Metchnikoff had scarcely recovered from all the emotions caused by his experiments on cholera, which he was still studying, when, in 1894, a work appeared by a well-known German scientist, Pfeiffer, bringing out new facts in favour of the extracellular destruction of microbes.
Whilst studying the influence of the blood serum within the organism and not outside it as his predecessors had done, he had found that cholera vibriones, injected into the peritoneum of a guinea-pig vaccinated against cholera, were nearly all killed in a few minutes and that they then presented the form of motionless granules in the peritoneal liquid. This granular degenescence, said Pfeiffer, took place apart from the phagocytes and therefore without their intervention. Metchnikoff repeated the experiment at once and ascertained that it was perfectly accurate.
The complexity of biological phenomena being very great, he fully admitted the possibility of other means of defence in the organism besides that of the phagocytic reaction. However, this new fact disagreed so much with his own observation, and seemed so isolated, that Metchnikoff supposed an error of interpretation must have been made and tried to throw light upon it. He spent sleepless nights seeking the conclusive experiment which might explain Pfeiffer’s phenomenon.
His excitement was all the greater that he was very soon going to the International Congress at Buda-Pest, where he intended to expose the results of his new researches, and he feared that he should not have time to make all the experiments which he required in support of his arguments. However, the general impression of the Congress was clearly favourable to the phagocyte theory. This is how M. Roux picturesquely described the scene at Metchnikoff’s Jubilee in 1915:
"I can see you now at the Buda-Pest Congress in 1894, disputing with your antagonists; with your fiery face, sparkling eyes, and dishevelled hair, you looked like the Dæmon of Science, but your words, your irresistible arguments raised the applause of your audience.
“The new facts, which had at first sight seemed to contradict the phagocyte theory, now entered into harmony with it. It was found to be sufficiently comprehensive to reconcile the holders of the humoral theory with the partisans of the cellular theory.”
This is how Metchnikoff had reconciled the apparent disagreement of Pfeiffer’s phenomenon with the phagocyte doctrine: he demonstrated, by a series of experiments, that the extracellular destruction of the cholera vibriones in the peritoneum of a guinea-pig vaccinated against cholera, did in no wise depend on the chemical properties of the blood serum, but was simply due to the digestive juices which had escaped from the inside of the leucocytes, damaged by the intraperitoneal injection. Those digestive juices, or cytases, poured into the peritoneal liquid were what killed the injected cholera vibriones and transformed them into “Pfeiffer’s granulations.” On the other hand, if by means of various precautions the phagocytes were left unmolested, the extracellular destruction did not take place and the vibriones were digested within the phagocytes.
Metchnikoff used other experiments to prove that the bactericidal property of blood juices did not exist without intervention from the phagocytes. For instance, in a guinea-pig vaccinated against cholera, the bacilli are not destroyed if they are injected into parts of the organism that are devoid of pre-existing phagocytes, such as in the subcutaneous tissue, in the anterior chamber of the eye or in an aseptically-obtained œdema. On the other hand, if, in the same medium, some exudate is injected containing damaged leucocytes from which the digestive juice is leaking, the vibriones introduced are destroyed. The same results are obtained in vitro.
All these experiments proved that the extracellular destruction of the cholera vibrio was accomplished by the digestive juices which had passed from the phagocytes into the humors and not at all through a special property of those humors. Once again the phagocyte theory rose triumphant from the test.
After having finally proved that it is by means of its phagocytes that the organism fights microbes, Metchnikoff wished to find out whether it was by the same process that it struggled with their poisons, or toxins. This problem, far more difficult to solve, took him many years’ study. Whilst every phase of the phagocytes’ struggle against microbes can be followed with the eyes, it is impossible to do so where poisons are concerned, since they are invisible; it is necessary to proceed by a different road.