PRACTICAL FIXATIVE SOLUTIONS 59 



Use at about 5° C. Fix pieces not exceeding 1 mm in thickness for 

 1 to 4 hours. Wash out in several changes of distilled water; or pass 

 directly to 50% or 70% ethanol. 



Palade's fixative and variants of it have been enormously used 

 in electron-microscopy, with great success. Since Michaelis's 

 buffer does not precipitate calcium from its salts, it is legitimate 

 to substitute 10 ml of calcium chloride solution for the 10 ml of 

 distilled water used in making up the buffer. ^^ The concentration 

 of this solution may be anywhere from 1% to 10% of the 

 anhydrous salt; the final concentration will be from 0-1 % to 1 %. 

 Calcium chloride is particularly valuable in the study of lipid 

 droplets. 



60, 23 



We turn now to fixative mixtures. 



CLARKE (1851)«2 



Absolute ethanol . . . . .3 vols 

 Acetic acid (glacial) . . . .1 vol 



(The constituents are not ionized and pH has therefore no meaning.) 

 Wash out in absolute ethanol. 



The formula for this fixative was given by the English neurolo- 

 gist no less than 35 years before Carnoy, the celebrated Belgian 

 cytologist, first published it.^^ In the intervening period it was 

 familiar in microtechnique as die Clarke' sche Vorschrift.'^^ This is 

 the most ancient of all fixative mixtures commonly used in micro- 

 technique today. 



The advantage that may be gained by mixing certain primary 

 fixatives cannot be more vividly illustrated than by a considera- 

 tion of Clarke's hardy centenarian. By themselves, ethanol and 

 acetic acid are both very bad fixatives (see pp. 33 and 53), but 

 each compensates neatly for the defects of the other. The shrink- 

 age that ethanol alone would cause is off"set by the swelling action 

 of acetic acid; the latter stabilizes nucleoproteins, which are left 

 unfixed by the former; acetic acid fixes neither cytoplasm nor 

 nuclear sap, but both are fixed by ethanol (the former in a rather 

 coarse coagulum). The mixture is admirable in routine micro- 



