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 sections for the puposs of demon- 
strating bacteria present in the tissues is recommended by reel (1891) asa 
substitute for the method of Kiihne. 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 toa 
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 aceton-celloidin solution is placed upon it by means 
of aglass 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 aceton-celloidin solution referred to is prepared by adding celloidin in 
small, dry pieces to aceton 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 keptin 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 aceton as obtained from dealers 
contains considerable water and must be dehydrated by adding to it red-hot 
sulphate of copper. 
he sections, attached to a slide or cover glass by one of the methods 
mentioned, are stained with Kiihne’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 microdrganism to be studied. When the 
development is deemed sufficient the cylinder of gelatin is removed from the 
test tube by gently warming its walls. Itis 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 percent. It is then cut into suit- 
able pieces, and these are attached to a cork in the usual manner and placed 
fortwenty-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 ef 
alcohol and oil of cloves. Clear the preparation with oil of bergamot. 
