254 
SUMMARY OF CURRENT RESEARCHES RELATING TO 
structure of which becomes materially altered by the slow contraction 
of the imbedding mass, and the other with material which has been 
previously well hardened and is in itself dense, so that the defects 
alluded to are unperceived. 
(4) Staining' and Injecting. 
Kiihne’s Methylene-blue Method of Staining Bacteria.* — This 
method is especially recommended for staining bacteria in sections of 
animal tissues, although it is equally applicable to cover-glass prepara- 
tions made from fresh tissues. The usual differences in the method of 
staining cover-glass preparations and sections are to be observed. 
The advantages to be derived from this method are found in its being 
applicable to all known forms of bacteria. It eliminates the use of 
special stains for certain micro-organisms where only their presence is 
to bo demonstrated. It possesses superior powers of differentiation 
between bacteria and the tissue elements. The method as given by 
Dr. Kiihne | is essentially as follows. 
The sections which have been cut by the ordinary method (although 
Dr. Kiiline recommends the freezing microtome for this purpose), are 
transferred directly from alcohol to a watch-glass containing carbol- 
methylene-blue. (1) The sections should remain in this staining fluid 
for about half an hour ; some bacteria, such as the bacillus of leprosy, 
requiring a longer time, one to two hours. If the sections remain in the 
staining fluid for a much longer period, the differentiation between the 
germs and tissue-elements becomes more difficult. 
After staining for the desired length of time, the exact period of 
which will have to bo determined by test experiments for the different 
germs and tissues, the sections are rinsed in clear water and then 
placed in acidulated water (2) until they become a pale blue. They are 
then washed in a weak watery solution of carbonate of lithium (3), and 
again placed in clear water. This part of the procedure is very 
important, and to insure good results should be performed with much 
care. The time that the sections should remain in the decolorizing 
agents varies with their thickness, histological structure, and the intensity 
of the stain, making it impossible to give any definite rule to be 
followed. The degree of decolorization can be very nearly determined 
at any moment by moving the sections about in the fluid by means of a 
glass rod. If the section is very thin, or if there are other reasons why 
it should take up very little of the stain, a momentary immersion in the 
acidulated water is sufficient. In all cases where the staining process 
is completed the sections should have a pale blue colour, for if darker, 
the over-stained corpuscles and cell-nuclei of the tissue would obscure 
the bacteria. In cases where it is feared that too much colour has been 
removed in the acid a drop of a saturated watery solution of methylene- 
blue should be added to the lithium-water. 
After the sections have remained in the water for some minutes they 
are dehydrated in absolute alcohol in which, in difficult cases, a little 
methylene-blue may be dissolved, and then transferred to a watch-glass 
* Amer. Mon. Micr. Journ., x. (1889) pp. 259-60. 
t Kiihne, ‘Praktisohe Anleitung zura inikro£kopisclien Nachweis der Bakterien 
iin tierischen Gewebe,’ p. 15. 
