Hydra 



SQUASH PREPARATIONS FROM SOLID BODIES 



79 



The tube containing the hydra is half 

 filled with 20% nitric acid. The specimens 

 may be left overnight for treatment the 

 next morning or, if one is in a hurry, the 

 tube may be very gently warmed (to a 

 maximum of about 50°C.) for twenty 

 minutes. In either case it will be found 

 that the hydra, which had become hard 

 in the fixative, are now flaccid and tender. 

 The acid is removed, either by pouring or 

 by withdrawing it with a pipet, and the 

 tube filled with the saturated solution of 

 potassium alum. The specimens will float 

 for a brief time but, as soon as they have 

 sunk to the bottom, the alum solution is 

 poured off and replaced with fresh solu- 

 tion. The tube is now shaken gently until 

 such time as each hydra has dissociated 

 into its constituent cells. If this does not 

 take place after shaking for a few minutes, 

 it is necessary to withdraw the alum solu- 

 tion, replace it with nitric acid, and to 

 continue macerating for a further period. 

 It is not to be anticipated that all hydra 

 out of a batch of 20 or 30 will disintegrate 

 at the same time, but any large cell masses 

 which remain may easily be picked out 

 with the point of a needle after the tube 

 containing the cells has been emptied into 

 a watch glass. * 



Having thus secured a suspension of the 

 cells in a solution of alum, it is necessary 

 ,, first to wash most of the alum from them, 

 ^' and then to get them into stain. For this 

 purpose rinse the tube into a larger one 

 which is filled with water, stir up, and 

 allow the cells to settle. Any of the alum- 

 carmine stains given in Chapter 20 (DS 

 11.21) may be used. Whichever one is 

 chosen is poured over the cells after the 

 supernatant water used in the last wash has 

 been poured off. The time for staining is 

 not important, three or four days being 

 usually enough. Though the cells cannot be 

 seen in the stain, it may be assumed that 

 they have fallen to the bottom. The upper 

 two-thirds of the stain is poured off before 

 filUng the tube with a weak (1 %) solution 

 of w^hatever alum was used in the prepara- 

 tion of the stain selected. The cells are 

 again allowed to settle, the supernatant 

 liquid poured off, the tube refilled with 

 alum solution, and so on, until the wash 

 solution is practically colorless. 



The cells now have to be dehydrated; 

 this is done by pouring off the alum solu- 

 tion, replacing it with, say, 30% alcohol, 

 which is itself replaced with 70% alcohol, 

 as soon as the cells have fallen to the 

 bottom. At this stage a few cells should be 

 withdrawn with a pipet, placed on a sUde, 

 covered, and examined under a high power 

 of the microscope. Each cell should show 

 the nucleus clearly stained dark red with 

 a faint pink cytoplasm; if they appear too 

 dark, a small drop of acetic acid should be 

 added to the tube, mixed with the alcohol, 

 and poured off after five minutes. The 

 washing is continued until the alcohol no 

 longer smells of acetic acid. It is easy to 

 overdifferentiate at this point and unless 

 the cells are grossly overstained, it is 

 better to take them through without fur- 

 ther differentiation occasioned by the 

 wash in alum solution which thej^ had im- 

 mediately after staining. The 70% alcohol 

 is now replaced with absolute alcohol in 

 which the cells should be thoroughly 

 stirred and then left overnight. A second 

 change of absolute alcohol should be 

 given; this should not be poured off but 

 should be withdrawn with a pipet, so as to 

 leave the cells accumulated in the least 

 possible quantity of absolute alcohol at 

 the bottom of the tube. The tube is then 

 filled with benzene and left until the cells 

 have again fallen to the bottom, when the 

 benzene is withdrawn and replaced with 

 fresh benzene, which is again replaced. 

 The cells, which are now lying at the 

 bottom of the tube in not more than a drop 

 or two of benzene, should be covered with 

 three or four drops of a strong solution of 

 Canada balsam in benzene. The specimens 

 should now be stirred up and left for an 

 hour or two until they are thoroughly 

 permeated with the balsam, a drop of 

 which may then be removed, placed on a 

 sUde, and covered. By this method, as 

 many slides may be made as there are 

 drops of balsam; and, if the cell concentra- 

 tion has been kept reasonaljly heavy, it is 

 usually better to use a very small {% inch) 

 covershp in order to get as many slides as 

 possible. A preparation of this size should 

 contain two or three hundred cells and will 

 give the student an excellent picture of all 

 of the cell types found in hydra. 



