenc I 



t 

 carbol- 



alcohol ^^'''le 



70% absolute 



alcohol alcohol TIT 



I (optional) 



95% t 



alcohol absolute 



I alcohol II 



fast green in f 



95% alcohol > absolute 



5-30 sec. alcohol I 



