434 



METHODS AND FORMULAS 



DS 22.10 DS 22.11 



tached to the slide in the usual way, 

 dewaxed, and run down tlirough the cus- 

 tomary alcohols to water. 



An alternative technique, which is not 

 nearly as widely used but is really most 

 successful, is to employ smears rather 

 than sections of the root tip. Though these 

 do not give, of course, a clear picture of 

 the relations of the cells to each other, 

 they at least insure that the majority of 

 mitotic figures will be so oriented on the 

 shde as to occasion no difficulty to the 

 students studying them. These smears 

 may be prepared by taking the tip of the 

 root, placing it on the slide, taking a sharp 

 scalpel, and smearing, quite hterally, the 

 root onto the glass with the pressure of 

 the flat side of the knife and a rotary mo- 

 tion of the hand. These smears are then 

 fixed by being placed face down, resting 

 across two glass rods, in a petri dish of 

 fixative. The depth of the fluid must be 

 such that the lower surface of the shde 

 bearing the smear is in contact with the 

 fixative, which must not, however, be 

 permitted to rise up until the upper sur- 

 face of the shde is wetted by it. After 

 fixation by this method the sUdes are re- 

 moved and washed in several changes of 

 50% alcohol until the fixative is removed. 



Two solutions are required for staining. 

 First is DS 21.11 Kedrovsky]l931. To pre- 

 pare this, dissolve 0.3 gram of rose bengal 

 in 50 milhliters of water; add one milliliter 

 of 1% phosphomolybdic acid to the boil- 

 ing solution, one drop at a time, with 

 constant stirring. Cool and add 50 milli- 

 liters of a 1.2% solution of orange G. The 

 only other stain required is a 1 % solution 

 of toluidine blue. 



Whether sections or smears are being 

 employed, the shdes are transferred from 

 distilled water to the rose bengal-phospho- 

 molybdic acid stain for from 20 to 30 



minutes. The time is not particularly 

 critical, and a, pcu'iod of as long as one hour 

 may be employed if it is more convenient. 

 Each slide is then taken separately, rinsed 

 very briefly in distilled water, and trans- 

 ferred to 1 % toluidine blue for 5 minutes. 

 It is transferred directly from toluidine 

 blue to 70% alcohol, in which it is waved 

 Ijackward and forward for about one min- 

 ute to remove the excess stain from the 

 section, and then transferred to 95% 

 alcohol, in which differentiation proceeds 

 relatively slowly. If differentiation in 95% 

 alcohol is too slow, the specimen may be 

 returned to 70% alcohol for a brief period, 

 and then put back in 95% alcohol. The 

 sections must be examined under a high 

 power of the microscope to determine 

 when they are properly differentiated, and 

 it is not safe to do this if they have been 

 removed directly from alcohol. Before 

 being examined, therefore, each section 

 should be dipped in bergamot oil (specified 

 by Kadrovsky) or in clove oil, which is 

 equally safe. The oil should be wiped from 

 the back of the slide and the slide then 

 examined. The differentiation should not 

 be watched with regard to the chromo- 

 somes themselves, but with regard to the 

 spindle fibers, which are far better shown 

 by this than by any other method. In a 

 properly differentiated specimen the spin- 

 dle fibers will be a clear pink against a 

 blue background, and when this condition 

 has been reached the bergamot oil should 

 be washed off thoroughly in xylene, and 

 the specimen mounted in balsam. If the 

 spindle fibers are properly differentiated 

 the chromatin will be a clear dark blue, 

 very readily differentiated from the blue 

 background of the cytoplasm, and the 

 students' attention will clearly be drawn 

 to the nucleoli in the resting nuclei, which 

 are stained a bright red. 



22.11 OTHER TECHNIQUES 



22.11 Backman 1935 20540b, 10:83 



This method, recommended by Gatenby and Painter 1937, 691, calls for a "saturated 

 solution of anthraquinone " ; anthraquinone is not soluble in water and it is not ap- 

 parent which soluble derivative was employed by Backman. 



22.11 Balbiani test. 1895 Maggi Maggi 1895, 174 



REAGENTS required: A. 1% osmic acid; B. water 100, acetic acid 1, methyl green 1; 



C. 0.2% ammonia 

 method: [protozoans] —* A, 1 min. B, 3 mins. — > C, till differentiated 

 RECOMMENDED FOR: nuclear detail in ciliates. 



