Various other methods have been used to bring about this beneficial change in hemoglobin. Gioelli (1896) used a medium made up of 1.1 per cent. hemoglobin and 21.5 per cent. malt extract. This is reddish brown in color, becomes clear when neutralized with potassium hydrate and remains so on heating. This added to agar is reported as very favorable in growing this bacillus. Ghon and Preyss described a medium made up of meat, peptone, salt and agar prepared in the ordinary way, but not filtered for at least a week, and then only roughly. This medium is favorable for symbiotic growths. He further used beef blood heated in a soda solution and blood heated in water as hemoglobin preparations to be added to agar. Thalhimer found an amorphous hemoglobin medium to be more favorable than when a purer hemoglobin was used. W. F. Robertson found a hemoglobin agar, prepared by allowing sheep’s blood to clot, decanting off most of the serum, freezing and then thawing what remains and adding 1 c.c. of this to an agar tube at about 60° C., to be very favorable for the growth of B. influenzæ. Cantani used a blood treated with pepsin and hydrochloric acid, digested some days in the incubator, filtered and made weakly alkaline. This mixture was heated for a few minutes, refiltered and added to the medium. He speaks of it as extraordinarily good for B. influenzæ. Blood treated with trypsin has been used by Matthews, Averill, Young and Griffiths, Harris, A. Fleming and others. Fleming further found that this alteration in hemoglobin can be brought about in a number of other ways. Blood boiled in agar (suggesting the Voges agar) and the tubes slanted while hot, blood boiled in water, the clotted blood precipitated and the clear fluid added to agar, or more rapidly by adding equal quantities of sulphuric acid to the blood and a similar amount of potassium hydrate he obtained altered blood suitable for media. He reported that by any of these methods he could obtain a medium very stimulating to the growth of B. influenzæ. By the addition of brilliant green (1 in 500,000) he inhibited the growth of staphylococcus, streptococcus and pneumococcus. For storing cultures of B. influenzæ Fleming found a minced meat medium with the addition of blood to be the best. I have found this medium without the blood to be an excellent one for keeping a great variety of cultures. Bernstein and Loewe have reported the use of gentian violet (1 in 5,000) for the same purpose as the brilliant green used by Fleming. Avery’s oleate blood agar medium he reported to be largely selective. It checked the growth of pneumococci and streptococci, but gave luxuriant growths of B. influenzæ. Pritchett and Stillman have used it with excellent results recovering B. influenzæ from a very high percentage of the cases studied.
The use of symbiotic bacteria has been extensively studied in investigations of the biology of B. influenzæ, and it has been shown, as noted elsewhere, that such accessory bacteria will bring to growth B. influenzæ on media otherwise quite unsuited to its needs. It has been further found that on various preparations of hematin agar, on which B. influenzæ refused to grow, such media could be rendered favorable for their growth by the addition of living or freshly killed cultures of staphylococcus and many other bacteria. And although the method is well known, it has not been extensively used for the purposes of isolation. Many of the workers, however, have pointed out the importance of looking for growth of the influenza bacillus in the neighborhood of the more easily grown bacteria which almost always develop in cultures from the respiratory tract. Grassberger has particularly studied this problem and has made practical application of the method. Accidental contamination of plates with air bacteria have made possible, in some instances, the isolation of B. influenzæ—as, for example, in the finding of Heyrovsky from a case of empyema of the gall bladder—while other workers have pointed out the difficulty of demonstrating growth where B. influenzæ is pure in the material cultured, and the comparative ease and relative luxuriance of growth where other bacteria are present. To just what this stimulating effect is due has been much discussed, and it is generally agreed that the hemoglobin is markedly changed and rendered more available by the action of these germs. It is to be noted that on a medium containing blood altered by heating or by the various methods as described by Fleming the foreign bacteria no longer show any symbiotic action on B. influenzæ. Grassberger considered the effect of the bacteria on the blood to be the same as that of heating. Allen laid particular stress on this symbiotic character. He used a staphylococcus, either living or killed, in making his cultures and noted the difficulty of growing B. influenzæ from material in which it occurred pure. W. F. Robertson made use of these facts of symbiosis for both isolation and stimulation of growth. He employed alternate drills of M. catarrhalis or pneumococcus with the B. influenzæ, and Brown and Orcutt used strains of hemolytic streptococci for the same purpose. The latter authors considered that the beneficial effect of the streptococci was merely due to the setting free of the hemoglobin. The fact that similar results are to be obtained by the use of non-hemolytic bacteria as well as forms giving green color changes to the blood makes this explanation untenable. In my own studies I have confirmed the results of several previous workers. I have found that B. influenzæ is stimulated in its growth by the presence near it of colonies of staphylococcus pyogenes aureus and albus, pneumococci, streptococcus viridans and hemolyticus and other bacteria. The largest colonies of the bacillus I have obtained were those growing near the periphery of a colony of an air nocardia. I have also noted that emulsions of a staphylococcus killed by boiling for five minutes, when added to ordinary blood agar, had a marked stimulating effect, although no evidence of hemolysis was present. This effect was practically absent if the emulsion was boiled for 15 minutes, or after being killed was left at room temperature for several days. There was no evidence of these stimulating effects by any of these methods when heated blood agar was used, the colonies on this medium growing equally large by themselves. Comparative studies of the effect of different bacteria can be simply carried out as follows: Smear evenly the surface of an ordinary blood agar plate with an emulsion of B. influenzæ. Seed this plate at various points with minimal amounts of the various bacteria. After various periods of incubation the size of the B. influenzæ colonies about the other bacterial growths can be estimated, and impression preparations on cover glasses will give very interesting pictures.
The growth of B. influenzæ in primary cultures from sputa and similar sources is to be explained by the probable presence of traces of blood or altered hemoglobin as well as the symbiotic relationship with other bacteria. Fichtner used fresh heated sputum (60 to 65° C.) in place of blood, and Richter a medium made with sterilized pus. Parker, in her study of a filterable poison produced by the B. influenzæ, found veal infusion broth with 10 per cent. defibrinated blood heated to 75° C. until the blood coagulated and settled on standing to be the best for the purpose. Jordan in his study of indol production by these bacteria used a meat infusion broth with 5 per cent. sheep’s blood added at 90° C. or over and filtered through cotton or paper. Wittingham and Sims noted that in using blood from influenza cases the bacteria frequently did not grow, more especially B. influenzæ; and Rivers found human blood poorer than cat or rabbit blood for growing this organism, as did Minaker and Irvine. It would seem clear from this review of some of the suggestive work on the methods of growing B. influenzæ that little attention should be given to the results of many workers, where ordinary media were used, particularly when the difficulties of isolation were not appreciated.
B. Influenzæ as a Pathogenic Bacterium
If B. influenzæ is the causative agent in clinical influenza, there is certainly ample evidence that it is pathogenic to man. The symptoms of toxemia, which are so manifest in the pandemic disease as well as in the sporadic cases, would indicate that the etiological agent is markedly toxicogenic. Animal experiments by Pfeiffer, and a long list of investigators following him, would seem to show that the majority of cultures of B. influenzæ do not have any power of establishing themselves in the animal tissue. Killed cultures showed equally as high toxic effects as the living, and so it was generally concluded that many of the general effects in influenzal infections were of a toxic nature.
There are many exceptions to the above-mentioned failures to produce infections in animals. Cantani obtained very constant positive results by subdural injections. He first clearly showed that killed cultures were markedly toxic and that virulence could be raised very definitely by animal passage. By injecting brain emulsion with a culture he obtained a subcutaneous abscess in a rabbit which after eight days still contained the living organism. Nastjukoff found that animals with a lowered resistance, or definitely ill from, for example, an artificial tuberculosis, became infected while others did not. Jacobson showed that B. influenzæ injected with streptococci caused a definite mixed infection, and that after six passages the influenza bacillus alone could produce a fatal infection. Saathoff (1907) confirmed Jacobson’s findings and found pneumococci equally effective. Davis (1915) also confirmed the principle established by Jacobson of the symbiotic relation of other bacteria to infection with B. influenzæ. He used a culture of a non-virulent staphylococcus pyogenes aureus, and was able to produce death invariably in guinea pigs after intraperitoneal injection. From the heart’s blood, as a rule, only the hemophilic bacillus was recovered. He also found animal passage increased the virulence, and further that M. catarrhalis and an avirulent streptococcus had the same effect as the staphylococcus. Slatineanu (1901) found that he could infect animals with B. influenzæ if the cultures were injected along with weak solutions of lactic acid, and that after animal passage by this method the bacillus became more virulent and would eventually kill by itself. It must not be forgotten in this connection that strains of B. influenzæ from meningitis cases are frequently definitely pathogenic for animals. The importance of considering these various factors in a discussion of infection by this organism is, of course, very evident. Ecker found his strains pathogenic for mice after subcutaneous injection, and the bacilli were readily obtained from the heart’s blood. Spooner and his co-workers from their results of more than a hundred intraperitoneal injections concluded that the organism is not pathogenic for mice.
In all animal experiments it is of the greatest importance that the bacteria be known which may interfere in the experiments through spontaneous infection (often liable to be induced by the injection) from the animal’s own flora, as well as the greater susceptibility of previously diseased animals (Nastjukoff). It would appear from the results of Bruschettini and Cornil and Chantemesse in the early days of the influenza bacillus, and those of Lamb and Brannin in their recent study, that these authors did not seriously consider the spontaneous infection of guinea pigs and rabbits with B. bronchisepticus or the bacillus of rabbit septicæmia, both morphologically, very similar to B. influenzæ. Rosenow in his experiments with streptococci from cases of influenza has also apparently failed to realize the importance of the lung lesions produced by the B. bronchisepticus in guinea pigs as reported by Theobald Smith, myself and many others.
Parker has found a filterable poison from the influenza bacillus which developed rapidly (6 to 8 hours) in a special heated blood broth medium, deteriorated rapidly even in the cold, and killed rabbits in quantities of 2 c.c. in from 1 to 3 hours. Rabbits could further be immunized against this poison, and their sera protected other rabbits against fatal doses. This is the first time that a true powerful toxine has been obtained. Couret and Herbert obtained toxine from B. influenzæ in Avery’s oleate broth. Huntoon and Ross also clearly demonstrated toxine production by this organism so that it would appear, with this confirmation, that the B. influenzæ can be definitely classed among the toxine producers. Toxemia being the most striking clinical characteristic of influenza, we have in these findings very strong evidence of the etiological importance of this hemophilic bacillus to the disease. A very interesting observation was made by Latapie that the serum of a goat immunized against influenza bacillus is toxic if it is used shortly after the injection of the microbes, but that this toxicity is absent three weeks after the last injection. It would appear to me that the evidence of a filterable virus from the secretions of the respiratory tract does not eliminate the very probable toxine from such materials. The production of toxine by this organism probably depends, as is the case with very many of our toxine formers, on the most favorable combinations of conditions. That it is not readily formed in artificial cultures, or that it is very unstable if formed, is evidenced by the frequent failures of a great many workers. It has been suggested that different symbiotic conditions in the respiratory tract determine the amount of toxine produced. Huntoon found a high toxine production in mixed cultures with streptococci. This, however, does not appear to be necessary, as there is ample evidence of severe toxemia from pure infections with B. influenzæ in various parts, such as the accessory sinuses of the head, the meninges, the lungs and other parts of the respiratory tract.
It is not fundamentally necessary that a toxine producing organism be present in overwhelming numbers before it can be accepted as the cause of the toxemia. Nor, on the other hand, must we have toxemia every time the organism is found. The prevalent idea among bacteriologists would appear to be the reverse of what I have just stated. It would, indeed, be extremely difficult to make bacteriological diagnoses of a great many of our diseases, where the etiological factor is well established, if these conditions were required. We do not do so, for example, in diphtheria, examinations of stools for typhoid, nor in infections with the tetanus bacillus. We recognize carrier cases of meningococcus, B. typhosus, hemolytic streptococci and many others, without detracting seriously from their importance in definite types of infection. Formerly the specificity of the different bacteria for definite disease processes was very rigid, but today we interpret more broadly the finding of gonococcus in endocarditis, the meningococcus in bacteremia, B. typhosus in osteomyelitis, streptococci and pneumococci in all manner of infections and many other bacteriological results. True it is that the various bacteria show predilections for attacking certain tissues, but the varying susceptibilities bring about the greatest variations in the manifestations of these infections.
The B. influenzæ is not confined to the causation of severe pandemic or epidemic influenza, but includes in its field purulent bronchitis, meningitis, sinusitis, conjunctivitis and many other pathological processes. It further should be recognized as a relatively frequent cause of complications in measles and other diseases.