464 



SCIENCE 



[N. S. Vol. XLII. No. 1083 



Probably nothing else in this comparison ia 

 so significant as the disagreement in regard to 

 the spore production and the corresponding 

 high thermal death point of our cultures. 



Beyerinck, in his original observations on 

 Azotohacter croococcum, failed to detect the 

 presence of spores. Nerertheless, in 1911 E. 

 Mencl^ demonstrated their presence in this 

 species. It was recognized early in the study 

 of Azotohacter that they are very resistant to 

 drying and other adverse conditions, which 

 fact aroused the suspicion that spores are pro- 

 duced. The morphological irregularity and 

 change of form under different cultural condi- 

 tions obscured the true nature of the case until 

 the date mentioned above. If all Azotoiacter 

 are to be regarded as one species, my observa- 

 tion of spores is, of course, only a repetition 

 of observations made recently by several inves- 

 tigators. 



In the cultures with which I worked the 

 organisms attained very great size, and showed 

 in many cases a striking resemblance to bud- 

 ding, similar to that observed in yeast cells. 

 At such times the cell is well filled with re- 

 fractive bodies which do not stain readily with 

 the analine dyes. Such bodies have generally 

 been looked upon as fat, but the fact that by 

 special effort they can be stained with methy- 

 lene blue led to some doubt as to their fatty 

 nature. Efforts were made to stain these bodies 

 with the ordinary fat stains, such as Soudan 

 3 and Sharlac red, but the results were all 

 negative. Azotohacter were then grown on 

 potato in suiScient quantities to secure a 

 sample large enough for ether extraction. 

 Potatoes were cut in thin slices and sterilized 

 in petri dishes, and then inoculated. The 

 growth was excellent, and a considerable mass 

 of this was secured by scraping from the sur- 

 face. 



The material thus secured was placed in a 

 separatory funnel and treated with pure ether 

 for twenty-four hours without heat. The 

 funnel was shaken several times during the 

 extraction and then allowed to stand for sedi- 

 mentation. Several cubic centimeters of the 

 clear solution were then drawn into a weighed 



I Arch. Protistenk., 22 (1911), No. 1, pp. 1-18. 



platinum dish and evaporated to drjmess. It 

 was found that a sediment detectible by weight 

 was left in the dish. This may have been a 

 mixture of fats, gums and resins, or possibly 

 any one of these. 



The funnel in which the extraction was 

 made was set aside with the residue of the 

 bacterial mass and a thin layer of ether which 

 overlay the mass. No attention was given to 

 it for about two weeks. When it was examined 

 there was a brownish layer on the surface of 

 the ether. This layer gave one the impression 

 that it consisted of a bacterial growth. Ether 

 has, of course, been regarded as a disinfectant, 

 and although it has never been thought of as 

 having great germicidal power, yet in a high 

 concentration one would certainly be surprised 

 if it should not be found sufficient to inhibit 

 all growth. 



Cover-glass preparations were made from the 

 scum on the ether in the funnel referred to, 

 and it was found to contain large numbers of 

 bacteria. These might have come from the 

 original mass from the potato and been dead 

 at the time they were taken from the ether. 

 At the same time that the microscopic prepara- 

 tions were made, however, inoculations on agar 

 were placed in the thermostat for incubation. 

 At the end of twenty-four hours, when the agar 

 tubes were examined, they were found to have 

 a good growth on them, stains from which 

 showed that it consisted of Azotohacter. It 

 was evident, therefore, that practically pure 

 ether had not killed this organism. It was 

 also made reasonably certain by this observa- 

 tion that the scum on the ether in the funnel 

 consisted of living Azotohacter. 



It remained to be shown that the organisms 

 were actually multiplying in the solution. 

 This was accomplished in the following way: 

 Small Ehrlenmeyer flasks were supplied with 

 sufficient Squibb's pure ether to make a thin 

 layer on the bottom of the flask and a small 

 particle of the potato culture was introduced 

 into them. These were set aside at room tem- 

 perature for development, and at the end of a' 

 week or ten days a decided growth could be 

 detected. 



Another method that was employed to test 



