84 



6 



Witte peptone (W) aiiswering to abt. 0.3% N. As will subsequently be seen, this cannot 

 compare with casein peptone (C) i. e. peptonised casein^). In certain cases, yeast extract 

 (Y) i. e. autolysed press yeast 2) proved the best source of nitrogen. It is somewhat dark 

 in colour, however, and we therefore used, as far as possible, C-Agar — and this, it should 

 be noted, with 0.5% N,i. e. with the same quantity of nitrogen as in milk. Witte peptone, 

 which evidently consists for the most part of albumoses, forms strong deposits with acid, 

 so that the stab is no longer visible in old W-Agar cultures of powerful acid formers; the 

 nitrogen sources I have suggested, on the other hand, are free from this disadvantage. 

 They are, moreover, rich in phosphates. A further 2%o dibasic potassium phosphate, and 

 1 "/oo magnium sulphate is, however, added. A sKghtly larges or smaller quantity, of 

 common salt does not as a rule affect the growth of lactic acid bacteria; this point will be 

 further referred to later on. 



For preservation of the bacteria cultures, we have also tried cane sugar solution and 

 other sugar solutions at various concentrations. Such solutions, which have been employed 

 with great success for the preservation of yeast cells, are as a rule not suited to bacteria. 

 An exception, however, is water with 2% soluble starch, which is gradually coagulated 

 by the action of acid. To 10 cm^ starch mixture is added 1 cm^ of the precipitate from 

 a broth culture. The starch solution, however, cannot as a rule compare with C-agar, as 

 far as concerns the preservation of true lactic acid bacteria, but is on the other hand to be 

 preferred to bacteria with surface growth (Coli-Aerogenes bacteria, micrococci, etc.) 

 and in particular to strong ammonia-formers, which rapidly die off in the highly nitro- 

 genous C-agar. 



No less important than the composition of the nutritive substrate is the temperature 

 at which the cultures are preserved. When kept on ice, however, the air is very liable to 

 become so moist as to further the formation of mould3)and we therefore restricted ourselves 

 to water cooling. As long as the temperature is kept below 18°, the great majority of bac- 

 teria can be preserved — without transference — for several years on the nutritive sub- 

 strates mentioned. To make sure, however, we transferred the strains investigated every 

 month, or every alternate month, according as they had proved more or less difficult 

 to keep alive. Bacteria, it should be noted, are very tricky things to deal with, and it was 

 found more than once that a bacteria strain suddenly weakened or died, while the same 

 strain under apparently identical conditions remained unimpaired for a long time. Any 



') 100 gr. (sugar-free) acid-casein was digested for a week at blood temperature with 1 liter water, 

 containing 4, G "lu HCl and 2 gr. pepsin. The solution formed contains, after neutralisation, sterilisation 

 and filtration, abt. 1 "/« N and 1.2 »lo NaCl. 



At öO"", the digestion is completed in 24 hours. The sugar disappears at the same time. The 

 highly acid solution contains abt. 2 "lu N. 



^1 As this laboratory is also used for teaching purposes, and a great deal of work is done witli 

 mould fungi, we have at times had great difficulty in avoiding infection by mould. As the mould 

 formation often proceeds from the labels, these were always moistened with sublimate solution, before 

 being stuck on, and the Fretdenreich flasks were of course sealed with scaling wax. When an agar 

 culture exhibits mould on the surface, it may easily be cleaned bj' letting it stand a moment witli 

 spirit over. Tlie surface, whicii is tlien no longer dusty, is peeled off, in a laj'er not too tiiin, and tlie 

 inoculation can then be made from the lower part of the stab, which is always free from mould 



