52 



THE ART OF MAKING MICROSCOPE SLIDES 



Narcotizing 



quantity of narcotic at the beginning and 

 increase tlie quantity later, adding the 

 fixative only after the cessation of move- 

 ment. This is easy to judge in the case of 

 motile forms, which may be presumed to 

 be naiTotized shortly after they have 

 fallen to the bottom, but in the case of 

 sessile forms it is necessary to use a fine 

 probe, preferably a hair, to determine the 

 end point of narcotization. 



Recommended Narcotics and Fixatives 

 for Specific Objects 



It must be pointed out that the primary 

 purpose of fixing an object before making 

 a wholemount is to retain as nearly as 

 possible the natural shape. The fixative 

 selected should, therefore, contain an im- 

 mohilizing agent as well as a hardening 

 agent. Gray 1933 (11360, 53:14), in a dis- 

 cussion of the principles governing the 

 selection of fixatives, came to the conclu- 

 sion that there were only two good im- 

 mobihzing agents. These were heat and 

 osmic acid. It is therefore necessary, when 

 dealing with highly contractile or imper- 

 fectly narcotized animals either to select 

 a fixative containing osmic acid (Chapter 

 18, F 1000) or to heat the fixative. Neither 

 osmic acid nor heat are good hardening 

 agents and should not, therefore, be used 

 alone. The best hardening agents for ob- 

 jects which are subsequently turned into 

 resinous wholemounts appear to be chro- 

 mic acid and formaldehj'de, used either 

 singly or in combination, and these solu- 

 tions are usuall}'' acidified with acetic acid 

 to assist in the preservation of internal 

 structures, particularly nuclei. Reference 

 to the classification of fixatives at the be- 

 ginning of Chapter IS will show a large 

 number of solutions fulfilling these re- 

 quirements and no specific recommenda- 

 tion need be made here. Mercuric chloride, 

 particularly in the solution of Gilson 1898 

 (Chapter 18, F 3000.0014), is another 

 good fixative to use before wholemount- 

 ing. The following recommendations, 

 drawn largely from Gra}- 1935 (Microsc. 

 Rec, 35:4), and Gray 1936 {Microsc. Rec, 

 37 :10), are to be taken only as suggestions 

 representing the author's opinion and 

 should be used as a basis for further 

 experiment. 



NoNCONTRACTiLE Protozoa. These do 

 not require nnrcotization and may be fixed 

 directly in a weak solution of osmic acid. 

 The writer, however, much prefers his own 

 technique (described at the end of 

 this chapter) for the handling of these 

 specimens. 



Individual Contractile Protozoans, 

 These are very difficult to handle. Ten per 

 cent methanol is quite a good narcotic for 

 Dileptus, but 1 % hydroxylamine seems 

 better for Spirodomum. It is the writer's 

 practice to try new forms with the follow- 

 ing narcotics in the order given: 10% 

 methanol, 1% hydroxylamine, 1% ure- 

 thane, AF 51.1 Hanley 1949, AF 51.1 

 Rousselet 1895, and AF 51.1 Cori 1893 

 (Chapter 19). There are many forms, how- 

 ever, which do not respond to these nar- 

 cotics and of which it appears almost im- 

 possible to make a good wholemount. 



Individual rhizopods, as Amoeba and 

 Difflugia, are best fixed to a coverslip in 

 the following manner. Take a clean cover- 

 slip and smear on it a very slight quantity 

 of fresh egg albumen. The solution of 

 Mayer 1884 (Chapter 26, V 21.1), which 

 is often recommended for the purpose, 

 should be avoided, for the glycerol and 

 preservative included in it inhibit the ex- 

 pansion of the animals. Each individual 

 protozoan is placed in the center of a 

 covershj) and left to expand. While this is 

 going on a flask (or kettle) is fitted with a 

 cork. Through this cork is inserted a glass 

 tube the outer end of which has been 

 drawn to a fairly fine point. The water in 

 the flask is boiled to produce a jet of 

 steam. As soon as the protozoan is satis- 

 factorily expanded, the coverslip is picked 

 up very gently and the underside passed 

 momentarily through the jet of steam. 

 This instantly hardens the protozoans in 

 position and at the same time cements 

 them to the coverslip through the coagu- 

 lation of the egg white. The coverslip 

 should then be transferred to any standard 

 fixative solution for a few minutes before 

 being washed and stored in alcohol. 



Among the Suctoria, Acineta and Den- 

 drocometes may be prepared by placing 

 them in a good volume of water, sprink- 

 ling menthol crystals on the surface, leav- 

 ing them overnight, and then adding suffi- 



