36 STAINING METHODS. 



Ziehl-Neelson Method, for the tubercle bacillus in tissues.- 

 Leave the sections for fifteen minutes in carbol-fuchsin solution 

 (No. 3) ; decolorize in sulphuric or nitric acid, twenty-five-per-cent 

 solution; wash in sixty -per-cent alcohol; place in a saturated aque 

 ous solution of methylene blue for contrast stain; wash, dehydrate, 

 and mount in balsam. 



The following- method of staining 1 sections for the purpose of demon- 

 strating bacteria present in. the tissues is recommended by Pregl (1891) as a 

 substitute for the method of Klihne. The results are said to be excellent, 

 and it is much simpler and more expeditious. 



The sections are made from tissues embedded in paraffin, and are attached 

 to clean glass slides with albumen-glycerin. Or they may be attached to a 

 cover glass by the following method when not embedded in paraffin : The 

 sections, completely dehydrated, are taken out of absolute alcohol on a thin 

 glass cover, upon, which they are extended ; a piece of filter paper is applied 

 to the side of the cover glass to absorb the alcohol, and before the section is 

 completely dry a drop of acetoii-celloidin solution is placed upon it by means 

 of a glass rod. The cover glass is now moved about in the air to promote 

 rapid evaporation of the alcohol, and is then placed in water. The section 

 now remains attached to the cover glass during subsequent manipulations. 

 The aceto)i-celloidin solution referred to is prepared by adding celloidin in 

 small, dry pieces to acetoii until a concentrated solution is obtained. A 

 large drop of this added to five cubic centimetres of absolute alcohol makes 

 a suitable solution for use. This must be kept in a glass-stoppered bottle, and 

 will require to be frequently renewed, as it is not suitable for use after hav- 

 ing absorbed moisture from the air. The acetoii as obtained from dealers 

 contains considerable water and must be dehydrated by adding to it red-hot 

 sulphate of copper. 



The sections, attached to a slide or cover glass by one of the methods 

 mentioned, are stained with Kuhne's carbol-methylene-blue solution, which 

 is dropped upon them from a pipette. Usually they will be sufficiently 

 stained at the end of half a minute to a minute, but in some cases a longer 

 time and the application of heat will be desirable. They are then washed in 

 water and immediately placed in fifty-per-cent alcohol, where they remain 

 until the sections have a pale-blue color with a greenish tinge. They are 

 now completely dehydrated in absolute alcohol and subsequently cleared up 

 in xylol. 



STAINING SECTIONS OF GELATIN STICK CULTURES. Fischl, Weigert, 

 and Neisser have given an account of methods for staining stick cultures in 

 gelatin of non-liquefying bacteria. The object of this is to show the mode 

 of growth and the association of individual cells in undisturbed cultures. 

 Neisser gives the following directions : The gelatin cultures are inoculated, 

 by several punctures, with the microorganism to be studied. When the 

 development is deemed sufficient the cylinder of gelatin is removed from the 

 test tube by gently warming its walls. It is then placed for several days- 

 one to eight, according to its size and thickness in a one-per-cent solution of 

 bichromate of potassium. While in this solution it must be exposed to the 

 light, which causes a change in the gelatin, rendering it insoluble. The 

 gelatin cylinder is thoroughly washed and then hardened in alcohol, first of 

 seventy per cent, and then of ninety-six per cent. It is then cut into suit- 

 able pieces, and these are attached to a cork in the usual manner and placed 

 for twenty-four hours in absolute alcohol. Thin sections may now be made 

 with a microtome, and these are attached to a glass slide and stained by 

 Gram's or Weigert's method or by the use of Loffler's solution (No. 4). The 

 decolorization should be effected by the use of alcohol and not with an acid 

 solution. When Gram's method 'is used decolorize by the alternate use of 

 alcohol and oil of cloves. Clear the preparation with oil of bergamot. 



