CHAPTER XXVII. THE STAINING OF PATHOGENIC MICRO-ORGANISMS IN TISSUES Rapid fixation and hardening are requisites for the successful staining of micro-organisms in sections. Alcohol, Zenker’s, mercuric chloride and formol give best results; Müller’s because of its slow action is not good, although formol-Müller’s may be used because of the more rapid fixation with this fluid. In the case of formalin-fixation staining with Weigert-Gram’s method may not give good results unless the sections are oxidized in potassium permanganate solution and then reduced in oxalic acid. (See Staining of Fibrin.) Preservation of the tissue for a long time in alcohol impairs the staining power of micro-organisms contained within it. The tissue should be imbedded preferably in paraffin, as very thin sections must be obtained. The freezing-microtome may be employed and the thinnest sections selected for staining. Celloidin stains very heavily with the aniline dyes and retains the color, so that bacteria in celloidin sections do not stand out very distinctly. On the whole paraffin sections, floated on slide or cover, and fastened by albumin-fixative, give the best results, though for the micro-organisms stained in carbol-fuchsin and decolorized in nitric acid it is best to float the sections directly onto the warm stain without removing the paraffin, and mount without the use of alcohol. This method may be employed for all stains that are taken out by alcohol. The stains used for film preparations are as a rule applicable to sections. The basic aniline dyes, particularly methylene-blue, fuchsin, methyl or gentian violet, kresyl-echt-violett, thionin, and Bismarck brown, either in saturated alcoholic solutions or dilutions of such, or in combination with alkalies, aniline oil or phenol, are usually employed. The various modifications of the Romanowsky method are very useful. The time required for staining in sections is usually much longer than for films; but the staining can often be accelerated or strengthened by warming over the flame or in the incubator. Contrast staining of the nuclei with lithium-carmine or Bismarck brown is advisable after the use of staining methods in which the nuclei are decolorized. Xylol or origanum oil should be used for clearing. I. THE STAINING OF BACTERIA IN TISSUES. According to their staining-reactions bacteria may be very conveniently grouped in three classes: 1, _Staining with Gram-Weigert’s method_; 2, _Not staining with Gram-Weigert’s_; 3, _Staining with the tubercle-bacillus method_ (_acid-resisting_). 1. BACTERIA STAINING BY THE GRAM-WEIGERT METHOD.