310 



METHODS AND FORMULAS 



DS 11.40 



rather more than the usual amount of 

 trouble. Each tube is now agitated until 

 the pollen grains (but not air bubbles) are 

 dispersed throughout the celloidin, one 

 drop of the dispersion being then taken 

 and allowed to run over the surface of the 

 cover. The most critical stage of the pro- 

 ceedings is the next one, in which the cel- 

 loidin is congealed without being per- 

 mitted to evaporate to dryness. If it is 

 allowed to evaporate to dryness, it will 

 almost invariably become detached, either 

 by curling in the dry state or in one of the 

 solutions which is subsequently applied to 

 it. 



The best method of congealing the cel- 

 loidin is with the aid of chloroform vapor, 

 a high concentration of which is easily 

 maintained at the bottom of a coplin jar 

 by pouring a few cubic centimeters into 

 the jar, placing the hand over the top, 

 shaking the jar once or twice, and then 

 setting it on the bench with a loose cover 

 in place. Chloroform vapor is so heavy 

 that it will remain in the jar for a long 

 period. Each drop of the suspension of 

 pollen should be spread uniformly over 

 the coverslip and then waved once or twice 

 in the air until it appears about to become 

 dry. Each coverslip is then, while held 

 with forceps, lowered into the chloroform 

 vapor at the bottom of the cophn jar and 

 held there for a few moments until it ac- 

 quires a slightly opaque appearance. The 

 coverslip is then dropped into a rack in a 

 jar of 70% alcohol, where it may remain 

 until sufficient have been accumulated to 

 permit staining. 



Two reagents are required for the stain 

 of Johansen 1940, the first being the 

 rather specially prepared solution of safra- 

 nin given under the technique of Johan- 

 sen 1930 (11.42 Johansen 1930) below, 

 and the other, developed by Masson 

 (Chapter 22, ADS 22.1 Masson 1942), a 

 65:35 mixture of a saturated solution of 

 picric acid in 95% alcohol, and 95% alco- 

 hol. The coverslips carrying the dispersion 

 of pollen are removed from 70% alcohol 

 when is ready to stain them, transferred 

 to distilled water until the diffusion cur- 

 rents have died down, and then placed in 

 a jar filled with Johansen's safranin 

 stain, where they may remain not less 

 than 24 hours or until next required. Each 



individual coverslip is now taken sep- 

 arately and rinsed in the differentiating 

 solution of Masson until examination un- 

 der the low power of a microscope shows a 

 clear differentiation of red nuclei against 

 the yellow background of the pollen cell. 

 Differentiation may be stopped immedi- 

 ately by transferring the covershp from 

 the picric acid to water. 



Several courses are now open to the 

 mounter. If it is desired only to show the 

 nuclei, the slide may be dehydrated in the 

 regular manner as far as 90% alcohol, 

 cleared in terpineol to avoid dissolving the 

 celloidin holding the pollen grains in place, 

 and then mounted in balsam. If the gen- 

 eral outline of the pollen cell is required 

 as well as the nuclei, the material may be 

 treated in the same manner up to the 

 moment of its clearing and then counter- 

 stained either with a solution of light green 

 or fast green in clove oil. If the latter 

 course is adopted, the excess hght green in 

 clove oil should be washed off with clove 

 oil and the oil itself subsequently washed 

 out in xylene as a preliminary to mounting 

 in balsam. The very common practice of 

 passing directly from a solution of hght 

 green in clove oil to balsam itself is most 

 unsatisfactory and results always in the 

 gradual removal of the light green from 

 the tissues as the clove oil diffuses through 

 the balsam. The intermediate washing in 

 clove oil is also itself insufficient^ since it 

 will result in the removal of additional 

 dye, and necessitate the third step recom- 

 mended, i.e. removal of clove oil with 

 xylene. 



Demonstration of chromosomes of the 



grasshopper in a smear preparation 



of the testes using magenta by the 



method of Henneguy 1891 



The grasshopper provides unusually fa- 

 vorable material for the demonstration of 

 chromosomes, for the reason that these 

 are compact and small, usually with the 

 sex chromosomes well differentiated. Any 

 species of grasshopper may be employed, 

 but the specimens should be collected 

 during the early months of tlie summer, 

 preference being given to young specimens 

 (Baker 1945, 182) in which the wings are 

 not yet fully grown. 



