STUDIES ON BACTERIAL NUTRITION. 1. GROWTB OF BACILLUSINFLUENZE ~HEMOGUIBLN-FBXE MEDIA. BY THRODOR TEJ~~A,M.D. (From t.40 EospitcJ of The Rockejdlc~ Insti#u& for Me&d Resuwch.) (Received for publication, March 8,1921.) Pfeifkr in 1892 fnst obtained growth of B. injluGnsa by the use of agar slants, the surface of which was smeared with a few drops of human blood. Pfeiffer showed that this bacillus grew ouly in media containing blood or hemoglobin. The influenza bacillus was thus brought into the group of the hemoglobiiophilic bacilli and has since always been considered an obligate hemophilic organism despite the fact that the literature shows that several investigators have been able to cultivate it in media free of blood or hemoglobii The first to show this fact was Cantani (1), who in 1901 obtained growth of B. &pknare on ascites agar in symbiosis with other bacteria, such as gonococcus, diphtheria bacillus, and several large cocci. B. it@mle grew in giant colonies in the vicinity of the other colonies. Cantani also obtained growth on the sur- face of agar to which had been added emulsions of bacteria hilled by heat at 6O"C. for 3 hours. Cantani excludes symbiosis and supposes that some factor in the bacterial cell induces the growth of B. in&enr.te and that this factor is more easily liberated from the dead than from the living cell. Neisser (2), however, demonstrated the growth of iufluenza bacilli in symbiosis with xerosis bacilli on plate cultures made from the in&.ned conjunctive of a child. Impure colonies were observed which could be transferred through many generations on plain agar without the disappearance of 8. injwsa. The latter, however, failed to grow on this medium if the xerosis bacillus was not present. Neiser ah tried to cultivate B. inpucnSa on media prepared by adding hilled emulsions of the xeroais bacillus or the diphtheria bacillusto plain agar. A slight growth ocxxrrecl for three or four generations, but the continued cultivation of B. irzjhunta on this medium was impossible. He therefore considered the growth of B. &?knsar in mixed cultures as a symbiotic phenomenon. Grassberger (3) observed that when 8. im$wnea was grown on blood or hemoglobin agar plates in sssociation witb other bacteria, especially staphylo- cocci, the colonies of influenza bacilli adjacent to the colonies of cocci were of un- usually large size. He assumed that the cocci exerted some elkt upon the blood medium that was beneficial for the growth of B. is*. 763 764 STUDIES ON BACTERIAL NUTRITION. I Luerssen (4) c&irmed the observations of Cantani by growiug B. iqknm on agar containing emulsions of dead staphylococci, B. coli, or B. prodigimus. Growth of B. i@emz did not occur in ordinary mixed cultures with these other organisms. The bacterial emulsions were sterilized by heating at 60oC. for 3 hours; if they were boiled the growth which occurred was not so good. Luerssen presumes that the bacteria contain a factor that exerts a stimulating effect upon the growth of B. i$-@, and that this is destroyed at high temperatures. He found that this growth factor is contained in the bacterial cell, since the carefully washed cells are still active. Growth of B. in&mm did not occur if the enrich- ing emulsion of dead organisms was smeared on the surface of the agar; it only occurred if the emulsion was incorporated in the medium. Luerssen also ob- served that B. in$m grew sparsely in sterile fikates of broth cultures of staphylococci, B. diphtherb, and B. w~olaaus. Ghon and von Preyss (5) attempted to grow B. itrpUe~r~ in media containing no hemoglobin, but the results were negative and they concluded that the reason other investigators had been successful was that in making the inoculations small amounts of blood had been carried over. They lihewise held the opinion that when B. infkunsce grows on plain agar, as occasionally happens, thii agar contains traces of hemoglobin. Recently Putnam and Gay (6) tried to con&m the experiments of the earlier investigators. They were unable, however, to obtain any growth of B. injhema on plain agar to which either hilled or livfng cultures of B. azrosis, B. diphthwicz, 8. coli, or staphylococci had been added. In spite of the observations concerning the growth of Bacdlw Gz..uensa in media free of blood or hemoglobin, the opinion that this organism is hemoglobinophilic has not been altered, and for its cultiva- tion media containing blood or hemoglobin in some form is always employed. EXPERIMENTAL. In a study on the growth of mucoid bacilli and the transformation of non-mucoid bacilh into mucoid ones (7) the writer undertook the cultivation of different microbes, Budhs puru#hm..v B and pneu- mococci, in broth containing mucus produced by mucoid bacilli such as Friedliinder's bacillus, the ozenabacillus, and other similar organ- isms. An attempt was also made to cultivate Bacillus in$wze in this medium. The development of this work led to the present studies on bacterial nutrition. TBJZODOR lXIJ6TTA 76.5 &p&ncnJ 1.-A culture of a mu&d Gram-negative bacillus (FrkUnder's) was grown on plain agar. After 24 hours iu the incubator the growth from each plate was suspended in a few drops of plaiu broth. 0.5 cc. of this emulsion was added to 5 cc. of plain broth, then heated for 1 hour at 6O"C., tested for sterility, and stored in the iee box. The reaction of the broth, pH 7.8, waa not altered by the addition of the bacterial emulsion. With the medium preparui in this manner, the foliowing tests were made. A tube of this medium was inoculated with influenza bacilli from a biood broth cuiture, the b&&i celis of which had compieteiy settled to the bottom of the TABLE I. c;rmufk of B. inf- in Plain Broth Aa'&d to .%nW of Fricdwndcr's P-k Badlus. Emulsion broth 1 " 4s 2 " I` 6 = I. - - - - 1 2 3 4 3 6 8 9 10 - - - -- - - - - 1 - k-l - - - - - - - tube. The following day 0.2 cc. of this subculture was transferred to another tube of emulsion broth. Siuitaneousiy a ioopfui of the first culture was streahed on the surface of blood agar to demonstrate the growth of B. in-. The last procadure was nSemary since the emulsion broth in itself was too cloudy to show growth. From the first culture there were made eight succesive transfers to emulsion broth, in all nine transfers from the original blood broth culture. The result of these eqeknents demonstrates that the infiueuza baciihrs grows well in the emulsion broth described. In this medium B. &$MMZ was found living and capable of multiplying after 10 days at 37oC. (Table I), No. of days. 766 STUDIES ON BACTERIAL NUTRITION. I Experiment 1 was repeated in emulsion broth prepared in the manner described from the mucoid growth of an organism class&xi as Baciuur ozana. fnthipexperimentthcgrowtbwaswashedofithe~~ofplainagarwith 1 cc. of normal saline solution and the bacteria were hilled by heating at 7O"C. for 1 hour. 0.5 cc. of this sterile emulsion was then added to 5 cc. of plain broth. The emuhlon broth was inoculated with 0.1 cc. of a blood broth culture of B. i&etzscz after all blood cella had settled to the bottom of the tube. 24 hours later 0.1 cc. of this first emulsion broth culture was transferred to a second emul- sion broth tube and simultamoualystreaked on the surface of blood agar to deter- mine growth. Tlils series of c&urea was carried on succ&ully for ten transfers in em&ion broth, and then was voluntarily discontinued. The ten consecutive cultures all showed typical colonies on the blood agar, and %LS of the last seven transfers showed typical bacilli and a few involution forma of B. i?@&r&r. TABLE II. D+siwminafh of Stnalkst Aewnnt of Em&h Ca#abZe of Stimdating Growth of B. in-. Tuba NP. Amount of broth. cc. 5 5 5 5 5 Amount of malLion. 01 0.3 0.1 0.01 0 GKwtb. -t-4-+ +++ +++ I + Ezpm Z.-After it had been shown that an emulsion of heat-killed mucoid bacteria, when added to pJain broth, is able to support growth of the influenza bacillus, it seemed desirable to learn how small an amount of the emulsion would m&e for this purpose. Accordingly, dilutions of the emulsion in broth were made and inoculated with comparable amounts of a culture of infiuenza bacillus. After 24 hours incubation subcultures were made on blood agar to co&m growth in the cukures containing various dihrtions of the emulsion. These results are recorded in TabIe II. 0.01 cc. is evidently the lower limit of the growth-stimulating sub- stance in this park&r emulsion of heat-kikd mucoid bacilli, since cultures containing this amount showed less growth than those in whkh larger quantities were used. THEODOR TIIJ&TA 767 l&#rimont 3.4.n order to determine whether the growth of B. in- in the emulsion broth was more pronounced in the bottom of the tubes or at the surface of the broth, tubes were inoculatai after all mucoid material had settled to the bottom. They were dowed to stand in the incubator for 24 hours and subcultures were made both from the thick residuum in the bottom of the tubes and from the superiicial layers of the broth. The growth was compared with the following result. Qrolvtll. (a) From bottom of tubes . . . . . . . . . . . . . . . . . . . . . . a.. . . . . . . . . . +++ (b) From surface . . . . . . . . . . . . . . . ..*............m.*......... + The fact that this accessory substance appeared to be more con- centrated in the immediate vicinity of the sedimented bacterial emulsion than in the upper portions of the culture fluid seems to in- dicate that the growGA.imulating factor is contained within the bacterial cell and slowly passes out into the surrounding fluid. It therefore seemed reasonable to attempt to extract this substance from the bacterial emulsion. `Phe addition of a clear bacterial extract to media, moreover, would have the advantage of making it possl%le to observe bacterial growth directly without the secondary transfer to blood agar. This was done in Experiment 4. RzpairnnJ k-The growth of a mucoid organism was collected from agar plates and emulsifkd in plain broth, 1 cc. of broth being used to each pIate. The em&ion was boiled for 5 minutes, and then centrifugA to separate the clear fluid extract from the bacterial bodies. Tks sterile extract was then tested for growth-stimdatlng action by the addition of decrea&g amounts to plain broth. The medium prepared in this manner was inoculated with one drop from an emul- sion broth culture (Table I, No. 6). The results of this experimat are recorded in Table III. TABLE III. Growth-lnduting Adim of an iWad of Jiucoid Bacteria on B. in..menxtt. Amountof- Gd. 0:; )r-f 6.0 ++ 0.1 2.0 ++ 0.03 1.0 + 0.01 0.2 0.001 0.02 0 0 768 STUDIIZS ON BACTERIAL NUTRITION. I It is evident from Table III that it is possible by simple boiling of an emulsion of mucoid organisms to obtain an extract which when added to plain broth is capable of inducing growth of Bwillzssdajm, That the first extraction of the bacillary emulsion does not completely exhaust it of this growth factor is shown in the following experiment. Ex#r&nt& J.-An emulsion of mucoid bacilli was made as previously de- scribed. A portion of this emulsion was heated at 60oC. for 1 hour and auotha portion was boiled for 5 minutes and then centrifuged and the clear supernatant extract pipetted off. The bacterial residuum was then washed in normal saline solution three successive times aud the following experiment carried out. GDWt.b. 5 cc. of plain broth + 0.5 cc. of unboiled emulsion heated to 60oC. for an hour . . . . . . . . . . . . . . . . . . . ...*..*..........*.. + 5 cc. of plain broth + 0.5 cc. of extract from boiled emulsion. . . -I- + 5 cc. of plain-broth + 0.5 cc. of residuum from boiled emulsion. ++ 5 cc. of plain broth (control) . . . . . . . . . . . . . . . . . . . ..*.......... - In this experiment &extract and the residuum from the boiled emulsion, when added to broth, gave even better growth than the emulsion heated at 60oC. for 1 hour. That extraction of the growth-inducing substance is obtained simply by allowing the bacterial cells to remain in contact with broth for some time is shown in the following experiment. EzpW &-To two tubes of pIain broth there was added 0.5 cc, of a b&I- lazy emulsion which bad been heated to 60oC..for 1 hour. The tubes were then left in the ice box for 1 week. After this time, one tube was centrifuged and the clear supematant fluid used as culture medium, while from the other, the super- natant fluid was pipetted off without being centrifuged. The two tubes were inoculated with 0.1 cc. of au emulsion broth culture of B. i&wmz. Good growth occurred in both tubes. This experiment indicates that the growth-inducing substance passes from the bacterial cells into the surrounding fluid and there exists apart from the cell. In the foregoing experiments it has been shown that it is possible to obtain good growth of Bacillus &&mm in plain broth to which emulsions and extracts of mucoid bacteria have been added. It seemed reasonable, therefore, to seek the same growth factors in other microorganisms, and BaciJk poteus wis selected as an organ- ism which normally shows an abundant growth on ordinary media. THZODOB TEJ&CTA 769 ExpeSmtM 7.-Agar plates were inoculated with B. prolcw. After 24 hours growth, to each plate 1 cc. of normal saline solution was added, and the growth washed off and collected in a sterile centrifuge tube. The emulsion was boiled for 5 minutes. The sterile emulsion was then centrifuged and the supernatant fluid pipetted off. This extract was clear, yellowish in color and had a reaction of pH 7.4. 0.5 cc. quantities of this extract were used to enrich plain broth. After the addition of this amount the beef infusion broth remained clear, so that eventual growth could be indicated by the turbidity of the medium. Control cultures, however, were always made from the extract broth on blood agar or oleate hemoglobin agar, and in plain broth. In the fluid medium thus prepared the following experiment was made. From a 24 hour culture of B. cnflumua in blood broth 0.1 cc. was transferred to PY&W Extract Broth 1; from this after 24 hours growth, to No. 2, and from thii to No. 3. These three cultures all gave good growth and upon transfer to blood agar showed the typical colonies of B. inpW~. Films also showed the typical wall Gram-negative bacilli. The experiment was vohmtarily discontinued after the third transfer. This experiment showed that Bacillus in@enzce would grow on a watery extract of Bacillus PO&US for at least three generations. An experiment was next made with the whole bacterial emulsion of protMcs in the same manner as described in Experiment 1. Here, however, growth of B&lZus injZuen~tz occurred only in the kst two transfers after the blood broth culture. A considerable difference between the emulsions of the mucoid bacilli and the emulsion of proteus, therefore, seemed to exist. The explanation of this has not been found as yet, but it is reasonable to seek this in the morpho- logical dikence between these two microbes. The large capsule of the mucoid bacilli may be a better growth-inducing factor than the capsule-free Qrokus. The possibility that the capsule may contain some nutritional reserve for the bacillus has already been put forth by Toenniessen (8), who finds that the capsule consists of a polysac- charide, gala&an, and that other bacteria grow better on the surface of cultures of FriedBnder's bacillus than on plain agar. Since it had been shown that it was possible to extract a growth- iuducing factor from Badus proteus, tests were made to determine the infknce of this extract upon the growth of Bczcilhcs in$um in various sugar solutions (1 per cent of sugar in peptone water with Andrade indicator (9)). 770 STUDIES ON BACTERIAL NUTRITION. I Ex$e&ment &-To each 5 cc. of sugar medium was added 0.2 cc. of #ro&us atract. The tubes were then inoculated with 0.1 cc. of culture (No. 2 in the foregoing exp&ment) and incubated. The following sugars were used: lactose, mannitol, maltose, dextrose, saccharose, ratlinose, inulin, and salicin. After 12 hours incubation a heavy clouding of the medium was visible in all tubes and the dextrose culture had turned slightly red. After 24 hours the culture containing dextrose was distinctly red, while those containing the other sugars remained colorless. At this point, control cultures on blood agar from all tubes showed pure growth of B. inflzccnt&. 4 days after inoculation the tubes showed the same reactions. On transfers to blood broth the cultures were all found to be Iiving and pure. Eqbetiment 9.-It was considered of interest to determine whether or not the clear extract of B. proteus could be atered through a Berkefeld filter without losing its potency. After the extract had been prepared as already descriid, it was passed through a Berkefeld titer N and tbe water-clear filtrate, after being proved sterile, was added to plain broth in the following amounts. Broth. Filtnte. GlUWth. cc. cc. 5 1.0 ++-I- 5 0.5 ++-l- 5 0.2 + 5 0.05 5 0.01 5 0 Growth was controlled by turbidity of the medium, by films, and by sub- culturea on blood agar and plain agar. These controls showed the growing organ- ism to have the characters of B. in$m. This experiment shows that the baciUa.ry extract in question can pass through a Berkefeld filter without losing its growth-inducing Property. SUMNARY. From the data presented iu the foregoing experiments it is evident that BadJZw in.umtkz will grow in a fluid medium consisting of plain broth to which have been added small amounts of emulsionsorextracts of mucoid bacilli or of Badhs proteus. The bacterial extracts may be made by simple boiling of the bacillary emulsions in broth or saline solution and centrifuging out the bacterial bodies; they may be filtered without losing their growth-inducing property. TEEODOR TH J&TA 771 Cultures of BaciClzcs &@WW in bacterial extract broth, if not too small doses of the extracts were employed, always showed heavier growth than the control cultures in blood broth, and growth occurred at a considerably earlier period than in blood broth. In many in- stances growth could be seen after 3 to 4 hours, and a bacterial whirl was always visible after 6 hours incubation. When the nature of the culture used for seeding is not stated, this was 0.1 cc. of the supematant fluid of a blood broth culture. All cultures were made in fluid medium; solid medium is much more diflicult to use in connection with the extracts. In explanation of the remarkable growth of BacilJw ht.&&mm in this blood-free medium, the idea is proposed that the growth-slimu- lating efiect of the bacterial extracts is due possibly to substances of the same nature as the so called vitamines. Further investigations on this principle of bacterial nutrition will appear in subsequent papers, together with a more thorough study of the sources and character of the growth-inducing substances. CONCLUSIONS. 1. .It is shown that BactXus infwnzce will grow profusely in hemo- globin-free media consisting only of plain broth and emulsions or extracts of mucoid bacilli and Ba&l~s jrof&us. 2. The emulsions and the extracts can be boiled and atered through Berkefeld filters without losing their growth-inducing property. 3. The growth-stimulating efIect of the bacterial e.xtracts is possibly due to substances belonging to the class of the so called vitamines. BIBLIOGRAPHY. 1. Cantani, A., 2. Byg. u. Injectiomkralrkh., 1901, XXIV& 29. 2. Neisser, M., Des&& med. Woch., 1903, xxix, 462. 3. Grassberger, R., Z. Eyg. u. I+#&nskrankh., 1897, xxv, 453. 4. Lnerssen, A., Centr. Bakt., lk AN., Orig., 1904, 434. sxxv, 5. Ghon, A., and von Preyss, W., Cmtv. Bakt., lte AM., Orig., 1904, IXN, 531. 6. Putnam, J. J., and Gay, D. M., J. Med. Research, 1920, dii, 1. 7. ThjWa, Th., and Eide, 0. K., J. Bad., 1920, v, 501. $8. Toenniessen, E., Cc&. Bakk, Ifc RU., CSg., 1920, lxxxv, 225. 9. Stillman, E. G., and Bourn, J. M., J. Ezp. Afti., 1920, xxxii, 665.