MS 30 



METAL STAINS 



543 



brain connective tissue, with the result 

 that these techniques are commonly em- 

 ployed for the demonstration of spiro- 

 chetes in tissues and in smears. In general 

 the techniques of class MS 33 are more 

 certain and more accurate than those of 

 the last class, in those cases in whicli it is 

 desired to show a specified structure, but 

 are less useful when it is desired to secure 

 a good general stain of nervous tissues. 



The final class, here designated as MS 

 34, is known sometimes as the "dichro- 

 mate-silver method," though other sub- 

 stances than dichromate have been em- 

 ployed. The techniques are experimental, 

 yielding the most brilliant results when a 

 successful impregnation is obtained, but 

 for which it is almost impossible to specify 

 conditions leading to success. Tissues are 

 fixed for varying lengths of time in either 

 potassium dichromate, dichromate-osmic 

 acid, or dichromate-formaldehyde mix- 

 tures. The length of time is entirely crit- 

 ical, but results cannot be predicted or 

 even reproduced with certainty. For this 

 reason, it is usual to start with a large 

 number of pieces in the prehminary fixa- 

 tive, remove groups of these to silver at 

 frequent intervals, and extract from each 

 group a number of pieces after varying 

 times in the silver. Among the numerous 

 pieces thus treated, one may be found 

 which will show tlie required condition. 

 Tliis condition is curious and depends 

 entirely upon the fact that there is not, 

 as one might anticipate, a uniform precipi- 

 tate of silver chromate or dichromate 

 throughout the tissues. In certain cases, 

 under conditions which it must again be 

 emphasized cannot be anticipated, iso- 

 lated nerve cells, with all their dendrites, 

 will become impregnated with a dark- 

 colored precipitate, while neighboring cells 

 and neighboring dendrites will remain 

 entirely unaffected. This is undoubtedly 

 the best method by which the structure 

 of such cells may be demonstrated. Un- 

 successful impregnations show either a 

 uniform granular precipitate throughout 

 the whole tissue or else no staining of any 

 kind whatever. The nature of the material 

 precipitated onto the cell is unknown, 

 though it has the pecuhar property of be- 

 ing unstable unless exposed to oxygen. For 



this reason such preparations cannot be 

 moimted in the conventional manner un- 

 der a coverslip, but must be placed upon a 

 slide and varnished either with balsam, 

 dammar, or one of the special media 

 recommended. 



The fugitive nature of the stain pro- 

 duced by this last class, and the moder- 

 ately fugitive nature of those obtained 

 from the previous two classes, may be im- 

 proved in either of two ways. The silver 

 complex may be changed into a gold com- 

 plex, or quite possibly to a colloidal dis- 

 persion of metallic gold, by a process 

 analogous to photographic toning, in 

 which treatment by gold chloride, either 

 alone or in some combination, results in 

 the replacement of silver with gold. This 

 technique may be apphed to any material, 

 but in the formulas which follow it has 

 only been specified if it is indicated by the 

 original author. The second, and more 

 satisfactory, method of securing perma- 

 nency is to change the silver complexes, 

 whatever they may be, to metallic silver 

 itself. There are many methods by which 

 this can be acliieved, the simplest being 

 to expose the sections to a weak (10%) 

 solution of hydrobromic acid until the 

 silver complex has been changed to silver 

 bromide. The sections are then exposed to 

 bright Hght to render the bromide un- 

 stable, and this compound is then reduced 

 to metalUc silver with ordinary photo- 

 graphic developers. Though Globus pro- 

 posed the use of hydrobromic acid for 

 quite another purpose, this technique is 

 often associated with his name. 



The most important consideration in 

 securing successful results by any silver 

 technique is the absolute purity of all the 

 ingredients used and the absolute cleanli- 

 ness of the glassware employed. Ordinary 

 distilled water is insufficiently pure ; triple- 

 distilled water should be used. In many in- 

 stances the shghtest trace of an impurity 

 at any stage of the proceedings will com- 

 pletely wreck what may be several weeks 

 of subsequent work. The use of com- 

 mercial formaldehyde, for example, in 

 prior fixation should be avoided, and the 

 grade sold as "analytical reagent" should 

 be used. Another important step is to re- 

 move all traces of one solution before plac- 



