7 



85 



sudden rise of temperature may, it need hardly he said, prove fatal lo the cultures, but 

 we have also seen cases (e.g. with Bacillus bulgaiiciis) where a sudden fall in temperature, 

 of only 5°, occasioned a serious weakening. 



In order to determine whether the bacteria in the keeping cultures were still in pos- 

 session of their full vitality, we compared the rate of growth with lliatinfreshly reinoculated 

 cultures. In the case of lactic acid bacteria growing in milk, all that is needed is to deter- 

 mine the length of time which elapses before the milk coagulates, and determine the quan- 

 tity of acid formed when this has attained its maximum, which will always be the case 

 after 14 days at 30°. The metiiod, however, is not without its sources of error. Ev'en where 

 the sowing out is done throughout with the same needle, it is impossible to avoid sowing 

 more cells at one time than at another, and what is worse, the cells themselves may 

 exhibit marked individual difTerences. Consequently, as will be seen from Table I, we 

 do not always find, as might be expected, an even decrease of vitality in course of time. 



For producing pure cultures of lactic acid bacteria and, investigating the same, we 

 likewise used substrates containing the mentioned nitrogen sources and salts. They dif- 

 fered from the keeping substrates only in containing more sugar. The sugar broth to be 

 used for determination of the quantity of acid formed should thus contain at least 2% sugar 

 as some of our strains can form over 1.5% lactic acid therein. It is also less easy to over- 

 look any development of gas when the substrate is not too poor in sugar. A slight devel- 

 opment of gas is best observed by close sowing in tall agar tubes (Burri's tubes). By using 

 certain species of sugar and litmus for the plates, we succeeded in isolating the species 

 which could ferment the sugar species in question. 



In order to isolate as far as possible all the lactic acid bacteria found in a starting 

 material, it was sown out both in tall agar tubes and on gelatin plates, so that both aero- 

 bic and anaerobic forms might develop. Besides neutral, sugar-containing gelatin (S. G.), 

 we also used alkaline sugar-free gelatin (A. G.) on which only certain species of lactic 

 acid bacteria grow strongly. We also had recourse to various enrichment methods. In 

 order to get at those which thrive at particularly high or low temperatures, the starting 

 material was added to sterile milk, and left to stand for a time at the temperatures in 

 question. The heat-resisting species of the milk we obtained by pasteurising it in various 

 ways, and its acid-resisting species by adding greater or smaller quantities of acid (lactic 

 acid or acetic acid) before placing to stand at the respective temperatures. 



The raw cultures obtained at the first spreading were cleansed by continued spreading 

 until all the colonies showed the same appearance and contained uniform cells. That the 

 pure cultures thus obtained really were pure cultures was apparent from the work itself, 

 which consisted in a constant checking of the isolated strains. As long as these remained 

 uniform year after year, and in particular, constantly exhibited the same negative 

 qualities, there can be no doubt as to their purity. Single cell cultures are only practicable 

 where but a few species of bacteria have to be dealt with; not, as in the present case, where 

 the object is precisely to study the greatest possible number of strains. As a matter of 

 fact, we had really no practicable method of obtaining single-cell cultures when I commenced 

 the work in 1907; it was not until 1914 that Gerd.\ troili-Petersson published a modi- 

 fication of the BuRRi Indian ink method, suitable for lactic acid bacteria i). 



•) Centralblatt f. Bactériologie, II. Abt. Bd. 42, p. 526. 



