Pasteur's critics maintained that if putrefaction and fermentation be caused solely by microscopic organisms, then these must be found everywhere and in such quantities as to encumber the air. He replied that they were less numerous in some parts of the atmosphere than in others. To prove his contention he set out for Arbois with a large number of glass bulbs each half filled with a putrescible liquid. The necks of the bulbs had been drawn out and hermetically sealed after the contents had been boiled. In case the necks were broken (to be again sealed immediately), the air would rush in, and (if it held the requisite micro-organisms) furnish the conditions for putrefaction. It was found that in every trial the contents of a certain number of the bulbs always escaped alteration. Twenty were opened in the country near Arbois free from human habitations. Eight out of the twenty showed signs of putrefaction. Twenty were exposed to the air on the heights of the Jura at an altitude of eight hundred and fifty meters above sea-level; the contents of five of these subsequently putrefied. Twenty others were opened near Mont Blanc at an altitude of two thousand meters and while a wind was blowing from the Mer de Glace; in this case the contents of only one of the bulbs became putrefied. While his opponents still professed to believe in the creation of organized beings lacking parents, Pasteur was under the influence of the theory of "the slow and progressive transformation of one species into another," and was becoming aware of phases of the struggle for existence hitherto shrouded in mystery. He wished he said to push these studies far enough to prepare the way for a serious investigation of the origin of disease. He returned to the study of lactic fermentation, showed that butyric fermentation may be caused by organisms which live in the absence of oxygen, while vinegar is produced from wine through the agency of bacteria freely supplied with the oxygen of the air. Pasteur was seeing ever more clearly the part played by the infinitesimally small in the economy of nature. Without these microscopic beings life would become impossible, because death would be incomplete. On the basis of Pasteur's study of fermentation, his demonstration that decomposition is owing to living organisms and that minute forms of life spring from parents like themselves, his disciple Joseph Lister began in 1864 to develop antiseptic surgery.