72 Botanical Microtechnique 



of time. The triple stain should be kept in its proper place among 

 the diverse tools ol the technician. 



The three stains used in the conventional process are the 

 following standard stock solutions: 



Safranin O, aqueous, or in 50% alcohol. 



Crystal violet, or gentian violet, 0.5 or 1.0% in water. 



Orange G, or gold orange, saturated solution in clove oil. 



Mordanting is necessary for some subjects. After killing in fluids 

 that contain osmic and chromic acids, mordanting is usually not 

 necessary. For materials that do not retain the stains, mordant for 1 

 to 12 hr. in 1% aqueous chromic acid or in an aqueous solution 

 containing 2% chromic acid and 0.57c osmic acid. 



Staining Chart V is intended primarily to show the sequence of 

 operations in a typical schedule. The variability of the time element 



STAINING CHART V 



Triple Stain 



Pre-Staining Operations and Intervals as in Chart I 

 Safranin resin and 



4-'^4 hr cover glass 



i ^^ 



"" xvlene 



water, 3 changes 2'chan<'^es 



i (jars) 

 crystal violet 'I 



10 luin. to 1 hr. rinse with 



I xylene (pi[)ette) 



t 

 water. ^ changes . ' . , 



" rinse with 



-l clove oil-xylene 



<lip into (pil)ette) 

 50% alcohol I 



I (lifTerentiate 



rinse with ^"i^l^;" microscope 

 95% alcohol I . 



(pipette) "^'"^1 ^^'^1^ ., 



, new clove oil 



i . ^ 



dip into jiiisr with used 



95% alcohol j,,^^. „ii (pipette) 



I .t 



2-3 changes > flood sections > drain off 



.inhydrous alcohol with orange G orange G 



in any of the operations hardly can be overemphasized. The suggested 

 intervals merely furnish a starling point for experimentation to 

 determine the o|)tiinuni linie schedule h)r any specilic subject. 



Many modihcations ol schedide have appeared in the literature. 



