THE BACTERIOLOGY OF EPIDEMIC INFLUENZA WITH A DISCUSSION OF B. INFLUENZÆ AS THE CAUSE OF THIS AND OTHER INFECTIVE PROCESSES
ByW. L. Holman, B. A., M. D.
ByW. L. Holman, B. A., M. D.
ByW. L. Holman, B. A., M. D.
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.
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 meningococcalwork. 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.
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.
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.
TABLE I.BACTERIOLOGY OF THIRTY-TWO AUTOPSIES FROM INFLUENZA CASES.
TABLE I.
BACTERIOLOGY OF THIRTY-TWO AUTOPSIES FROM INFLUENZA CASES.
Autopsy Number.Date.Day of Disease.Hours P.M.DIRECT SMEAR—GRAM’S METHOD.B. InfluenzæPneumcocci.Strept. Mococci.Hemolytic Strept.S.P.A.Other Cocci.Other G—B.Other Bacteria.NOTES.Bronch.Lung.Pleural Fluid.7411918 Oct. 9316G +staph. Few pneumo-like. Few chains of elong. cocci.0+0+G+ diploc.Nine plates used to isolate B.I. Sp.a. overgrew all cultures. B.I. seen in blood smear agar in 24 hours.7431158Br. G—bac. from coccoid to short threads. Mostly scattered. Some phagocyted. Fewer G +cooci in short chains.+0–Pleural fluid and seen as diplos in direct smear.Br. G + diploc. not like pneumo.Br. lux. white almost coccoid.Pericard, fluid and liver juice, no growth.74411711Br. G—bac. moderately stout about in small groups and scattered. G+diploc (pneumo) also G— threads. Phago. of both in a few cells.+–0Lung +M. tetrag. in Br. M. pharyng. in Br.Pneumococcus from lung. No attempt after first plate to isolate B.I.74512106Br. G +–large bac., strept. short, G—B, few, very short, no threads.+0–Pleural fluid, also seen in smear.Pl. fluid, also seen in smears.Swab from ruptured rectus. Sterile. No material from lung.746125½Br. G—B very short, no threads. Irregularly scattered. More seen in left bronchus. A few cells phagocyted.+–0Strept. viridans from bronchus.B. coli from bronchi and lung.B. xerosis from bronchus.The overgrowth of B. coli in lung material prevented further attempts to isolate B.I.7471363Br. G+diploc, fairly numerous. G—B tiny, as diplos and in long threads scattered or in small groups. Pleural fluid and lung no bacteria seen.+––Bronchus and pleural fluid.Strept. viridans from bronchi and lung.B. subtilis group from pleural fluid.Five picks from blood agar plate failed to recover B.I. from lung.7481344Br. nothing like B. I. seen. G+ small elong. diplo. Numerous G + diploc. in lung. Comparatively few Q-B, very short.–+0Lung+, not isolated from bronchus.Strept. viridans from bronchus.B.I. not seen nor isolated from the bronchi.74914415Br. G+large pneumo like, many G+large bacilli, single and in pairs. Few G—B very tiny and widely scattered; lung, heavy mixture as in bronchi.––0Bronchus Lung?Bronchus and lung.B. coli from bronchus and lung.The overgrowth of B. coli prevented any further attempts to isolate B.I.7501496Br. G+B large like B. welchii, G—rather stout coccoid forms, G+C in pairs and short chains. Tiny G—coccoid forms like B. I. Lung G+ pneumo-like and caps, chains; no B. I.––0Bronchus? Lung?B. coli from bronchi and lungs.B. coli again present as in No. 749. Direct smear suggests heavy contamination.7511476Br. G +cocci large elong.? caps, also G +C in flat pairs. G—coccoid forms. Lung, numerous bacteria. G+strept. with flattened cocci. Some G-short forms?––+Pleura. Lung. Bronchus.M. tetragenous from bronchus.Spore-bearer with tiny cols, pleur. B. xerosis from bron.752151315Br. G+pneumo-like. G+B smaller than B. welchii, occasionally tiny G -diplobacillus. Lung, G+chains of cocci Gram weak. Few G—tiny bacilli scattered or in groups.–+0Bronchus and lung.Strep. viridans from bronchus and lungs.B.I. like seen in original culture on blood agar but not isolated from bronchus.75616818Br. numerous G+B. B welchii like. G—B large and few tiny. G+round diploc. Pl. fluid almost pure pneumo-like, few G-forms probably the same.–0–Pleural fluid.Strep. viridans from bronchus.B. coli from bronchus and pleural fluid.B. xerosis from bronchus.Compare No. 749 and 750. Fluid from lung not obtained for culture.75716614Br. G—B tiny, to medium. G—like M. catarrhalis. G+cocci, pairs and chains. Few B. W. like. Lung, many G—B like B. I. Some cells filled, also G—cocci. M. catarrhalis like and rare B. welchii like.++0Bronchus and lung.Bronchus and lung.M. tetragenous? from lung.B. xerosis from bronchus.This case 14 hours P. M. gave B.I. from all the material.758161416Br. pneumo-like in excess. G—B from tiny to forms stouter than B. I. Few strept. rare M. catarrhalis. Lung, pneumo-like. Phago.+?0Bronchus and lung.M. catarrhalia-like from lung. Strep. viridans from lung and bronchus.B. coli from bronchus.The B. coli did not prevent the isolation of B.I. like seen in original blood agar cultures of lung.76117719Br. pneumo-like. B. I. like common, M. catarrhalis like. Both B.I. and M. catarrhalis phagocyted. B.I. single or in threads. Some typical groups. Lung, pneumo, caps, rare, M. catarrhalis like.+–0Bronchus and lung.Bronchus and lung.B. coli from bronchus.Even after 19 hours P. M. the B.I. was isolated.762171012Br. numerous B.l. like typical, also many pneumo. and M. catarrh. Lung same. M. catarrh. phagocyted. B.I. smear, many phagocyted, many pneumo.+++Pleural fluid and bronchus.M. catarrh. like from lung and bronchus.B. xerosis from lung. B. subtilis from bronchus.763171113Lung, pneumo-like, slight phagocytosis. Pl. fl., pneumo and few strept., slight phagocytosis.0––Pleural fluid.No growth from lung on plate. B.I. like seen in original culture from pleural fluid. No material from bronchus.7641796Br. B.I. smear. Cells crowded. Pneumo-like fewer, occasional G—stouter thread.+00Bronchus.Staph, albus from bronchus.Material only from bronchi.76517916Br. pneumo. B.I. few scattered. G+flattened diploc. Phago. of B.I. and pneumo. Lung, pneumo-like, rare strept. very questionable G—B free and in cells.++0Lung.M. catarrh. from bronchus and lung.B. coli from bronchus and lung.767181014Br. rather round pneumo-like with caps. B.I. few. Scattered, also in cells. Lung, few bacteria. G+strep. often phagocyted.––0Bronchus.Lung.Bronchus.Blood culture1510gave pure growth of pneumo. mucosus.77019119Br. crowded with B.I. like. Few G+cocci and fewer M. catarrh. like. Pl. fluid G+flattened pairs, pus cells, phagocyted.++–Bronchus.Bronchus, lung, pleural fluid.7732120 Recurrence.3Br. few bacteria G+and G—pneumo-like. Rare G+–thread. Lung, pneumo and rare strept. Pl. fluid, pneumo-oat shapes, etc.–––Bronchus.Strept. viridans bronchus. Sarcina albus lung.B. xerosis from bronchus. G + B lux. white pleura. fluid.No growth from lung except sarcina. Only 2 colonies from pleural fluid on blood agar plates.778242317Br. B.I. smear. Fewer large pneumo. Lung, G + small diploc. Few B.I. like. Pl. fluid, few cells, no bacteria.++–Bronchus and lung.Lung.Non-motile, non-fermenting, lux, white from bron.Ten plates and 30 picks were done for the isolation of B.I.7812654Br. crowded with staph. like. Fewer G—B, larger than B.I., few M. catarrhalis like. Lung G+ small staph. like, caps, cocci in pairs and chains. Few tiny G—B. Pl. fluid pneumo-like and elong. cocci in chains capsulated.–+–Lung and pleural fluid.Bronchus and lung abscess.Staph. albus and sarcina from pleural fluid.B.I. like seen from 24 hour Ht. blood agar from bronchi and lung but only isolated from lung on replating. Bl. culture2510sterile.7822683Br. numerous B.I. like scattered, some phagocyted. Fewer G+ flat pairs with capsule.+–0Bronchus and lung.No B.I. like on 24-hour Ht. blood agar from lung.7832681Br. G+small caps, pneumo-like. Lung poor smear, occasional pneumo-like.+–0Lung.Bronchus.M. catarrh. like bronchus.No B.I. like on 24-hour Ht. blood agar from lung.7842886Br. capsulated pneumo-like, few strep. Lung, chiefly pneumo-like. few G—B like B.I., also G—pneumo-like.++0Bronchus and lung.Bronchus and lung?M. catarrh. like bronchus.Numerous B.I. like on 24-hour Ht. blood agar of bronchi and fewer from lung. Isolated by replating.7862942Br. G+cocci in round pairs and rather flat chains, suggested caps. Tiny G—B very rare. Lung streptococci flattened, often phagocyted.+–0Bronchus.Bronchus and lung.Staph, albus from bronchus.Pleural fluid not collected sterilly, Haemol. strept. isolated.7872982Br. numerous pneumo-like, bacillary forms. A rare suspicious B.I. like, some of these in cells. Lung, caps, elongated diplos, and chains of elong. cocci.++0Bronchus and lung.B.M.C. from bronchi.All the bacteria isolated were seen in 24-hour Ht. blood agar cultures from bronchi and lung.791Nov. 166Br. few bacteria. G+pneumo-like round, G—B and threads, size varies, like B.I. Lung, G + caps, pneumo. G+Large B. few suspicious G—coccoid forms. Pl. fl. caps, pneumo and caps, elong. chains.++–Bronchus and pleural cavity.Bronchus and lung.Replated from Ht. blood agar to isolate B.I. from lung.792263Br. caps, pneumo-like bac. forms and chains. G-caps, pneumo-like. Few G—B. questionable. Lung. caps, pairs and chains of elong. cocci, in cells. Pl. fluid, numerous caps, chains of diploc.++–Bronchus, lung and pleural fluid.Staph. albus, lung, strept. viridans lung, M. catarrh. like lung and bronchi.B.I. like seen on 24-hour Ht. blood agar from bronchi and lung but not pleural fluid.79341032Br. M. catarrh. and G+cocci, few bacteria, few G—B. Ear, G+cocci.–––?Throat.Bronchus, lung, arm vein, spleen ear.Throat, ear and bronchus.Strept. viridans from throat.B. coli from throat.B.I. like never seen except from throat which may have been B. coli.Total20132206416B. influenzæ found—Percentage66½4614Total percentage for B. influenzæ78%EXPLANATORY NOTE.B.I.—B. influenzæ.S.P.A.—Staphylococcus pyogenes aureus.M. pharyog—Micrococcus pharyngis siccus.Br.—Bronchus.Phago.—phagocytosis.Ht.-Heated blood agar.B. W.—B. welchii.
Staphylococci develop opaque, paint-like colonies of varying size, with or without hemolysis, and so do other less frequently found bacteria give more or less distinctive colonies. The heated blood agar does not show these differences.
The colonies most liable to be confused with those of B. influenzæ are, therefore, those of B. xerosis, immature colonies of the Gram negative cocci and certain colonies of the streptococcus viridans group. Transfers should always be made to heated blood agar of all colonies suggestive of B. influenzæ, or when the growth of the B. influenzæ has only occurred in the more crowded portions of the plate, and it is difficult to pick pure cultures, attempted pickings should be made to this medium for further platings. It is frequently necessary to make further blood agar plates from the original blood agar, blood broth or heated blood agar cultures after longer incubation periods, depending on the findings in smears from these media. The heated blood agar is the best of these to encourage the growth of B. influenzæ. It must, however, be used at once, or within a very few days of its preparation, and cannot be kept on hand as a stock medium. I have not found it as useful for plating because of the difficulty of differentiating colonies. The phenomenon of the star-like and more luxuriant growth of the colonies of B. influenzæ about colonies of other bacteria has often been noted, and will be referred to in a later portion of this report. Here it may be said that this is at times a marked feature of certain mixtures and must be recognized in studying the plates. The finding of B. influenzæ in picks from apparently isolated colonies of other forms is not uncommon, and is the same type of difficulty which I have discussed in papers on streptococci. It is important to recall, in connection with cultures taken from the lungs at autopsy, the experimental work of Norris and Pappenheimer, who showed that B. prodigiosus put in the mouth immediately after death could be recovered from the lungs in over 50 per cent. of the cases studied.
In Table I are shown my results from the 32 cases which came to autopsy. The B. influenzæ was isolated from one or more sources in 25, making a total of 78 per cent. Most of the negativecases probably also had this organism, but I did not grow it from the material which I used for culturing. The work of others would indicate that it may have been present in other regions, such as the sinuses of the head or other portions of the lung and respiratory tract. The positive results show B. influenzæ present in 20 out of 30 cases from the bronchi; in 13 of 28 from the lungs; in 2 of 14 from the pleural cavity; in 9 of 26 from both bronchi and lung where both were cultured; in 8 of 26 from the bronchi with the lung negative; in 3 of 26 from the lung with the bronchi negative; once of 10 from the pleural cavity with both the bronchi and the lung negative, and once from all three sources.
The negative results occurred in seven cases. In three of these (749, 750, 756) B. coli overgrew the cultures from the bronchus, in two also from the lung, and in one, without lung culture, from bronchus and pleural cavity. The mere presence of B. coli, however, did not preclude the isolation of B. influenzæ, as is seen in cases 746, 758, 761 and 765. The finding of B. coli would suggest a post-mortem invasion. The hours after death before the autopsy was done were in these seven cases, ½, 15, 6, 18, 16, 19, 16, respectively. That delay in performing the autopsy, as emphasized by Spooner, Scott and Heath, adds to the difficulty is self-evident, but successful isolations of B. influenzæ have been obtained after even longer periods than in the negative cases (761). In the fourth negative case (763) the bronchus was not cultured. A pneumococcus was grown from the pleural cavity and no growth was obtained from the lung. In the original culture from the pleural cavity influenza-like forms were seen but could not be isolated. In the fifth case (767) a blood culture three days before death gave a growth of pneumococcus mucosus which was also grown from the lung at autopsy. Direct smear from the bronchus showed very few influenza-like forms. Our sixth negative finding was in a case of 20 days’ illness, the patient having had a recurrence (773). Staphylococcus pyogenes aureus, streptococcus viridans and B. xerosis were grown from the bronchus. Only a sarcina form grew from the lung, and a further probable air contamination occurred on the media from the cultures of the pleural cavity. The B. xerosis colonies were confusing, picked as possible B. influenzæ,and, before this was discovered, the overgrowth prevented further attempts to isolate the influenza bacilli. The last unsuccessful case was one with a general infection of a hemolytic streptococcus from an acute otitis media. The streptococcus was isolated from the bronchus, lung, spleen, arm vein and the middle ear at autopsy.
It will be seen that in these seven negative cases technical difficulties prevented the isolation of the B. influenzæ, even if it had been present. I would not, therefore, conclude that the organisms were necessarily absent, but rather that we have failed either to secure material from the focus of infection or on account of the other reasons mentioned.
It is very evident that a variety of secondary organisms very frequently overgrow the field and become numerically predominant. In our first case staphylococcus pyogenes aureus overgrew all the other organisms present in cultures from the lung material. B. influenzæ was, however, seen in the original 24-hour blood agar culture. It required 9 blood agar plates before the organism could be isolated. In another case 10 plates were used for the isolation.
The findings of the bacteria in the lung sections are particularly interesting and instructive. The entire series of cases have not been completely studied, so I am unable to tabulate the findings. In cases 761 and 762 sections of the lung showed influenza-like bacilli to be almost pure in the earlier stages of the process, while in areas with purulent foci pneumococcus-like and other Gram positive cocci were also numerous. In some cases B. influenzæ-like organisms were to be seen in overwhelming numbers. In others they were scarce, while in some nothing resembling B. influenzæ could be found in the sections. Positive cultures were often independent of whether the influenza-like forms were to be seen in smears or sections or not, although they were found in the great majority of the cases. The findings in the direct smears and the bacteriological results make useful material for comparison.
Swabs from the nasopharynx were cultured from 31 individuals; nearly all of these were cases suspected of diphtheria or as carrying the diphtheria bacillus, and no particular effort was made to isolate the B. influenzæ. They were seen in themixed culture occasionally. In the last eight cases the heated blood agar, ordinary blood agar and Loeffler’s serum were seeded from the throat swabs. B. influenzæ practically overgrew all the other bacteria from seven of these cases on the heated blood agar medium and was isolated without difficulty; all eight showed M. catarrhalis. The two other media gave little or no evidence of the presence of B. influenzæ. As I have said above, our attention was concentrated on the autopsy material. These cultures from the throat were simply made to demonstrate the usefulness of the heated blood agar.
TABLE IIBACTERIA SEEN IN DIRECT SMEARS FROM NASOPHARYNX
TABLE IIBACTERIA SEEN IN DIRECT SMEARS FROM NASOPHARYNX
TABLE II
BACTERIA SEEN IN DIRECT SMEARS FROM NASOPHARYNX
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.