( 845 ) 
vaseline; this is preferable to thickened olive oil and diapalm, as 
previously indicated. 
The coverslips are not fastened to the moist chambers by means 
of vaseline, but by a mixture of 20 volumes of vaseline with 1 volume 
of paraffin, which can resist a temperature of 37° without melting. 
I should gladly have limited myself to the above as regards the 
technique of my method, had I not thought it necessary to add 
some remarks concerning a paper by Mr. A. W. NrevwenNuuis, in 
the Proceedings of the meeting of Nov. 26 last, entitled: “Method for 
growing micro-organisms out of a single cell’. I will not refer to 
the principal features of the method, as they are quite the same as 
those of my own. I only propose to consider the objections, which 
NIEUWENHUIS raises against my method, and the modifications which 
he has hence thought necessary to introduce into the more delicate 
manipulations. . 
Niguwennuis applies four tests to an isolation method and says 
there is no method satisfying these tests. The first of these is that 
isolations can he carried out at magnifications of 300 and higher 
(N. uses magnifications of 300 and 350). My method of course complies 
with this test, for all my isolation experiments with bacteria were 
carried out with a */,, oil immersion Lerrz, therefore at a magnification 
of about 1000. 
The second test is that the organism to be isolated should not be 
harmed by either chemical or physical stimuli. As regards chemical 
stimuli, Nrieuwennvis asserts, that when my eye-shaped needles have 
been desinfected with sulphuric acid and ammonia, substances remain 
behind which irritate the cells, and that I myself give the proof of 
this, on p. 115 of my dissertation. In reality however I pointed out 
there, that this is only the case if the sterilisation is not conducted 
properly, i.e. if the needles are placed more deeply in ‘the sulphuric 
acid than in the ammonia, so that the superfluous acid ascends 
towards the point of the needle. I added explicitly: “When I again 
“isolated the bacterium, while avoiding these mistakes, its power of 
“liquefaction was found equally strong after the isolation. The absence 
“of growth of the colonies then caused no more trouble”. That this 
sterilisation should have harmful consequences, I have never been 
able to discover, although I have specially investigated the question 
by making parallel experiments with sterilized and non-sterilized 
isolation needles. Moreover the needle is always washed before use 
in a sterile drop on the isolation coverslip, and beforehand the 
needle may be held for a moment in a tube with sterile water at 
100°, in which any trace of ammonium sulphate, should it have 
