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a drawback, that the shape of the bacteria is not always 

 clearly visible ; therefore a methyleneblue preparation Avas often 

 made at the same time. But since GiNS taught us how to avoid 

 this difficulty, the double-stained preparation will do. 



In some laboratories a second film is made and stained after 

 Gram, thus making use of the property of the diphteria bacillus 

 of being Gram-positive. Wanting to know if the addition of 

 this manipulation, which would not delay the diagnosis but only 

 cause a great extension of the routine work, would be a benefit. 

 Miss Smit has made pure cultures of a great many cases in 

 which diphtheria bacilli were diagnosed by us and investigated 

 their behaviour towards Gram's stain. Only one of them was 

 not immediately stained after Gram. According to these 

 investigations I cannot find sufïicient reason for extending our 

 working-method in this way. 



So the shape and the relations to different stains not being 

 able to procure any new information, let us now have a look 

 at the other characteristics mentioned above. Before proceeding 

 in this direction we must bear in mind that a pure culture of 

 the microorganism we want to examine, is absolutely necessary 

 and also that for investigating these characteristics we want 

 some time, for the bacilli must grow in their new medium in 

 order to show their characteristics. 



In many a case and especially in those of a doubtful nature 

 the diphtheria bacilli or what looks like them, are overgrown 

 by other microorganisms to such an extent, that no distinct 

 separate colonies are formed on the medium. If we want 

 to get a pure culture it is necessary to make a new culture 

 on an other plate of the most suspected places and we have 

 got a separate colony then in at least 20 hours. But this 

 may not always be possible, not seldom we have to repeat 

 this operation and every one, who does this work regularly, 

 knows by experience that the isolation often fails altogether. 



It will be clear now, that there are some cases in which an 

 almost pure culture, suitable for further investigations is obtained at 

 once, but there are a great many cases in which we only 

 succeed in 1 or 2 or more days. Now the question has to be 

 raised as to whether a delay of 24 hours at least, but as a rule 

 of two or more days, gives so much more certainty, that the 



