number of red cells in a cubic millimeter. Thus the proportion is as follows: The number of red blood-cells in a given number of fields is to the number of bacilli in the same number of fields as 5.500,000 red blood-cells (Colorado altitude) is to X. X equals the number of bacilli in a cubic millimeter. From this the number of bacteria in a cubic centimeter is obtained by multiplying the number in a cubic millimeter by 1000. The test-tube containing the bacterial emulsion is again sealed and placed in the hot-water bath at 60°-62° C. for one hour. It is necessary to destroy the vitality of the microorganisms but to preserve at the same time their toxic properties. Too short a period of sterilization will fail to kill the bacteria, while if this is too prolonged the preparation will be rendered inert. After the process of sterilization is completed the bacterial emulsion is diluted with sterile salt solution, and a computation made of the number of bacteria in each cubic centimeter. Control cultures are made in all cases to demonstrate that the vitality of the bacteria is actually destroyed. The tubercle bacilli vaccine, in contradistinction to the above, is estimated by weight rather than by enumeration, and is given in initial doses of from one ten-thousandth to one one-thousandth of a milligram.

The determination of the opsonic power of an individual consists of an estimate of the relative number of bacteria ingested by washed white blood-cells under the influence of the patient's serum, in comparison with the number ingested by the same number of cells in the presence of normal blood-serum under precisely similar conditions. It follows, therefore, that as essential factors there must be blood from the patient; blood assumed to be normal; and washed leukocytes. In addition, there must be at hand an emulsion of the specific bacteria concerning which the opsonic action is sought. After cleansing of the finger the patient's blood is withdrawn by means of capillary attraction into the curved extremity of a glass capsule, the fine capillary ends of which have been broken. The straight end of the capsule is sealed by holding in a mildly burning flame. It is important to have the flame hot enough to seal the end of the tube quickly, in order to avoid heating that portion of the сарsule containing the blood and thus modifying the opsonic power. If the straight end of the capsule is not too short, the bulging portion wherein the blood is to be shaken from the curved extremity is not likely to be heated by the sealing of the other end. Should this take place, a portion of the blood is forced from the curved end by the expanding air within the tube. The blood is to be shaken from the curved extremity into the bulging portion. Pouring of cold water upon the distal or straight end produces such contraction of air as to draw the blood quickly beyond the elbow. The normal or control blood is obtained in the same manner. The two capsules, after identification marking, are allowed to clot and are then centrifuged in order to separate the serum. The ends of the capsule are broken and the serum is ready for

extraction with a fine pipet.

The washed leukocytes are prepared as follows: About twenty large drops of blood are placed in several times this volume of normal salt solution, containing 1.5 per cent. of sodium citrate. After thorough mixing and centrifuging the supernatant liquid is aspirated with a fine pipet. Normal (85) per cent. salt solution is added to the corpuscles to remove traces of serum. The process of centrifuging is repeated, and the supernatant liquid again separated. Thus the serum is removed and the blood-cells remain in the bottom of the tube, the leukocytes forming the superficial layer or cream. To produce the bacterial emulsion the cultures are diluted with a small amount of salt solution until a milky appearance is secured, and are centrifuged in a small tube to throw down the clumps.

A commercial product of tubercle bacilli emulsion may be secured containing a suspension of bacilli in glycerin. This is washed off by salt solution, filtered, and the residue ground with 0.5 per cent. salt solution until a milky emulsion is produced. The essential constituent factors are now ready for utilization. Fine capillary pipets, made after the direction of Wright, with an even caliber, are marked in such a way as to designate a fixed volume or unit of measure. An equal volume of washed leukocytes, patient's serum, and bacilli emulsion are withdrawn into the same pipet and deposited upon a sterile slide for the purpose of more thorough mixing. The combined liquid once more is drawn into the pipet which is sealed and placed in the opsonic incubator for fifteen minutes at a temperature of 37° C. An identical process is pursued with the normal blood. After the incubation period is finished a film or smear is prepared in each instance upon a slide in such a manner as to insure even distribution. The specimen is now ready for staining, carbol-fuchsin being used for tubercle bacilli and the so-called Leishman stain, consisting of eosin and methyleneblue, for nearly all others. In lieu of Leishman's stain the specimen may be fixed with a saturated solution of corrosive sublimate and stained with thionin or any suitable anilin dye.

After selecting a proper field with the low power the oil-immersion lens is used to count the number of microorganisms found in each of 50 to 100 polymorphonuclear neutrophiles. The average number of the bacteria contained in the whole number of cells counted, constitutes the phagocytic index. This, for the normal blood, is regarded as unity, though some variation exists among healthy individuals. The phagocytic index of the patient as compared with that of a healthy individual gives the opsonic index. Thus, if the average number of bacteria contained within a given number of normal bloodcells is eight, and the number in the blood of the patient is four, based upon the count of an equal number of cells, the opsonic index would be one-half that of the normal and would be expressed as 0.5.

The opsonic index, as previously stated, is subject to considerable variation in different individuals suffering from the same disease, and in the same person according to the degree of systemic infection, and as claimed by some according to certain external conditions, as exercise or excitement. If the index of any given microorganism is continuously low, it is assumed that there exists a localized focus of bacillary invasion. If the index is high above unity, or if a decided fluctuation is found in successive examinations, the evidence points to a pronounced systemic infection. It is suggested, therefore, that repeated observations of the opsonic index should possess a considerable degree of diagnostic merit, and afford approximate indications with reference to vaccine therapy.

There has been much convincing testimony presented by Wright and his followers concerning the practical utility of his method of attempted artificial immunization. The general consensus of opinion among scientists and clinicians is to the effect that vaccine medication is founded upon rational grounds and is destined to represent a great advance in the therapy of the future. The precise regulation of the dosage, based upon the determination of the opsonic index, however, is open to controversy. Adverse opinions are freely expressed concerning the general impracticability of his work, on the score of the many opportunities for error and confusion in the detailed application of the intricate technic. In addition to the difficulties attending the technic, due cognizance should be taken of the possible sources of error inherent to differences in the susceptibility of the microorganisms to agglutination in a comparatively large volume of serum, and to the variations in the effect of the pathologic sera upon normal phagocytes. It does not follow, however, that for these reasons alone the method of Wright is unworthy of recourse by those qualified, through training and equipment, to take advantage of his contributions.

Wright and Bullock have called attention to one of the difficulties in securing immunization in pulmonary tuberculosis from the employment of tuberculin. They attribute considerable importance to the histologic and pathologic structure of the pulmonary tubercle as offering a barrier to the antibacterial forces of the organism. They believe that the toxins, otherwise stimulating to the machinery of immunization, are locked up within these foci of infection, and, per contra, if artificial aids to the immunizing process are introduced into the circulation, that the bacilli remain protected to some extent behind a wall of non-vascular connective tissue. It thus appears that inoculations with bacilli emulsion are regarded as dangerous for one class of consump

tives suffering from an excess of toxins, and as non-effective on account of structural conditions for those exhibiting a deficiency of these protective substances. This hypothesis of the defense of the bacillus is opposed to usually accepted ideas concerning the inclosure of the bacillus, its possible exclusion from the organism as a result of encapsulation, and the protection accruing to the individual by this means. The theory of Theobald Smith as to the defensive rôle of the cellular outlying breast works in the interests of the bacillus contained within its tubercle abode, is somewhat in accord with that of Wright, although inspired by no acceptance of a special relation of opsonins or blood leukocytes to immunity. He regards the tissue reaction concerned in the process of tubercle formation as an important element in the mechanism of defense, both for the host and the parasite. Thus a quiescent focus is secured for the indefinite and undisturbed sojourn of the bacillus, but opportunities at the same time are denied for its multiplication or escape. He also advances the theory, as previously stated, that the bacilli are at times provided with a protective envelope, which he believes, in contradistinction to Wright's hypothesis, to remain intact. when the opsonic power is low, and thus exert a protective influence upon the organism by preventing multiplication. An apparent immunity is supposed to exist at such a time, to be succeeded by removal of the envelope, multiplication of bacilli, and greater tuberculous activity in proportion as the opsonic power is elevated. It would seem, in the midst of conflicting views entertained by many eminent authorities, that general clinical observations should be worthy of presentation.

CHAPTER C

PERSONAL OBSERVATIONS UPON THE USE OF
BACTERIAL VACCINES1

THE published reports of Wright and his fellow-workers indicate the value of vaccine medication in localized tuberculous infections of the bones, joints, glands, and portions of the genito-urinary tract, but suggest that the results of its employment in pulmonary tuberculosis are likely to be disappointing. He recognizes elements of danger if the bacilli emulsion is administered indiscriminately to pulmonary invalids, and particularly in the presence of fever and a widely fluctuating index-curve. Under such conditions the patient is already undergoing a continuous infection from an improperly adjusted and interspersed dosage of the toxins. At such a time the employment of the vaccine only adds to the burden of the individual, and diminishes any effort on the part of the organism toward autoimmunization. This objection to the use of the vaccine in pulmonary tuberculosis, obtains in the event of an existing surplus in the blood of toxins emanating from tuberculous foci, and as well from centers of secondary infection. The

1 A portion of this chapter was written for the annual meeting of the American Climatological Association, held in Washington, May, 1907, but was not read, owing to unavoidable absence. A supplementary report is appended, embracing the results of subsequent observation.

thought naturally arises whether the existence of a severe mixed infection in pulmonary tuberculosis materially alters the indications for tuberculin, or suggests the expediency of resorting to an autogenetic vaccine.

It is very essential to establish somewhat definitely if vaccine medication is of clinical value in pulmonary tuberculosis, and to what extent its employment should be based upon the determination of the opsonic index.

In view of the unreliability of the serum preparations sometimes employed to combat the mixed infection of pulmonary tuberculosis, and the many disadvantages attending their use, I was actuated, in the early part of 1907, to institute a clinical inquiry concerning the results possible of attainment with the bacterial vaccines. It was also my purpose to determine, if possible, the effect of the bacilli emulsion upon cases of pulmonary tuberculosis uncomplicated by mixed infection. It was recognized that a purely scientific investigation along these lines would involve such frequent estimates of the opsonic index as to be prohibitive of the observation of more than a few cases. In view of the supposed range of variation in the indices of many pulmonary invalids, it was felt that approximately correct conclusions from an opsonic or laboratory standpoint would demand the observations of the index once daily. I did not feel, however, that the scope of my investigation should relate merely to the recording of indices, and the assumption of an increased power of resistance in a very few closely observed cases, but rather to the clinical study of a comparatively large number of patients conforming to the same general class. This has necessitated the taking of the index of each patient at quite infrequent intervals. A degree of compensation for this discrepancy has been secured by the careful selection of cases, and the fact that the patients were kept under the closest practicable supervision in order to avoid diurnal fluctuations of the index from external causes. I beg to express my obligation to Dr. W. C. Mitchell for his careful performance of the technical portion of the work, including the determination of the opsonic indices and the preparation of the bacterial vaccines. An appreciative recognition is also accorded Dr. E. W. Emery for valuable assistance rendered in the opsonic work. In all instances the index was secured prior to the first dose, and thereafter at intervals of from ten days to a few weeks. In the beginning an effort was made to take the indices more frequently, but this practice was discontinued for several reasons. The evident futility of such spasmodic efforts to obtain an approximate estimate of the mean daily opsonic power was apparent. In view of the large number of patients undergoing vaccine treatment, frequent observation of the indices was impossible. The increased financial burden incident to their oft-repeated determination represented a factor of considerable importance. The clinical study was undertaken solely for practical purposes, and despite its deficiencies resulted in an instructive experience.

The total number of patients undergoing vaccine therapy, in the first four months of 1907 was 67, who were divided into three widely differing groups. In work of this character a proper classification of cases constitutes a feature of the utmost importance.

Group 1 comprised 42 cases of chronic pulmonary tuberculosis without symptoms referable to mixed infection. It was recognized that all patients should represent, if possible, the same general type and

stage of the disease, and conform more or less closely to a fixed régime. It seemed highly desirable that there should be eliminated all sources of confusion arising from climatic influence or change of environment. To this end patients included in this group were selected with extreme care. In view of the uncertainties attending its employment, no individual was permitted to undergo the treatment, whose general condition and previous progress had been entirely satisfactory, or who exhibited appreciable temperature elevation. On the other hand, an effort was made in the selection of cases to include only those who, in spite of a continued residence in Colorado under appropriate conditions of daily life, had failed to secure an entire arrest of the tuberculous process. It was believed that more definite information concerning the effect of the treatment, could be secured by limiting its application to those whose condition had been almost stationary for prolonged periods. Of all the cases comprising this group, the average period of residence in Colorado, with practically unchanged environment, was two years and eleven months, the longest being ten and one-half years and the shortest six months. A remaining activity of the tuberculous process was present in all cases, as evidenced by physical signs, cough, expectoration, and bacilli. A new method of treatment was hailed with enthusiasm by these patients as precursory of possible future recovery, thus introducing a psychic element impossible of elimination. The injections were administered at regular intervals of two weeks. This periodicity of dosage was decided upon in order to conform as far as possible to the expected expiration of the positive phase. The initial dose was usually one ten-thousandth of a milligram. Both a low and high index were considered suggestive of a small dose in the beginning.

It was noted that the initial opsonic index of several patients, upon the basis of Wright's conclusions, suggested a non-tuberculous condition. The variation in health is supposed to range from 0.8 to 1.2. Wright has assumed that any persisting deviation from these limits is fairly indicative of a bacterial invasion, and has regarded a normal index as suggestive of its probable exclusion. As a matter of fact, in this series many patients exhibiting a normal index were individuals displaying pronounced physical and subjective evidences of advanced. tuberculous change. There was shown a wide range in the opsonic index, even among a group of patients especially selected with a view to securing approximate uniformity of conditions. It may be stated. parenthetically that the subsequent clinical results in a few patients with high indices, indicated their favorable response to the tuberculin injections, quite as much as in others with a beginning low opsonic power. During the entire period of observation a disparity was noted between the clinical and opsonic findings. Many patients displaying conspicuous improvement as a result of the tuberculin injections were found to exhibit trifling variations in the opsonic index. Upon the other hand, several whose index curve was found to undergo a satisfactory elevation, nevertheless failed to respond favorably to the specific medication. The early discrepancy between the clinical and opsonic results suggested that the lack of parallelism, occasioned presumably by reason of the difficult and intricate technic was sufficient to vitiate any practical deductions based upon the observation of the index. It soon became questionable if any reliable information was furnished by the opsonic index either concerning the clinical progress or the size