(505 ) 
Of a culture, grown on a solid nutrient substratum with copious 
access of air, some material is put on a glass-slide and killed in 
such a way that eventually present enzyme remains unhurt. This 
may be done by immersing the material in strong alcohol, in 
which it should remain at least 24 hours to be quite sure that the 
microbes are killed, or by exposition to ether-, aleohol- or chloroform- 
vapour +). In the latter case the microbe-material is placed in a 
glass-box beside a vessel with chloroform, where ferments moulds, 
and most bacteria die after '/, to 1 hour already, while the enzymes 
in the cells remain unhurt. 
If a small lump of killed microbes is put in an indican-solution, 
poured out to a thin layer in a white porcelain vessel floating on 
water of circa 45° C., then only those microbes will become blue, 
which contain indigo-enzyme, while those, acting by katabolism, don’t 
cause decomposition. If in the latter case not all but only most of 
the microbes have been killed, there will at first be no manifest 
decomposition, but it will set in as soon as the living individuals 
have sufficiently multiplied, which is at the same time a good control . 
of the experiment. 
The microbes containing enzymes can be dried and powdered 
after killing and such “crude enzymes’, when kept dry, preserve 
their activity very long. By the little dissolubility of the indigo-enzymes 
in water, glycerine and salt-solutions, it was not possible by extracting 
the crude enzymes and precipitating with alcohol, to obtain more 
active preparations from them. 
It has been proved that all examined bacteria, blastomycetes 2) 
and moulds, which decompose indican, do not effect this by enzymes 
but by katabolism, while among alcohol-ferments both cases occur. 
So indican is decomposed katabolically by Saccharomyces ludwigi 
and Monilia candida, while Saccharomyces sphaericus *), S. apicu- 
latus, S.muciparus*), S.tyrocola®) contain indigo-enzymes. One of 
1) In aleohol vapour many microbes die sooner than in strong alcohol, this having 
a water absorbing power and thus acting protectingly. 
2) Blastomycetes have the shape of yeast-cells but produce no alcohol. To these 
belong e.g. the red “yeasts” Blastomyces glutinis, B. roseus, B. granulosus (of which 
the last colours deep blue with jodine), and which all decompose indican vigorously. 
3) Under this name, given by NAcext, | united the various forms of aethyl-acetate 
yeast. (Verhandelingen 5e Natuur- en Geneeskundig Congres te Amsterdam, 1895, p. 301). 
4) This name I give to a saccharose-yeast, very common in pressed yeast and which 
does not ferment maltose, 
5) §. tyrocola is a lactose-yeast, not rare in Edam cheese, [ts cultures on wort- 
gelatine are sometimes rose-coloured. 
3 
Proceedings Royal Acad, Amsterdam. Vol. IL. 
