Part 16

_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_

The material used in the study I am reporting included swabs from the large bronchi and fluid from the lungs and pleural cavities of 32 autopsies, as well as blood cultures from 22 patients and swabs from the nasopharynx of 31 individuals. Fifteen sera were tested for fixation of complement with an antigen made from several strains of B. influenzæ. Fourteen other sera were tested for agglutinins. Complement content was determined in the sera of 25 patients. Skin tests after the Von Pirquet method were done on 14 convalescents, and carefully stained nasopharyngeal smears without cultures were studied from 48 patients.

The chief attention was given to the study of the autopsy material and we concentrated on the isolation of B. influenzæ. At the same time we did not neglect the other bacteria making up the flora of the bronchi, lungs and pleural cavity in these cases. The various types were isolated and most of them fully identified.

_Technique_

Direct smears were made on sterile slides of all material studied and stained by Gram’s method. The counterstain was always alcoholic fuchsin diluted 1-20 in distilled water. Direct cultures were made on a human blood agar plate containing 5 per cent. blood, which was further smeared just before use with defibrinated blood. This latter procedure was later discarded, as it did not appear to assist to any marked extent the growth of B. influenzæ. Blood broth containing a few drops of defibrinated blood and blood agar slants smeared with blood were also used. Heated blood agar (2-3 c.cm. of defibrinated human blood added to 100 c.cm. of ordinary agar at a temperature of from 90 to 100° C., or as the agar comes from the sterilizer) was used in the last nine cases to replace the blood agar slant in the direct cultures and as the medium of choice for transfers of the B. influenzæ.

I prefer the ordinary blood agar plate to the heated blood plate because the former gives readings which are very helpful in distinguishing colonies of various types. B. influenzæ appears as clear, tiny, pinpoint, inert colonies. B. xerosis or the pseudodiphtheria group gives more opaque but often rather similar colonies. Gram negative cocci as M. pharyngis siccus have dry, raised, soon becoming wrinkled, inert colonies, varying greatly in size; M. catarrhalis, more moist, inert colonies. The cocci of the streptococcus viridans group appear as very small colonies with greening, or are not infrequently inert, while thin, flattened colonies with central thickening may sometimes be noted. Those of the streptococcus hemolyticus group occur as small, frequently nipple-like colonies with clear, wide zones of hemolysis; pneumococci as moderately small, moist, dewdrop-like colonies with center collapsing early and with greening; streptococcus or pneumococcus mucosus as larger, watery, sticky colonies with greening and frequently an early clearing near the colonies.

TABLE I.

BACTERIOLOGY OF THIRTY-TWO AUTOPSIES FROM INFLUENZA CASES.