Part 20

Spectroscopic Studies.—Sera obtained from 20 post-mortem bloods were examined spectroscopically. In eight an absorption band in the red was observed. In some instances such a band was observed in blood obtained shortly after death and before coagulation had occurred, while other similar bloods, as well as bloods obtained at longer intervals after death, exhibited no such band. A similar band was observed in one case from blood obtained from a patient about 12 hours before death from pneumonia following influenza. Medication was not a causative factor. To ammonium sulphide the band in the red reacted as methemoglobin and the position (as estimated by Dr. Menten) corresponded with methemoglobin. Oxyhemoglobin bands in such bloods occupied normal positions as determined by Dr. Menten. On diluting such bloods with water no abnormality in character or position bands was observed, save in one instance (No. 778 below). This does not, however, disprove the possibility of such abnormality in the hemoglobin within the cells, for moderate dilution only of serum rendered the band in the red invisible, presumably by dilution.

Detailed examination of the absorption bands was made with a direct reading wave-length Hilger Spectroscope (which was calibrated by line spectra derived from salts added to an alcohol flame) by Dr. Menten. This spectroscope had an accuracy of about two Angstroms. In all, seven post-mortem bloods were examined, viz. autopsy numbers 756, 761, 763, 773, 778, 784, and 787. In five of these, sufficient serum was obtained to make readings. All gave the two characteristic oxyhemoglobin bands in the blue-green with centers of the bands at λ 758μμ λ and 542μμ. The second oxyhemoglobin band varied slightly in width in the different samples. In addition to the two oxyhemoglobin bands in each of four of the above sera, viz: Nos. 756, 763, 767 and 787, an absorption band in the red was found with the center of the band as follows: Number 756 at λ 627μμ, number 761 at λ 634μμ, number 763 at λ 625μμ, and number 787 at λ 634μμ. These bands varied considerably in intensity and could only be identified when the two oxyhemoglobin bands were merged and appeared as one broad band. As controls for the position of the oxyhemoglobin bands two normal bands were examined, which showed two bands with centers also at λ 758μμ and λ 543μμ. For comparison of the methemoglobin bands of the above post-mortem bloods, a sample of this hemoglobin compound was made by adding potassium ferricyanide to normal blood until the solution became brownish in color. The center of this methemoglobin band was found at λ 634μμ. In blood from autopsies number 773 and number 778 sufficient serum could not be obtained to make a reading. To each of these bloods distilled water was added. The laked blood of 778 gave a methemoglobin band with the center at λ 632μμ on examination 24 hours after autopsy. Similar treatment of corpuscles five days subsequently gave no indication of the presence of any methemoglobin spectroscopically.

From the serum and from the laked corpuscles of number 784 no trace of methemoglobin was found when the blood was examined a few hours after removal at autopsy.

Oxygen Capacity.—The total oxygen capacity was determined by the Van Slyke method (by Dr. Rohde and Mrs. Macklin). At this stage the more pronounced type of influenza had subsided, but in early convalescence the capacity was within normal ranges.

Other studies using different technique gave concordant results, but there were indications that oxygen was more slowly absorbed than normally.

Oxygen Content of Venous Blood measured by the Van Slyke method (by Dr. Rohde and Mrs. Macklin) on the same bloods examined for total oxygen capacity seemed to indicate a mild deficiency as compared to normal bloods.

Gases, Kinds, Quantity and Rate Yielded to Vacuum.—In general it may be said that quantitative differences observed are not considered fundamental, but that the studies indicate abnormal slowness in oxygen absorption.

Gases, Quantity and Rate of Absorption on Exposure to Air After Extraction by Pump.—The results emphasize slowness of oxygen absorption as compared to normal blood.

The material to be examined was exhausted for three minutes in the receiver of the Van Slyke apparatus. One c.cm. was then transferred, with as little exposure to air as possible, to a small empty bottle, which was then closed and placed in communication with a calibrated, horizontal tube, containing a segment of alcohol, which served the dual purpose of a seal and an air volume change indicator. (See Fig. 1.) The apparatus was made in duplicate and mounted on a common base, so that simultaneous readings on different samples could be made. After establishing the zero position of the alcohol segment, the base on which the bottles were mounted was vigorously shaken in a uniform manner. Ten seconds after the period of shaking, the volume readings were taken. Successive periods of shaking and reading were conducted at 30-second intervals, until the test was completed. Actual volume changes were then calculated, tabulated and plotted.

The greater confidence is placed on the results obtained by observing the color of the blood, as described below; but since then the method has been checked up and the results indicate that the findings were of sufficient accuracy to warrant their inclusion in this report.

Footnote 1:

Studies along this line are being made with improved apparatus, the results of which, together with the description of the apparatus, will be published elsewhere. (See Am. Gr. Physiol., 1920, li, 195.)

Effect of Addition of Serum on Behavior on Exposure to Air.—The persistence of venous hue of blood exposed to air was noted above. It was observed that the addition of serum from the same blood conspicuously shortened the time required for such blood to acquire an arterial hue. The addition of normal serum was more effective in this respect than pathological serum. Measurements of the rate of absorption of such blood after the addition of serum indicated acceleration of oxygen absorption. From this it would seem that the oxygen transmitting capacity of the serum was diminished.

Effect of Addition of Dry Sodium Bicarbonate on Behavior on Exposure to Air.—The addition of a small quantity of dry sodium bicarbonate to a blood refractory to arterialization on exposure to air enormously accelerated the process, as judged by the color. To what extent the change in color may have been due to causes other than oxygen absorption was not determined.

Comment

The most significant positive findings were evidence of deficiency of serum oxygen transmitting capacity or rate, and the detection in serum of an absorption band in the red corresponding to methemoglobin. The presence of the abnormal substance giving rise to the absorption band is considered of special interest as indicating abnormal chemical conditions in the blood, rather than material change in hemoglobin oxygen capacity.

THE BACTERIOLOGY OF EPIDEMIC INFLUENZA WITH A DISCUSSION OF B. INFLUENZÆ AS THE CAUSE OF THIS AND OTHER INFECTIVE PROCESSES

By W. L. HOLMAN, B. A., M. D.

Introduction

In a study of the bacteriology of a respiratory disease such as influenza, the technical difficulties encountered are very great and must be overcome before we can draw useful conclusions from the results obtained or attempt to determine the etiological factors. The important methods of attacking such a problem include: (1) the study of stained smears and cultures from the various available materials, along with the demonstration of the bacteria in the lesions found in the disease by a study of sections; (2) tests with the various materials to determine the presence of the causative agent, which includes experiments on man and animals and is more inclusive than the mere study of the bacteria isolated; (3) immunological studies of man suffering from the disease, or of man and animals treated with the materials from the disease; (4) pathological, clinical and epidemiological studies linked with the above.

Many of the difficulties and sources of error in these methods are manifest to all, but certain points may be indicated as more important in the phases of the work on which I am to report.

General Methods of Investigation

Stained smears from the material available. The choice of the material is of first importance. Sputum to be of any real value must be obtained from the deeper portions of the respiratory tract, should be as free as possible from the secretions of the buccal cavity, and should be washed in saline before it is used. These are considered among the first requirements in the study of lung infections by the pneumococci and are equally important in influenza. Swabs from the nasopharynx should be obtained with the same precautions as are demanded in meningococcal work. The other available material—such as blood, lung puncture fluid, pleural fluid and spinal fluid—must be collected with the greatest care.

The staining methods should, naturally, include those which will bring out the various types of bacteria, and must include the Gram method, using dilute alcoholic fuchsin (1-20) as the counterstain. The varying morphology of the B. influenzæ and its frequent minute size make it difficult to detect. It is not the only Gram negative small bacillus seen in smears from the throat, but when it occurs in the typical schools, or where there are numerous bacilli to be seen, its characteristics are quite definite. I have recently isolated an anærobic Gram negative bacillus from a series of swabs from the buccal cavity which suggests in many ways the morphology of the B. influenzæ, which will indicate one of the many difficulties to be met with in the study of stained smears. They are, nevertheless, of great use as a control on cultures, and most helpful in the study of the material from sources other than the respiratory tract.

Cultures of the bacteria from the various materials. Here we have the greatest difficulty of all. The medium chosen determines the bacteria which will appear to predominate, and there is no single medium that will answer all purposes. Streptococci will appear to be in excess when serum broth is used, as I have previously shown; pneumococci with Avery’s pneumococcus medium; and staphylococci, the Gram negative cocci, and the diphtheria group with Loeffler’s serum. Ordinary blood agar is perhaps the best general medium for direct and secondary plating. There have been many special media devised for growing the B. influenzæ, but the one I have used most and found particularly helpful is heated blood agar made after the general method of Voges.

The extremely tiny colony of B. influenzæ on ordinary blood agar makes it particularly difficult to detect, and one is apt to get the wrong impression of its numbers from the macroscopic appearance of the plate. In attempts at isolation there must be a liberal use of media in picking colonies, as many suspicious ones will turn out to be immature growths of B. xerosis, M. pharyngis (or M. catarrhalis), streptococci, or more rarely pneumococci and other organisms. Replating from such picks is frequently necessary, and further plates, from the original culture on heated blood agar, must often be made before the B. influenzæ can be isolated. The care required in all stages of the isolation of this organism, the unstinted use of media for plating and for picks, the number of stained smears to be studied, and the further transfers necessary to verify results, all these limit the amount of material which can be studied with any degree of accuracy. If further the streptococci, the pneumococci, the Gram negative cocci, the capsulated Gram negative bacilli and many others are to receive any attention, it can readily be appreciated that a few cases carefully studied are of far more value than a large number hurriedly examined in an uncertain routine.

The pathological study of the same cases on which I have done the bacteriology will be found in Dr. Klotz’s paper in these communications, and I will merely refer to some of the bacterial findings in the sections of the lungs and bronchi. The more inclusive methods which have been used in attempts to determine the etiological factor in influenza we have been unable to attempt, but I will refer later in this paper to the findings of the investigations of others. Immunological studies have been limited to a few investigations on the presence of agglutinins, complement binding substance, skin reactions and the amount of complement present in the sera of certain patients. The epidemiological and clinical studies are reported by Drs. Johnston and Lichty in this series of reports.

Material Studied