933 
red or jet-black spots will originate. 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 Actinomyces. Streaks on new culture plates of the said com- 
position to obtain a pure cuiture, give the surprising result, thatthe 
organism can vigorously grow on the tyrosin but produces no pig- 
ment 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 Actimomyces and prevents them from producing spores, which 
does take place’ on that part of the mycelium, which develops out- 
side the bacterial cover. If from this layer the bacterium 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 beside the tyrosin, for which glucose 
and peptone proved particularly useful. On the other hand, additions 
of an ammoniursalt 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 be- 
tween. 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-blaek wherever they were near 
Actinomyces. The following must therefore take place: Actinomyces 
decomposes the tyrosin and produces from it a colourless chromo- 
gene which is converted into melanin by the bacterium and easily 
61 
Proceedings Royal Acad. Amsterdam. Vol. XY. 
