FIXING AND HARDENING 21 



In such material as mammalian testis or molluscan ovotestis, it is 

 certain that more tubules are properly fixed than in any prepara- 

 tions hitherto made over a period of many years by the writer. 

 It is remarkable that Hirsch and Jacobs {Zeit. f. Zellf., 1926) 

 came to the conclusion that in just such mixtures as were used 

 by Young, Boyle and the writer, the addition of salt was of no 

 value. Hirsch* and Jacobs used crayfish mesenteron in which the 

 difficulty of penetration is obviously not so great as in liver or 

 testis, for example. We agree with Hirsch and Jacobs that cells 

 on the outside of a tissue are not visibly better fixed by fixatives 

 containing NaCl, but this is not true of the cells or tubules more 

 deeply situated. Therefore we advocate trying the addition of 

 0-75 to 0-9 per cent. NaCl to chrome-osmium, chrome-formalin and 

 formalin mixtures. There is, however, one point that should be 

 mentioned. Boyle has found that Flemming's and Champy's 

 fluids disintegrate in a few weeks when salt is added. Under the 

 separate paragraphs giving formulae this matter has been further 

 treated. 



Since the addition of NaCl to fixatives of the chrome-osmium 

 formalin types improves the penetration, it would seem unfruitful 

 to review some of the recent work on penetration of single sub- 

 stances in water which has been done without the addition of 

 NaCl. Refer, for instance, to the interesting and valuable paper 

 by Miss B. M. L. Underhill (J. Roy. Micr. Soc, lii, 1932). 



32. Period of Fixation. The beginner is often puzzled by the 

 varying directions given as to length of time the fixative is to 

 be used. For example, the variations of Flemming's fluid are used 

 sometimes for one day up to as long as a week. The late Professor 

 Doncaster always used Flemming acetic for one hour only. The 

 explanation is, of course, that if you merely want chromosomes, 

 the chromic and osmic will fix proteids in a short time — ^while 

 the worker who wants to study the fats * must needs prolong the 

 immersion until all these subtle bodies are sufficiently hardened 

 to resist the solvent effects of alcohol and xylol or toluol. 



In the same way there are many failures in the Regaud method, 

 because the proper times are not adhered to strictly. Most of 

 the tribe of corrosive acetic alcohol fixatives do quite well when 

 used for an hour or so ; it is conventional to use most ordinary 

 fixatives overnight. Some well-known mixtures, such as Da 

 Fano's formol-cobalt and Aoyama, are really good preservatives, 

 and we constantly use them to store material. The beginner 

 must read the directions for time of fixation given under each 

 formula. 



For routine zoological work Bouin's picro-formalin-acetic is 

 recommended. Gilson-Petrunkewitsch is a fixative which is easy 

 to work and generally better than corrosive sublimate acetic. 



* But see § 734. 



