18 FIXATION PROCEDURES 



SH groups, or sugars, are to be studied, oxidizing agents such 

 as osmic acid must be avoided. If amino groups are to be 

 studied, formaldehyde must be avoided, etc. When enzymes 

 are of interest, the limitations are even more severe. A sub- 

 stance like osmic acid must be avoided completely. 



During the action of chemical fixatives, artefacts may arise 

 due to precipitation, diffusion, and adsorption, and also due to 

 activation and inactivation of enzymes. The endeavour with 

 regard to precipitation is not to avoid precipitation — indeed 

 the whole object of fixation is to secure proper precipitation — 

 but to see that the size of the particles precipitated is less than 

 the resolving power of the light used. Diffusion and adsorption 

 artefacts present a source of great difficulty. One method of 

 detecting these artefacts is to use a variety of fixing agents of 

 widely different physical properties. For example, if the same 

 structure or distribution of a substance is observed with fixatives 

 which are acid, neutral, basic, and anhydrous, it is rather un- 

 likely that the observed cytochemical pattern is an artefact. 

 On the other hand, the use of a wide variety of fixatives is not 

 an absolutely safe guarantee against the formation of artefacts. 

 Moreover, it frequently happens, especially when enzymes are 

 being studied, that the range of fixing agents which can be used 

 without destruction of enzymes is too restricted to permit this 

 method of detection of artefacts. 



When the cytochemical method is applied to the analysis of 

 a physiological activity, e.g., enzyme action, it is very difficult 

 to assign a precise significance to the degree of activity which 

 is observed in the fixed preparation. If one knows the extent 

 of enzyme activity at a particular part of the preparation and 

 can be sure this has not been modified by the fixation procedure, 

 it is still not possible to estimate the degree to which this enzyme 

 is active under physiological conditions. Before that degree of 

 activity can be determined, it is necessary to have a precise 

 knowledge of the physio-chemical environment of the enzyme 

 under physiological conditions. This means knowing what other 

 colloids are present in the immediate environment of the enzyme, 

 since enzyme activation is greatly affected by the presence of 

 other colloidal molecules or surfaces on which the enzyme may 

 be adsorbed. Then it is necessary to know the hydrogen-ion 

 concentration, the ionic strength, the substrate concentration, 



