1890.] MICKOSCOPICAL JOURNAL. 161 



BACTERIOLOGY. 



By V. A. MOORE, M. D. 



WASHINGTON, D. C. 



Methods of Staining Spores.* — The spores of bacteria were 

 first observed and described, but not rightly understood, by Perty in 

 1853. Then Pasteur made a sharp distinction between the biology of 

 an organism and its spore without quite solving the question morpho- 

 logically. Colin was the first to describe biologically and morphologi- 

 cally the formation and germination of spores. Further peculiarities 

 were observed by Koch, Brefeld, Buchner, and especially Prazmowski, 

 who clearly described the different forms of the germination of spores. 



The spores in the unstained condition appear, especially in the hang- 

 ing-drop, as strongly refracting round or oval bodies either within the 

 less refractive bacteria, or free near these. Sometimes they are situ- 

 ated near the middle, sometimes at the end. The cells in which they 

 appear are sometimes unaltered, sometimes peculiarly swollen. Since 

 condensed bacteria-protoplasm, according to Prazmowski, strongly re- 

 fracts light, it is to be concluded that a body still more strongly light- 

 refracting is to be regarded as a spore. 



Here belong, together with the foregoing morphological changes 

 which regularly appear under certain biological conditions in spores, 

 their great resistance to chemical agents, and especially to high tem- 

 perature, and the fact that in the use of watery or dilute alcoholic so- 

 lutions they are not stained, but appear as unstained refracting spaces 

 within the stained bacteria. 



An accidental observation showed how the spores could also be 

 brought to view stained. Koch saw, in staining the tubercle bacilli 

 with aniline-water-methyl-blue, that the spores of a species of large 

 bacteria were stained blue at the same time, while the bacteria them- 

 selves were stained brown by the subsequent treatment. Gaft'ky w^as 

 not able to stain the spores of other bacteria in the same manner. On 

 the contrary, Neisser succeeded in staining the spores red and the bacilli 

 blue when he used warm aniline-water-fuchsin and subsequently stained 

 with methyl-blue. Buchner discovered a means for the isolated stain- 

 ing of spores. Because the staining of the living bacteria was not suc- 

 cessful, but those killed by drying and heating were readily colored, Buch- 

 ner thought that the reason the spores were not stained was because of 

 the greater resistance of the spore membrane. He endeavored, there- 

 fore, to destroy the membrane of the spores of the bacillus subtilis, and 

 thus make them accessible to the staining fluid. In this manner he 

 succeeded in staining spores in dried cover-glass preparations which 

 had been heated from one-half to one hour at a temperature of 210° C. 

 in a dry-oven or one hour in a steam-kettle at 120° C. A successful 

 result was also obtained when the preparations were dipped in concen- 

 trated English sulphuric acid for fifteen seconds', and afterward care- 

 fully washed, or when they were subjected for a longer time to a con- 

 centrated solution of caustic soda. In preparations thus treated, es- 

 pecially in the use of methyl-blue the spores alone are stained while 

 the bacteria themselves no longer take up color. 



* Hueppe's methods of Bacteriological Investigation translated by Biggs, p 74. 



