In any case, to render the bacteriolog ical diagnosis complete, it would be nec essary to obtain the diphtheria. bacilli in pure culture and test their virulence by inoculation of susceptible animals.
In routine practice this is done by in oculating half-grown guinea-pigs with from 1/4 to 1/, per cent. of their body weight of a forty-eight hours' milture of the bacilli grown at 37° C. in simple nutrient or glucose alkaline broth. In carrying out such experimentation many precautions are necessary to render such work accurate and trustworthy. Aluch time and labor are consumed in the proc ess. For our purposes it is sufficient to know that the great majority of those who have carried on such experiments uncier proper conditions with bacilli de rived from pseudomembranes and pre senting the morphological and staining characters of diphtheria bacilli have found the bacilli fully virulent.
So long as the bacteriological diagnosis is reinforced by clinical evidence of the presence of false membrane and the symptoms of diphtheria, we can safely trust to the examination of these cover glass preparations.
We find, however, that the examina tion of healthy throats has led to some remarkable results. In the throats of those who have been exposed to diph theria, but have remained perfectly well, we may find characteristic and fully viru lent diphtheria bacilli; in others we may find the pseudodiphtheria bacillus al ready spoken of, or a bacillus which, while presenting the cultural and mor phological characters of the diphtheria bacillus, proved in inoculations to be non-virulent.
Thus, in a series of 330 healthy throats examined by the New York Board of Health, in 8 virulent characteristic diph theria bacilli were found, in 24 non-vir ulent characteristic diphtheria bacilli, and in 27 non-virulent pseudodiph theria bacilli. Since Hoffmann's obser vation of these bacilli, so closely resem bling the Loeffler bacillus, but devoid of virulence, a great deal of attention has been given to this subject. Opinion is still divided as to the relation of these non-virulent bacilli. On the one hand,
they are regarded simply as degenerate or attenuated forms of the diphtheria bacillus; on the other, they are repre sented as a distinct species.
The identity of the pseudodiphtheria bacillus seems to be now established. In form these are smaller, shorter, and thicker than the diphtheria bacillus. When seen in stained smears the bacilli are often observed to be lying parallel to one another, in contrast to the irregu larly-angular disposition of the diph theria bacillus. In their growth in broth the pseudodiphtheria bacilli de velop alkali, where the Loeffler bacillus forms acid. They are never virulent. These differences are, by most authori ties, considered sufficient to warrant the belief that they are a separate species.
The other class of non-vinilent bacilli found in the throat present all the char acters of the Loeffler bacillus except their virulence. Roux and Yersin believed these bacilli to be simply attenuated forms of the diphtheria bacillus. It was shown that they are particularly likely to be met with in the throats of those who have had diphtheria some time be fore, or have been exposed to diphtheria. It was also found that the diphtheria bacillus could be so attenuated by vari ous methods of growth as to deprive it of its virulence. No one, however, has yet been able to restore virulence to any of the non-virulent forms met with, and the question must be considered as still open.
There are 70 varieties of diphtheria and pseudodiplitheria bacilli from the standpoint of agglutination by anti diphtheritic scrum. This property is an inconstant characteristic of the true Elebs-Li;ffler bacillus, and is in no way related to its virulence. Certain varie ties of this organism can be aggluti nated by the serum of horses immunized by cultures, while they do not react to the serum of horses immunized by tox ins. The pseudobacillus conducts itself toward these sera precisely as does the true organism, and by this means can not be differentiated from it. Ch. Lesieur (Comptes Soc. de Biologie, Aug., 1901).