DS 13.50-DS 13.51 DYE STAINS OF GENERAL APPLICATION 



3G3 



and brought down througli the customary 

 graded alcohols to 70% alcohol. Plant 

 material is far more likely to become dis- 

 torted through insufficient attention being 

 paid to grading alcohols than is animal 

 material. 



When all the slides have been accumu- 

 lated in 70% alcohol, they may be trans- 

 ferred to the first stain, which is of 1 % 

 safranin in 50 9o alcohol, and may remain 

 in this for any time desired l)y the mounter. 

 The customar}^ limits are from 2 to 24 

 hours. On removal from the safranin, the 

 slides should be cheeked under the low 

 power of the microscope to make sure that 

 the nuclei of the cells are densely stained. 

 Provided these are stained, all other struc- 

 tures will have acquired the necessary 

 safranin coloration. On removal from the 

 safranin, the slides should be washed in 

 distilled water until no more color comes 

 away. 



A single slide is then withdrawn, and a 

 saturated solution of crystal violet is 

 poured over the section on the slide and 

 left for about one minute. After being 

 stained with crystal violet, the sections 

 should be rinsed in water until no more 

 color comes away, and then dehydrated 

 as rapidly as possible through two changes 

 of absolute alcohol. This is a rather criti- 

 cal stage in the staining, since the alcohol 

 will remove the violet very readily and 

 one is, in effect, engaged in a race between 

 the minimum time required for dehydra- 

 tion and the minimum time in which the 

 stain will be lost. As soon as the slides 

 are judged to be dehydrated, they are 

 dipped briefly in 1 % fast green dissolved 

 in absolute alcohol in a copliii jar. Now 

 place the slide in a saturated solution of 



orange II in clove oil. It is essential that 

 the alcohol fiom the dehydration and from 

 the fast green stain be removed as rapidly 

 as possible so that it is desirable to have 

 at least two coplin jars of the orange II- 

 clove oil solution. The first of these is used 

 e.xclusively for rinsing the slide backward 

 and forward until all the adhering alcohol 

 has left it. The slide may then be trans- 

 ferred to the next coplin jar of orange II- 

 clove oil, in which it may be left, while the 

 remaining slides of the batch are brought 

 from 70 '^y alcohf)l through the successive 

 stages of the technique and accumulated 

 in their turn in the orange Il-clove oil. 



The orange Il-clove oil acts as a differ- 

 entiating agent and the slides may now be 

 observed at intervals, and, if differenti- 

 ation is insufficient, allowed to remain till 

 the required structures can be clearly seen. 

 As soon as difTcrontiation is judged to be 

 complete, the slides are transferred to a 

 fresh jar of clove oil. Here the orange Il- 

 clove oil is thoroughly washed from them 

 and they are then taken to xylene in which 

 the clove oil is removed. The omission of 

 the last clove-oil wash is a cause of fre- 

 quent failure, since any adherent orange- 

 Il-clove-oil solution which is carried over 

 into xjdene will lose the orange II by 

 precipitation, producing a veil over the 

 finer structures. The slides may be left in 

 xj'lene for as long as required before being 

 mounted in balsam in the customary 

 manner. 



It must again be emphasized that this 

 method is not intended for the class 

 demonstration of skeletal structures, but 

 only to bring out the finer details of the 

 cytoplasmic and nuclear constituents of 

 any plant material. 



13.51 OTHER TECHNIQUES 



13.51 Conant lest. 1940 Johansen safranin-crystal violel-fast gr((n-(>i(tii(i< II 



johansen 1040, 87 

 REAGENTS REQUIRED: A. 1% Safranin in 50% ale; B. sat. sol. {circ. 1%) crystal viukt; 



C 1% fast green in abs. ale; D. sat. sol. orange II in clove oil 

 method: [sections] — ► 70% ale. -^ A, 2-24 hrs. -^ water, rinse * H, 1 niin. — > water, 

 rinse -^ abs. ale. till dehydrated-^ C, 5-10 dips -^ I), till altohol removed-^ D, 

 fresh solution, till differentiated — > balsam, via xylene 

 note: A detailed description of the use of this stain is given under DS 13.50 above. 



13.51 Foley 1929 safmnin-orange G-crystal violet 76:3,43:171 



reagents reqcired: A. 1% safranin O; B. N /AO liydrochloric acid; C. 0.3% crystal 

 violet in 70% ale; D. ADS 12.2 Lugol (1905); E. 1% mercuric chloride; F. sat. sol. 

 orange G in clove oil 



