112 



In somewhat extensive culture experiments, similar to the above, it may with 

 certainty be expected that at some places brownish red or jet-black spots will ori- 

 ginate. The brown spots are caused by the oxidising action of some common soil 

 bacteria, which produce a red or brown-red pigment from tyrosin ; the black ones, 

 caused by melanin, which will be more exactly considered here, have quite another 

 origin. 



In or near the centrum of these black spots always lies a colony of Actino- 

 myces. Streaks on new culture plates of the said composition to obtain a pure cul- 

 ture, give the surprising result, that the organism can vigorously grow on the tyrosin 

 but produces no pigment at all. A more minute examination shows further, that the 

 black plants of Actinomyces lie under a thin, glassy, transparent layer of fine rod- 

 bacteria. This layer covers like a crust the jet-black sods of Actinomyces and 

 prevents them from producing spores, which does take place on that part of the 

 mycelium, which develops outside the bacterial cover. If from this layer the bac- 

 terium is brought into pure culture, which is easily done on brothgelatin- or broth- 

 agarplates, it proves to be an extremely delicate polar ciliate rodlet, which forms no 

 spores and strongly liquefies culture gelatin. Streaks of the pure culture on a tyrosin 

 plate produces no melanin at all, so that in this respect the bacterium resembles 

 Actinomyces. 



It is obvious that we here have a case of pigment formation reposing on the 

 symbiose of the two organisms. Experience shows that this supposition is right: 

 their combined streaks on a new tyrosin plate produce beautiful black spots of any 

 extension. As they can both be very well grown on better media, such as broth-agar, 

 the experiment is, the first isolation effected, easy and interesting. The experiment 

 may be improved by providing the culture plates with a better source of carbon be- 

 side the tyrosin, for which glucose and peptone proved particularly useful. On the 

 other hand, additions of an ammoniumsalt or of nitrates had no effect. 



In order to ascertain which of the two organisms is the real cause of the 

 melanin production, the following experiment was made. 



On an agar-tyrosinplate of the said composition, parallel streaks of both 

 organisms were drawn with some millimeters, distance between. The result 

 was not dubious ; after a few days the streaks of Actinomyces vigorously developed 

 and covered with snow-white spores, but for the rest were quite colourless. The 

 bacterial streaks, on the other hand, which had developed to a thin, hardly visible 

 transparent layer, had become jet-black wherever they were near Actinomyces. The 

 following must therefore take place: Actinomyces decomposes the tyrosin and pro- 

 duces from it a colourless chromogene which is converted into melanin by the bac- 

 terium and easily diffuses through the agar, evidently without spontaneously oxidising 

 at the air. 



From the foregoing it is clear that Actinomyces, as well as the bacterium, can 

 only be found in garden soil when germs of both species occur in each other's 

 immediate vicinity. To promote this occurrence I have tried first on fit agarplates to 

 grow Actinomyces and later floated them with a tyrosin solution, in which the 

 melanin bacterium was present in so great quantity, that it could develop anywhere 

 on the plate, after the tyrosin had diffused. 



As the various of Actinomyces are very vigorous, polyphagous microbes, which 



