R. Kudo 223 
case, however, was the gall bladder found to harbour any Myxosporidia. The 
same has been true with other organs except the kidney. The observations on 
fresh smears were further confirmed in stained smears and sections. 
L. ohlmacheri was only found in the kidneys. Isolated spores were also 
found m the ureters, cloaca and urinary bladder of the host whose kidneys 
were heavily infected. 
In order to get accurate knowledge of the organisms in the fresh state, 
large numbers of hanging drop preparations were made by mixing fresh spores 
of the Myxosporidian with the fluids taken from the stomach, small and large 
intestines and the urine of R. pipiens, physiological solution, and several 
diluted solutions of pepsin hydrochloric acid, lecithin and sodium glycocholate. 
At the same time, many smears were made on slides from the kidneys, 
infected as well as normal. As my experience taught, in preparing the smears, 
it is very advantageous to have an extremely thin and a more or less thick 
region of film on each slide. In the former part, the number and division of 
the nuclei of trophozoites can be distinctly seen, while in the latter portion 
the general appearance, shape and size of the trophozoites can be clearly 
recognized. 
The rest of the organ was fixed in toto, and sectioned. The serial sections 
varied from 2 to 5/x in thickness. 
The fixatives employed are Schaudinn’s fluid, Flemming’s weak solution 
and Bouin’s mixture, as used in my previous studies except the last named 
one (Kudo, 1916, 1917, 1920a and 1921). The first mentioned fixative proved 
to be most satisfactory. The methods of staining are the same as I have used 
before, i.e. Heidenhain’s iron haemotxylin, Delafield’s haematoxylin and 
Giemsa’s stain. The advantage of mounting Giemsa-stained smears or sections 
in cedar oil for immersion, after dehydrating by acetone and acetone-xylol of 
different proportions, is noticeable compared with those mounted in the neutral 
Canada balsam or any other media. According to my experience, typical 
Giemsa staining in sections as well as smears, has beer preserved perfectly in 
cedar oil for more than three years while those in neutral balsam have hardly 
lasted over six months. 
As to the extrusion of the polar filament, I have recently discussed this 
elsewhere (Kudo, 1921a). It was easily induced by means of potassium hydrate 
or mechanical pressure. Moreover, a weak solution of sodium glycocholate 
gave good results in five to ten minutes in hanging drop preparations. 
The gastric as well as intestinal fluids were collected by means of fine 
capillaries from the digestive tract after the frog was dissected. As the 
manipulation was done in November and December, the quantity of fluids 
obtained were usually so small as to suffice only for two or three preparations. 
For artificial infection per os, small pieces of infected kidney or of a 
sterilized cork which was made porous previously by means of a needle, and 
immersed in an emulsion of fresh spores in physiological solution, were fed 
to the frog. 
15—2 
