526 



METHODS AND FORMULAS 



MS 11.0 



chromate cleaning solution, thoroughly 

 rinsed in tap water, soaked for at least an 

 hour in two changes of large volumes of 

 distilled water, and then dried under such 

 conditions that dust cannot reach them. If 

 either bottle has previously had its stopper 

 greased for any purpose, it is better to 

 reject it outright than to endeavor to clean 

 it. 



In the first bottle, place 10 to 15 milli- 

 liters of Mann's 1894 osmic-dichromate 

 fixative, whjch will be found under the 

 classification F 1300.0000 in Chapter 18. 

 In the other bottle place a layer of about 

 3 milhmeters of 2% osmic acid. Before 

 pouring the osmic acid from the bottle in 

 which it is kept, wipe the neck of the bot- 

 tle with a rag soaked in alcohol, and then 

 rinse off the alcohol with distilled water, 

 which is subsequently dried off with a hnt- 

 less cloth. This is necessary, since the os- 

 mic acid will become reduced on the 

 surface of any organic material. It is also 

 necessary to provide six small dishes of 

 about 10- to 15-milhliter capacity, filled 

 with triple-distilled water, for the inter- 

 mediate wash between the two reagents. 

 These dishes should be as clean as pos- 

 sible, but need not be chemically clean, as 

 must those used for the fixative and for 

 the stain. Also required are a chemically 

 clean pipet of the eye-dropper type, a 

 sharp scalpel, and a pair of very fine 

 pointed forceps. If the latter can be of 

 stainless steel, so much the better, but it is 

 not absolutely essential in this technique. 



Next, remove the ovary from the earth- 

 worm. First, identify the female genital 

 aperture in segment fourteen. This estab- 

 lishes the ventral side of the earthworm. 

 Then wrap the earthworm round the fore- 

 finger, holding its back end between the 

 first and second fingers, so that the genital 

 aperture is towards the tip of the finger. 

 The worm will then be lying on its left 

 side, provided the operator is right-handed 

 and thus has the worm in his left hand. 

 Next, take a sharp scalpel and make an 

 incision covering two segments posterior, 

 and one anterior, to the fourteenth seg- 

 ment about one miUimeter to the left of 

 the genital aperture. Apply considerable 

 pressure and spread the hps of the wound. 

 The ovary will then appear as a small. 



white, pear-shaped body, which can be 

 removed without difficulty by taking hold 

 of it with fine forceps, just at the point of 

 its insertion, and pulling gently. Now 

 lay the ovary against the inside of the 

 stoppered bottle containing the osmic- 

 mercuric mixture, placing it against the 

 side of the bottle, a good centimeter above 

 the level of the liquid. Under no circum- 

 stances should the tip of the metallic for- 

 ceps be brought into contact with the 

 liquid. The ovary will adhere to the glass 

 surface, thus permitting the withdrawal 

 of the forceps, and it is then only necessary 

 to shake the tube gently, so that the ovary 

 is washed into the fixative. It should re- 

 main in this hquid from a half to one hour 

 (the time is not critical) and should be 

 shaken gently at intervals. 



After fixation the ovary should be re- 

 moved with the pipet, together with the 

 least possible quantity of fixative, to one 

 of the dishes of wash water. The pipet 

 should then be used to suck water in and 

 out rapidly, so as to mix the contents of 

 the dish. After about five minutes the 

 ovary is removed to the next dish, again 

 being thoroughly rinsed backward and 

 forward, and should remain in each dish 

 for about 30 minutes, with occasional agi- 

 tation. It is better, but not absolutely 

 essential, that it should remain in the final 

 wash water overnight. Remember that if 

 any of the osmic-mercuric mixture is taken 

 over into the osmic stain, there will be a 

 tendency to overfixation, which will make 

 the ovary brittle. 



When the ovary has been sufficiently 

 washed, transfer it to the osmic acid using 

 the same technique which was used to 

 place it in the original bottle; that is, lay 

 it against the inner wall of the bottle and 

 then remove the forceps. This avoids car- 

 rying over any water and thus diluting 

 the osmic-acid solution. There should be 

 enough osmic acid in the bottle to cover 

 the ovary so that the outline of the tissue 

 shows on the surface. The specimen is now 

 placed in a cupboard in the dark for from 

 ten days to two weeks; but it should be 

 examined daily to make sure that no con- 

 tamination is causing the reduction of the 

 osmic acid anywhere except on the ovary. 

 If, in any of these examinations, the osmic 



