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.

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.+–0 Strept. 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.++0 Lung. 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 culture 15
10 gave 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. culture 25
10 sterile.
7822683Br. numerous B.I. like scattered, some phagocyted. Fewer G+ flat pairs with capsule.+–0 Bronchus 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.++0 Bronchus 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.
7934103
2		Br. 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.
Total20132206416
B. influenzæ found—Percentage66½4614
Total percentage for B. influenzæ 78%

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.

Results of the Author

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 negative cases 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.