Blood cultures were done on 22 cases. Pneumococcus mucosus was grown from one patient who three days later came to autopsy (Case 767). In another case pneumococcus-like organisms were seen in smears from the dextrose broth flask after 24 hours’ incubation. These, for some unknown reason, did not grow on blood agar plates. After 48 hours smears made on blood agar from the original flask gave a growth of B. influenzæ and a M. catarrhalis-like organism. I consider this result a very unsatisfactory one, being quite unable to explain the failure to grow the pneumococci-like forms on transfer. Possibly the acidity developed might account for it.
TABLE III
AGGLUTINATION TESTS WITH SERA OF CONVALESCENT INFLUENZA PATIENTS
| Dilution of Serum | + | +– | – | |
|---|---|---|---|---|
| Convalescents | 1-1 | 3 | 0 | 2 |
| 1-10 | 5 | 2 | 7 | |
| 1-40 | 2 | 3 | 9 | |
| 1-80 | 0 | 1 | 13 | |
| 1-160 | 0 | 0 | 14 | |
| Normal Controls | 1-10 | 1 | 2 | 0 |
| 1-40 | 0 | 1 | 2 | |
The complete agglutination as would be indicated by +++ or ++ was not seen.
Agglutination tests were carried out with the sera of 14 convalescents and 3 normal individuals. A polyvalent emulsion of strains of the influenza bacillus isolated from our cases was used. The results are shown in Table III. Tubes were incubated at 37.5° C. The results did not indicate anything in the nature of a specific reaction. Dr. Frost carried out this work during the height of the epidemic, but we were unable to continue it further. A short review of the work of others will be found near the end of this paper. Miss Thompson and Mr. Mock studied complement fixation, using the sera of 15 convalescents against an antigen of B. influenzæ. Their results were negative. The antigen appeared to be slightly more anti-complementary than were emulsions of staphylococcus or B. coli. Huntoon also noted this anti-complementary character of emulsions of B. influenzæ.
Attempts were made to estimate the amount of complement present in the fresh blood serum of influenza patients. The technique was to use a 1-4 dilution of the patient’s serum, adding measured amounts of this to a 1 per cent. blood emulsion, with 1 unit of amboceptor and determine the smallest amount necessary to bring about complete hemolysis. This test was carried out on eight patients ill for only a few days. The average amount of the dilute serum was 0.181 c.cm. Fifteen patients, convalescent after a moderate illness, gave an average of 0.276 c.cm. Two patients seriously ill with temperatures of 104.3° F. and 105° F. required 0.4 c.cm. to bring about complete hemolysis. We would not like to draw any very definite conclusions where we are dealing with such small fractional differences. This lessening of complement has been noted in other infectious diseases and may be important in the questions of immunity in influenza. Dr. Frost carried out a number of cutaneous tests after the method of Von Pirquet, using a polyvalent, weakly alkaline emulsion of influenza bacilli in 25 per cent. glycerin. Eleven convalescents were tested and none of them showed any local or general reaction. The suggestion that these results may indicate an increase in resistance is discussed in another place. A number of strains of pneumococci which we had isolated from our autopsy cases were differentiated by the agglutination method. Type I was found 3 times; type II, 10 times; type IV, 9 times. Four showed agglutination with both type I and type II sera. Type IV pneumococcus was isolated in one case from the right and left bronchus as well as the lung. In another case the same type pneumococcus was recovered from the lung and pleural fluid. These results are similar to those found by numerous workers.
The Hemophilic Bacteria
The discovery by Pfeiffer of the hemophilic character of the bacillus found by him in cases of influenza opened up a new group of micro-organisms known as the hemophilic bacteria. Davis (1915) has laid particular stress on the group character of these bacilli, and the more they are studied the more clear does it become that there are several distinct members. The B. influenzæ is by far the most important as well as the most frequently found of the group and is considered as the type organism.
All these bacteria require for their growth the presence of some form of hemoglobin. The actual amount necessary may be very small, and Davis suggested that it may have a catalytic action. A great deal of work has been done in attempts to discover just what portions of the hemoglobin are necessary to bring about this phenomenon. In our discussion on media for the influenza bacillus we will briefly describe some of the various hemoglobin preparations that have been used successfully. It must at this point be emphasized that blood is very useful in many media to stimulate the growth of a great variety of bacteria, and the transfers made from such luxuriantly growing cultures may grow very poorly or not at all on ordinary media, and this might easily lead to erroneous conclusions on the hemophilic character of the organisms studied. There are certain bacteria which grow so much better on media containing blood that such media are sometimes necessary for their isolation, although after a few transfers they will grow on ordinary media. This is true for bacillus pertussis, and throughout the literature a good deal of confusion has arisen in not recognizing this temporary hemophilic character of certain bacteria. The true hemophilic bacteria do not grow except in the presence of hemoglobin in some form or other. The problem becomes almost academic when we consider the small amounts of hemoglobin that are necessary. Davis has shown that a dilution of 1 in 180,000 is sufficient, and in the interesting discussion between Cantani and Ghon and Preyss it was demonstrated that hematin or other hemoglobin product was necessary in the agar before B. influenzæ would grow in the presence of other bacteria, and that this hematin could be derived from the blood in the meat which was used in making the basic infusion.