Part 22

BACTERIA SEEN IN DIRECT SMEARS FROM NASOPHARYNX

════════════╤═════════╤═══════════════╤══════════════════╤═════════════════ Type of │Number of│ B. │ │ M. Disease. │Patients.│Influenzæ-Like.│Pneumococcus-Like.│Catarrhalis-Like. ────────────┼─────────┼───────────────┼──────────────────┼───────────────── Early │ 24│ 14│ 17│ 6 Serious │ 13│ 13│ 13│ 9 Convalescent│ 11│ 8│ 11│ 6 ────────────┼─────────┼───────────────┼──────────────────┼───────────────── Total │ 48│ 35│ 41│ 21 Percentage │ │ │ │ of │ │ │ │ positives │ │ 73│ 86│ 43 ────────────┴─────────┴───────────────┴──────────────────┴─────────────────

Direct Smears from Nasopharyngeal Swabs

It is recognized by most of the modern investigators that little reliance can be put on the finding of B. influenzæ-like bacilli in direct smears. The organism is markedly pleomorphic, occurring as extremely small coccoid forms up to threads of various lengths. Notwithstanding these morphological variations the organisms are usually seen as tiny bacilli, and these are considered as the typical form. We carried out a series of microscopical examinations of carefully made smears from the throats of patients with influenza. Particular attention was given to the occurrence of organisms resembling in morphology and staining B. influenzæ, pneumococci and M. catarrhalis. We have divided the cases roughly into three types—early, serious, and convalescent. Table II shows our results. The term B. influenzæ-like was used for the typical morphological picture so often described. Dr. Frost and Mr. Scott carried out this portion of our work and their results are interesting.

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.

Symbiosis.—The fact that other bacteria can bring to growth the influenza bacillus on media otherwise unsuited to its needs brings up the interesting problem of symbiosis, which is one of the most important characters of the influenza bacillus. Not only do other bacteria make possible the growth of B. influenzæ on media on which the influenza bacillus will not grow, but they stimulate a better growth on blood agar and other more or less favorable media. Grassberger first noted this stimulating character of other bacteria and described and illustrated the very large colonies of B. influenzæ which develop in the neighborhood of colonies of staphylococcus and other bacteria. Staphylococci killed by heat were found to have a similar effect. Meunier nicely described this phenomenon by using the term satellites for the circles of B. influenzæ colonies which develop about the colonies of other bacteria. A great number of workers have since noted this characteristic relationship between B. influenzæ and other bacteria, and occasionally have laid stress on its importance in the problems of the infections by the influenza bacillus. Allen particularly emphasized the probable importance of this in discussing the problem of carriers of B. influenzæ as sources of danger. There seems no doubt that this symbiotic relationship depends on so altering the hemoglobin products as to render them more readily available for the influenza bacillus. This is indicated by the fact that on various media containing hemoglobin, altered so that it encourages the growth of B. influenzæ, no such symbiotic stimulation can be demonstrated. This phenomenon is quite peculiar to this bacillus, distinguishes it from most of the other members of the group, and should be always determined before an organism is classed as B. influenzæ.

Other Hemophilic Bacteria.—The question of a pseudo-influenza bacillus was first raised by Pfeiffer and has been studied by many workers after him. Grassberger, who carefully investigated this problem, worked more particularly with two strains showing the extreme of variation between the small characteristic morphology of the B. influenzæ and the thread forms supposed to be characteristic of the so-called pseudo-influenza bacillus. The great majority of workers have agreed with him in concluding that this morphological variation is not sufficient nor constant enough to justify separating two such groups. Nevertheless many reports indicate peculiar tendencies of certain strains toward thread formation. There seems to be suggestive evidence that the organism described by Cohen in 1909 under the name B. meningitidis cerebrospinales septicemicus is different from true B. influenzæ. Although the cultural characters were apparently identical, this organism was definitely pathogenic for guinea pigs and rabbits. The involvement of joints in the cases reported by Longo and others would suggest a greater pathogenic power for these strains. Prasek and Zatelli reported a similar bacillus from meningitis, and Davis found that his meningitis strains were more pathogenic for rabbits than were others. Wollstein has studied this question very carefully and found a marked difference between the strains from the meninges and those from the respiratory tract in their pathogenicity for rabbits. The strains with a tendency to thread formation were usually also those grown from the meninges, but she concluded from the results of serological tests that all strains of B. influenzæ are of one race, irrespective of their origin or virulence. The question is still an open one, as Batten and others described strains from the meninges which are non-pathogenic, and Ritchie found his strains from meningitis pathogenic for guinea pigs but not for rabbits. The irregularity and wide divergence in the results of blood cultures may have a definite relationship to these differences in the pathogenicity of strains.

Other hemophilic bacteria include the bacillus described by Friedberger under the name of B. hemoglobinophilus canis. This organism is to be found in the preputial secretion of dogs. It does not show the phenomenon of symbiosis, and I have found that it grows rather more freely and is more resistant to drying than is the influenza bacillus. Krage has confirmed Friedberger’s findings growing this bacillus from 60 per cent. of his dogs, and believed it a pyogenic organism just as B. influenzæ may be.

The hemophilic and hemolytic organisms described by Davis, which he isolated from pathological urine, were non-symbiotic and non-pathogenic. Koch has described a similar organism from puerperal infection. Whether the hemophilic organism described by Thalhimer from the uterus in a case of puerperal infection, those found by Cohen in urethral discharge in one case and the pelvic exudate of another, and the findings of Kretz in pyelitis, Wright in pyelonephrosis and Klieneberger in cystitis cases, possibly refer to this same bacillus is, of course, uncertain. Pritchett and Stillman found a somewhat similar bacillus, which they called Bacillus X, from the mouths of 24 persons. It was hemophilic and hemolytic, stouter than B. influenzæ and showed long tangled threads in blood broth. It was non-pathogenic and is probably the same as Davis’ organism.

Davis described another hemophilic bacillus from a patient with purulent foci which was non-hemolytic and non-symbiotic. It was grown from an abscess of the shoulder joint, the blood and the bronchial secretion of an infant. Cyanosis was a marked feature of this case. Paranhos described a hemophilic bacillus from meningitis, which, however, was Gram positive, and Moon reported an anærobic hemophilic bacillus from an infection of the ethmoid sinus. The work of Jordan would suggest that there may be two groups of B. influenzæ based on the indol production.

Morphology.—The morphology of B. influenzæ has received more than usual attention. In what we consider its characteristic form, it is an extremely small bacillus, usually single but sometimes in pairs, and not infrequently exhibiting polar staining. In direct smears, where there are many bacteria present, they are frequently arranged in the schools so frequently described. The development of thread forms is today considered quite characteristic for B. influenzæ. The organisms vary from moderately long bacillary forms to very long twisted or curled threads suggesting leptothrix. In such cultures chains of tiny bacilli are also quite often noted. At the other extreme we have exceedingly tiny coccoid forms, resembling in size the B. bronchisepticus, which, as Ferry has shown, are small enough to pass through many grades of filters.

It is the thread forms, as discussed above, that have received most attention in relation to the so-called pseudo-influenza bacillus. The observations of Wollstein, Lacy and many others showed these forms to be common in meningeal infections and that, as a rule, they are more pathogenic for animals than other strains. Another interesting and important observation is that emphasized by Dick and Murray of the possible confusion of these forms with Gram negative leptothrix. That this confusion is liable to occur is illustrated by reports such as Macdonald finding leptothrix in a meningeal infection, now looked upon as an example of influenzal meningitis, and the probable B. influenzæ reported by Dick, and, as quoted by Dick and Murray, the finding of a Gram negative leptothrix as the cause of broncho-pneumonia by Kato. The 2 per cent. leptothrix reported by Nuzum and his co-workers from the recent epidemic may be still another example. Equally important is the recognition of the great frequency of this thread development in the majority of B. influenzæ cultures on ordinary blood agar media, or even in the water of condensation of fresh blood agar tubes. The delayed growth of this bacillus on ordinary blood agar would lead to its being frequently overlooked unless smears are made, and the irregular thread forms are recognized as being the B. influenzæ. This development of thread forms was particularly noted in my work before pickings were made to the Voges heated blood agar, but because I had been forewarned by discussing these morphological variations with Lacy, I was able to recognize them as forms of B. influenzæ. Most of my early isolations showed these predominating, and they were also noticed in cultures sent from the Public Health Laboratory at Washington. These cultures on further transfer, however, showed in 24 hours the typical small form on ordinary blood agar as well as on the Voges medium. On the latter the development of thread forms was greatly delayed and frequently did not appear at all, although after long periods other abnormal, swollen and irregular shapes sometimes developed.

Media in Growth of B. Influenzæ

The discovery of the hemophilic character of B. influenzæ has been confirmed by a long list of investigators. The agar smeared with pigeon blood as used by Pfeiffer has not, however, been found fully satisfactory and many modifications have been made. The fact that hemoglobin in some form is necessary for the growth of these bacteria has led to a great deal of study in attempts to discover the chemical part, or parts, essential for this purpose. Hemoglobin in very small amount, as shown by Davis and others, is sufficient to make media suitable for growing B. influenzæ. This fact has led to much confusion, owing to the difficulty of eliminating all possible sources from which some form of hemoglobin might enter the media. Kitasato used a glycerin agar and succeeded in growing the influenza bacillus for 10 transfers. Pielicke, however, did not consider that Kitasato was actually dealing with the influenza bacillus, but that he as well as Babes, Bruschettini and Markel had most probably streptococci in their cultures. Besson held the same view of Kitasato’s organism. It would further appear from the illustrations of Klein that he also grew streptococci and not the B. influenzæ. The first culture of the influenza bacillus was probably obtained by Bujiwid in February, 1890. He grew on agar smeared with the spleen pulp of an influenzal patient a tiny bacillus which he was unable to grow on blood free medium, but he did not appreciate its importance until Pfeiffer’s article appeared. Teissier in his book on “L’ Influenza en Russie” mentioned this culture.

The hemophilic character of these bacteria indicates that they are rather strict parasites, and despite the researches of Nastjukoff with various egg media, and Cantani with a number of supposedly non-hemoglobin additions to the agar, as well as the studies on symbiosis, with other bacteria, by Cantani, Neisser, Luerssen and many others, it still remains true that some form of hemoglobin is necessary for their growth. Fresh blood either incorporated in the medium or smeared on the surface is not the best medium for these bacteria. Altered hemoglobin is much more favorable, and a variety of methods have been devised to bring about those alterations which stimulate the growth of B. influenzæ. One of the earliest, as well as one of the very best, of these is the method of Voges, who added blood to melted agar at a temperature of about 100° C. I have found this medium exceptionally suited to growing B. influenzæ, and I consider it excellent for the primary culture from the original material, for pickings from plates and to obtain a heavy growth of B. influenzæ for any purpose. The medium was used by Delius and Kolle (1897), Grassberger (1898), who spoke very highly of it, and Paltauf (1899), who said that the use of this medium made the demonstration of B. influenzæ possible when only a very few were present. A great many other workers have used it with success, and during the recent epidemic it has gradually found its place. Levinthal’s medium (1918) is practically the same, although he boiled and filtered the agar after the addition of the blood. The growth of B. influenzæ on the Voges agar can properly be described as luxuriant, and to anyone only accustomed to the use of ordinary blood agar it is an agreeable surprise to see this supposedly delicate bacillus growing so remarkably well.

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