Tapwater 100 



Tyrosine o.i 



Natrium carbonate .... o.i 

 Dikalium phosphate .... 0.05 

 Agar 2 



The superfluous water is allowed to flow off the plate, which is cultivated for 

 some days at 30 C. 



It is true that here the tyrosine is at the same time source of carbon and of 

 nitrogen, but the method is now a separative one, as competition is excluded. 



On the second or third day peculiar black spots are seeen to appear around some 

 colonies and slowly extend over a distance of some millimeters 1 ). The black pigment 

 proves able to diffuse only to a rather short distance, whilst the enzyme itself 

 remains bound up with the bacterial bodies as belonging to the endo-enzymes. That 

 here we have indeed to deal with a true enzyme, is more easily shown in the species 

 of the sea than in the fresh-water microbes. To this end some material cultivated 

 on broth agar is killed by the vapour of chloroform, then transported to a culture 

 plate of the above composition, or to a nutrient liquid of the same preparation, but 

 with omission of the agar. At a temperature of 40 C. the black-colouring is then 

 rather quickly perceived but, of course, without development of he germs. As endo- 

 enzymes may be considered as constituents of the protoplasm, it is not surprising 

 that the reactions with such preparations, containing only dead material, are feeble, 

 for the enzyme itself is for the greater part annihilated. Hence, in my opinion, 

 endo-enzymes are best studied when still within the living cells themselves and by 

 considering them as an essential part of the living protoplasm. Taken in this sense 

 tyrosinase may be called a respiration enzyme, and it is remarkable that as a 

 product of repiration, beside the carbonic acid, ammonia is formed, instead of water 

 as in the ordinary respiration. 



In sewage water only a small number of tyrosine bacteria are found per cM 3 . This 

 number may be a little increased by leaving the sewage water for some time at room 

 temperature, then making on plates streaks of the microbes accumulated in the layer 

 at the surface. This microbe layer, very rich in infusoria and flagellates, produces, 

 in particular as it seems in late summer, many more tyrosine bacteria than the sewage 

 water itself. Nevertheless, as said above, it has not been possible to find a really 

 good accumulation method of these tyrosine bacteria, although many trials have 

 been made. 



The black pigment can be prepared in great quantities by cultivating the pure 

 microbes at 30 C. in large Erlenmeyer flasks of the said feebly alkaline solution 

 of natriumtyrosinate with the required anorganic salts. The conversion is relatively 

 slow, so that it is complete only after some weeks, but then a liquid is obtained which 

 may be used as ink. Traces of ferrisalts favour' somewhat the formation of melanine. 



') As the so generally distributed fluorescent bacteria likewise attack tyrosine under 

 production of a light red-brown pigment, there are always found spots of that colour 

 on such culture plates, which can, however, by no means be mistaken for those of 

 tyrosinase. 



