MFFERENTMKTIM OF MEMBERS 04" W2 GENUS WROMOBACYERIUM BERGONZIN! Thesis for the my” a? Ph. D‘ AMCMGM STATE UNIVERSFW Robert .L Ham W56 ‘5 T" g If G‘" This is to certify that the thesis entitled Differentiation of Members of the Genus Chromobacterium Bengonzini presented by Robert Joseph Hans has been accepted towards fulfillment of the requirements for Doctor of Philosoplgz degree in Microbiology and Public Health “(1% Yt\mfl1mfl# ‘ \!"\N\ Major rotessor Date May 22, 1956 0-169 DifffilENTIATION OF TEIBLiS OF THE GENUS CHROMOBACTEHIUM'RERUUNZINI By ' . \ Robert Jr'hans A THESIS Suomittei to the Sonool for Advanced Graduate Studies of Micuigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for tne degree of DMWQiW‘HflMBMfiY Department of Microoiology and Public Health 1956 VITA Rooert J. Hans candidate for tne degree of Doctor of Philosophy Final Exanination: may 22, 1955 Dissertation: Differentiation of Memoers of tne Genus Chromooacterium Bergonzini Outline of Studies: Major Subject: Microoiology Minor Suoject: Nycology Biograpnical Items: Born December 1/, 1926 in Detroit, Micnigan Undergraduate Studies: University of Michigan, 19u6-b7, 1946-51 Dearborn Junior College, l9h7-n8 Graduate Studies: Wayne University, 1951-53 University of munich (Germany), l95b—55 Experience: Teaching Assistant at tne Dental School, University of Detroit, 1951-5); Research Assistant at Parke, Davis and Company, summers of 1953, l95h; Research Assistant at Michigan State University, l953-5h, 1955-56. Affiliations: Society of American Bacteriologists Society of tne Sigma Xi ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Professor W. L. Mailmann for his patient guidance and counselling during the course of this investigation. Deep appreciation is also expressed to Dr. P. H. A. Sneath of the National Institute for Mbdical Research (London) and Brigadier Frank E. Buckland of the Microbiological Research Department, Experiment Station, Pbrton, Wilts, England, for providing many cultures and helpful information. The author is indebted to Dr. C. K. Smith who provided the filters, many carbohydrates and reagents used in this study, and to the Tierhygieniscnes Institut of Ludwig-thimilian University in Munich and its past director, Professor M. Rolls, for providing laboratory space during the author's sojourn in Germany. Finally, the author wishes to express his appreciation to all investigators around the world who submitted cultures for this study. Without their cultures, this study would have been impossible. This investigation was supported in part by a fellowship from the Deutscner Akademischer Austauschdienst, under the auspices of the Institute of International Education, Inc., New York. ‘ “ \\ d..., . Us... ‘f.’ 13 U) (‘n (‘T ABSTRACT A search was made for biochemical characteristics useful in differentiating members of the Genus Chromobacterium Bergonzini. It was shown that these organisms are alkaligenic in peptone media, so special emphasis was placed upon utilization of various carbohydrates in two peptone free media. The organisms were found to be very sharply divided into two groups by growth temperature ranges and optimum temperatures. These characteristics were closely correlated with fermentation of four carbohydrates in peptone media, the utilization of 11 carbohydrates in peptone free media, hydrogen cyanide production, pigmentation, and gelatin liquefaction. methylene blue thiocyanate reduction, nitrate reduction, ammonia production, tryptophane utilization, MRVP reactions, hydrogen sulfide production, and urea hydrolysis were found of no dif- ferential value. 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Md L'lph gm, .nu.” J -+,\ n ) . . . , - ¢ . ‘ 1 .-4 -,I 'I' ‘ " . . t/ uv +\ U A... “Jr ..,~) ljfifs IT‘LLDQ— b) C ._ 4 of :11 '~ -.\- 7' ‘7'3 :71. at pzch L9” fr‘m 1 L~ .. 3.1 ‘-._.1-_ Ln“ 4;l WQJLUDQ— L ‘ "fi “."1 .- " .‘XU‘JO'ZtlJ‘.L4.t4 3“ ,. ‘.4-- ' ~r~.‘-'.VV.‘- Ulru3abapdur1um spy. cuau-A¢u W 1-5--“ ,.‘ <7" y, lTTCLLQJ- +v > U '1__ L) «1‘ Lu.) ‘15) A148 INTRODUCTION In the United States the Genus Chromobacterium Bergonzini is considered to consist of the gram negative bacteria producing a non- photosynthetic violet pigment. These organisms are aerobic, motile, non-spore forming, occasionally pathogenic rods, and occur in water, soil and pathological processes in both man and domestic animals. Relatively ignored and in a confused taxonomic state ten years ago, these bacteria have received considerable attention this past decade. These recent investigations have dealt with taxonomy, physi- ology, and pathogenicity, and the pigment per se; but those dealing ‘with the taxonomy and physiology have been little more than repetitions of previous studies. Bacterial classification is based largely upon biochemical char- acteristics, chief among which on the species level is the production or the lack of production of acid or acid and gas from various carbo— lxydrates. It has been shown, however, that members of the Genus Sflgromobacterium, like many other Gram negative bacilli, produce alka- liJue reactions in peptone media. Despite this fact, investigators rurve continued to employ such media in attempts to arrive at suitable tmuxonomic criteria. In this study much attention is given to carbo- hyttrate utilization in peptone-free media, using a much larger number or £3trains and isolates than examined by any single worker to date. Additionally, such other oiocnemical determinations were made as seemed duplicable in other laboratories. .i. . x... ..H.....U‘ HISTORICAL REVIEW many authors have described supposedly new species of the violet pigmented organisms since the original description of Schrbter in 1872. Many of these differed only in motility, apparent flagellation, size, morphology, gelatin liquefaction, action on litmus milk, pigmentation on various media and similar unreliable criteria. These characters are recognized today as highly variable and depend both upon the cultural conditions and upon the method employed. In addition, some have even been described as different by virtue of spore formation, but recent evidence indicates that these supposed spores were artifacts. Accordingly, little is to be gained here from an exhaustive description of all existing species descriptions. Instead, only those ‘will be discussed which have been seriously considered and widely accepted at one time or another. A gram negative, non-motile organism isolated by Schrbter in 1872 atnd.named Bacteridium cyaneum produced a water soluble violet pigment axui is thus not a member of the genus Chromobacterium. Both organisms ixsolated and described as Micrococcus cyaneus by Cohn (1872) and "fiLauer Coccus" by maschek (1887) have been considered identical to Elioteridium gyaneum Schrgter (Migula, 1900; Godfrin, 1931:). In 1872, Scnrgter isolated and described a bacterium, Bacteridium Vixilaceum, producing a water insoluble violet pigment (Breed, Hurray arxi Hitchens, l9h8). This is perhaps the first true member of the Serums Chromobacterium to be described, and it is so accepted by Bergey's M3rnral.of Determinative Bacteriology (Breed, Mbrray and Hitchens, l9h5)3 .. Mn. .. viz., as Chromobacterium violaceum (SchrBter) Bergonzini. Also in 1872, Cohn described a Micrococcus violaceus, now con- sidered to have been the same as Bacteridium violaceum Schr8ter (Breed, Murray and Hitchens, 19h8). Bergonzini (1881) isolated a gram negative rod producing a chromo- parous violet pigment from an egg white solution and designated it Cromobacterium violaceum (sic). This is the origin of the generic name Chromobacterium. In 1885, Zopf characterized Bacterium janthinum (sic), a suppos- edly indol and hydrogen sulfide producing, nonemotile gelatin lique- fying organism isolated from "pieces of pig's bladder floating in badly contaminated water." In 1886, Schrgter again isolated a violet organism, designated Bacillus violaceus (Breed gt a1., l9h8), said to be identical with Bacterium Janthinum Zopf (Schrgter, 1886; Lehmann and Neumann, 1896). Schrgter (1889) and Toni and Trevisan (1889) described Bacillus lacmus from fresh greenhouse paint and Streptococcus violaceus from water respectively, both said to be the same by Godfrin (193k). Breed 33 31. (1988) consider this Streptococcus violaceus Trevisan the same as Bacillus violaceus Schrgter, Bacteridium violaceum Schrgter, and Qhromobacterium violaceum Bergonzini. Organisms subsequently isolated and designated Bacillus violaceus Mace (1887), Bacillus violaceus Frankland and Frankland (1889), and Bacillus violaceus Laurentius Jbrdan (1890) were considered the same as Bacterium ianthinum Zopf by Chester (1901). Godfrin confirmed this in.so far as he wrote Bacteridium violaceum Schrgter, Bacillus violaceus Frankland and Frankland, and Bacillus violaceus mace are probably the same species. Migula (1900) stated that Bacillus violaceus Mace and Bacillus violaceus lutentiensis Kruse (Flugge, 1886) show no significant differences, and along with Bacillus violaceus Berolinensis (sic) Kruse, are identical with Bacteridium violaceum Schrgter. Chester (1901) likewise pointed out that Bacillus violaceus Berolinensis and Bacillus violaceus lutentensis Kruse are similar. Thirey (1900), like Chester, felt that Bacillus violaceus Mace and Bacillus ianthinus Zopf were similar, as were organisms described as Bacillus lividus Plagge and Proskauer (1887) and Bacillus membran- aceus amethystinus Eisenberg (1891). Lehmann and Neumann (1912) regarded Bacteridium violaceum Schrgter _the first of its type to be described and considered it to be the same as Bacterium ianthinum Zopf. They further stated that Bacillus viola: ceous Mace, Bacillus violaceus Laurentius Jbrdan (1890) and Bacillus violaceus Berlinensis Kruse differ very little and agree with Zopf (188h) that the latter is identical with Bacillus lividus Flagge and Proskauer. Finally, they stated that Bacillus membranaceus amethyse tinus Eisenberg (1891) and Bacillus membranaceus amethystinus mooilis Germano (1892) are, along with the "sometimes motile and sometimes non-motile" organism isolated from the Themes by Ward (1898), closely related to those just mentioned. These relationships are perhaps best seen in Table I. It should be noted here that it was a custom of the time to apply trinomials and tetranomials to organisms which were considered slightly different from previously described binomial species (Novy, 1953). Accordingly, much of the above synonomy, such as between the various forms of Bacillus violaceus, is understandable and gains even more significance when viewed in the light of modern day knowledge of variability in bacteria. In 1890, Claessen described an indigo blue pigment producing bacterium designated Bacillus indigonaceus. The pigment of this organ- ism was soluble in both water and chloroform, however, and it is there- fore not a member of the Genus Chromobacterium. Beijerinck (1891, 1892) isolated from mucilage and described a motile, water soluble blue pigment producing bacterium which was also found to produce a condition of cheese in Holland known as “b1eu.” The natural habitat, however, is said to be soil and water (Godfrin, 193h). Since the pigment is water soluble, neither is this organism a member of the Genus Chromobacterium. Voges (1893) and Smith (1887) described almost blue-black pigment .producing bacilli under the name Bacillus coeruleus; but the two organ- isms were substantially different. Mbst significant of these differ- ences is the water soluble pigment of Bacillus coeruleus Smith, while that of Voges' organism is insoluble in water. Also in 1093, Voges described a supposedly spore forming, Violet Ibigmented organism designated Bacillus indigoferus, isolated from ‘Water at Kiel. It is significant that the organism was also reported (XV Vegas to be killed by exposure to 60°C for 15 minutes. 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Ammmwv “Amway when flammav ecea Humanow maaanoa Inseam macapm Ixnmnm new nonnav mmsmm Aawwav escammznmosm Lwamsm msmom fillmmmwwmmnm nmmmHv coma. mamcoHonmm nomounbm mdooomceupEma .m LamenEms .m .mseomaoa> .m Awwmavmsmomaoab .m msoomaoa> .m mseoaH0H> .m AHWmHV Acmefiv emeeoe poemxmoem Aeneas meg Amanda Heaaeomimm nmwmav snow. msapcopsmw new mmmmdm mammopmopsw mmmfl>mmm_ snow. msmomaowb .2 msoomaoa> .m usuabaa .m msmomaofl> .m msoomH0H> .m ImHOflb .o J a m w e mi aw _uu w n“ .. mm .. he mm“. nu . nu nu . m. . mm a n.wu u m a, n 3%: Lustig (1893) isolated an organism which he called "Bacille Bleu Indigo" wnich according to Godfrin is essentially the same as Bacillus indigonaceus Claessen. Godfrin (193k) briefly described a Bacillus pavoninus Forster, studied by Kraal (1599), Bownill (1899), Thomas (1930) and Van der Slenn (l89h), wnich produced opaque colonies, blue by transmitted light. This cannot be considered a violet pigment producing organism. Jobling and Nooley (Nooley, 1905) were the first to isolate a violet organism from a lesion. They isolated Bacillus violaceus manilae from the lymph glands of caraboas dying of fatal septicemia in the Philippine Islands. This organism was pathogenic for guinea pigs and rabbits and produced lesions which healed in dogs, cats and calves. No soluble toxin could be demonstrated. Subsequently, Gaudecheau (1907) and Minnett (1911) isolated strains, pathogenic for animals, from water supplies in Indo-China and British Guiana. More recently, Anderbaud gt El' (195h) reported infection of the liver in the monkey Cercopethecus cephus, and Sippel 32 El. (1958) reported fatal infection of cattle and swine. Lesslar (1927), Martin (1931), da Silva (Sheath, 1953), Black and Shanan (1938), Souls (1939), Schattenberg and Harris (19hl), Hetnerington (Sheath, 1953) and Sheath (1953) reported twelve cases of human infection, seven terminating fatally and two reported as recovered, With the termination not recorded in the other three cases. Symptoms of these various infections included .pyaemia, regional adenitis, liver or cutaneous abscesses, septicemia, lxrinary infection, rectal bleeding, and mild diarrhea. In 1905, Harrison and Barlow described Bacillus violaceus visco- furnitus (or Bacterium viscofucatum) from water, now thought to be a -10.. Pbeudomonas sp. by Tobie (Breed, Murray and Hitchens, 19h8). Breaudat (1906) isolated and named Bacillus violareus acetonicus (sic) from water in Saigon. This organism was said to produce acetone in a peptone-sucrose-potassium carbonate medium. Bampton (1913) studied 18 strains of Bacillus violaceus and h strains of Bacillus membranaceus amethystinus, considered by him to be the two main types in the Genus Chromobacterium. Of the latter species, he designated his strains Bacillus membranaceus amethystinus I, II, III and IV. thee, in 1913, isolated and described Bacillus lilacinus. In 1920, a Committee of the Society of American Bacteriologists, headed by C. -E. A. Winslow, proposed that the generic name Chromo- bacterium Bergonzini be accepted for the group of violet pigmented, non-photosynthetic bacteria. Cholkevitch (1922) isolated Bacterium cristillino violaceum from peat near Leningrad. This organism was reported to produce violet, yellow or red crystalline pigment(s) and is obviously not a member of the genus under consideration. In 1927, Creuss—Callaghan and Gorman described a Bacterium jgiplaceum amgthystinum, now considered to be identical with Basillus gggmbranaceus amethystinus Rosenberg (Breed, Mbrray and Hitchens, l9h8); and Grimes (1927) described but did not name a violet bacterium from Lnatter now designated Chromobacterium viscosum (Breed, Murray and Hitchens, 19h8). This organism was shown by Hans (1953), Gilman (1953) aliCl Sneath.(1956) not to be a member of the Genus Chromobacterigm. Grimes, in 1930, described Chromobacterium hibernicum and Chrome- Qéflzjagrium cohaerens from well water. _ 11 - Creuss—Callaghan and German (1935) examined 2h strains of violet bacilli and concluded that they represented 3 species; viz, Bacterium membranaceum amethystinum Eisenberg, Bacterium violaceum Schrgter and Bacterium ianthinum Zopf. Waeldele (1938) reported Bacillus violaceus sartoryi, a supposed spore—former from dental pus. The following year, Davis (1939) isolated and described Chromo- bacterium iodinum from milk, shown by Tobie (1939) to be probably a Peeudomonas. Chromobacterium maris-mortui was isolated from the Dead Sea by Elzari-Volcani (19h0). The last two organisms to be placed in this genus were Chromo- bacterium chocolatum Knutsen and a variant of this, Chromobacterium orangium Knutsen, both isolated and named by Knutsen (Lasseur and Giabicani, 1932-hh; Lasseur, Dupaix-Lasseur and Celcion, 19b2-hba, 19h2-hhb, l9h2-hhc). These two organisms have been shown not to be members of the Genus Chromobacterium (Hans, 1953; Gilman, 1953). Thus it is obvious that the taxonomy of this group is confused. Efrem the table on page 8, however, one might suSpect that a single 1irue species, by virtue of the frequent encounters of these mutually Siindlar organisms, is represented by that group. ChromObacterium ‘rixilaceum Schrgter appears to be the logical type species for this EHPOUpu and this name is used in Bergey's manual of Determinative Bacteriology (Breed, Murray and Hitchens, 19h8). Then, from the investigations of Bampton (1913) and Creuss- callaghan and German (1935) one might conclude that a second species .. 12.. is represented by Chromobacterium amethystinum Bisenberg. Chromooac— terium ianthinum Zopf, thought by Greuss-Callaghan and German to be a third species, has been too often likened to Chromobacterium . n , . . ... . Violaceum Schreter to ee cenSidered Signiiicantly different. The "Chromobacterium ianthinum" of Gilman (1953) was later found not to be Chromobacterium ianthinum (Gilnan, personal communication). This theory, viz., that the Genus Chromobacterium consists of two distinctly different species, is largely proved in the present Study I F.7'K'lmfb -4I“‘1a.n".ni Ilé'.‘: -_ 0.1 _ l3 _ GENERAL I‘ETHQ‘DS mm I'ATERIALS Source of Cultures The cultures for this investigation included ooth strains from established laboratory collections and isolates obtained by the author from soil in the United States and Europe. Fbrification and Maintenance of Cultures All cultures were tested f.r purity by streaking on nutrient agar plates containing 3 per cent yeast extract (Difce). No contam- inated cultures were received from cooperating laboratories. Each stock culture was maintained by Weekly transfer from a typical isolated colony on a nutrient agar streak plate. Plates were incubated hd hours at room temperature (2590) and were then stored at PC. Isolation Method The rice enrichment method of Cerpe (1951) was used for obtaining iiSelates from soil. Five grams of soil in a sterile petri dish was <30vered with sterile distilled water. Steri 3 preceoked rice grains (Tiinute Maid) were sprinkled on the surface, and the plates were illcubated at room temperature for five days. Approximately 50 per <3en1t of the plates prepared showed one or more areas of violet bacteria é§rWDwing on the rice grains after incubation. These grains were trans— feFred to a mortar with a small amount of sand and 5 ml. of distilled filly. III III I! lull -1)4.. water and were mascerated. One loopful of this suspension was then streaked on each of two nutrient agar plates, subSequently incubated at room temperature for hd hours or more, until violet pigmented colonies appeared. In some instances it was necessary to return to the original soil sample and repeat the isolation procedure when no violet colonies appeared on the initial agar plates. Biocnemical thhods Lhdia for biochemical determinations were employed in 13 by 100 mm., cotton plugged test tubes. With the exception of media con- taining carbohydrates, ammonium hydrogen phosphate, ammonium sulfate, or urea, sterilization was effected at 121°C in 15 minutes. Carbo- hydrates (except aesculin and dextrin), ammonium hydrogen phosphate, ammonium sulfate, and urea were sterilized in concentrated solution 'by filtration through ultra fine sintered glass filters. These concentrates were aseptically added to flasks of autoclaved basal nedium, and this was aseptically distributed to dry heat sterilized, cotton plugged test tubes. Aesculin and dextrin, because of their ‘peer solubility, were added to the basal medium before autoclaving. IVon-peptone containing media were employed in 2.5 ml. amounts and pep— ‘tcuw media in 3.5 m1. amounts. All media were incubated hS hours at Ifieom temperature to assure sterility. pH determinations were made with a Beckman Model G meter standard- jdaed with pH 7.0 phosphate buffer. All inorganic reagents were of C.P. grade. Organic materials were products of well known concerns. ['37- 'u' E “-1 'fl’;u*.sxc -guma‘iz'j ‘ v r-.\'r Vii! Ineculum Inoculum for solid media was obtained from typical isolated colonies on nutrient agar plates, incubated at room temperature (2500) for us hours. Inoculum for liquid media consisted of 0.1 m1. of a 70 per cent transmission saline suspension of cells from h8 hour old nutrient agar slants. Seventy per cent transmission was measured on a 6 volt, Cenco-Sheard-Stanford Photolometer, Industrial Type B—2, employing the blue filter and operating on a 6 volt Sears Roebuck motorcycle battery. Plate counts on 1h such 70 per cent transmission suspensions were made in nutrient agar to determine the approximate number of viable cells being employed. Incubation Tests were incubated at room temperature (25°C), and readings, lxnless otherwise stated below, were recorded every 2h hours for one vneek. Carbohydrates To illustrate the alkaligenic character of the organisms under Ccnasideration, all strains were tested in Phenol Red Broth Base (Difco), cOntaining l per cent glucose, mannese, maltose, sucrose, and lactose. ThHB final pH of these cultures after 7 days incubation was determined and recorded. To show, additionally, that this alkaligenic reaction could not J“ -16.. be avoided by employing a semi-solid medium to allow partial anae- robic fermentation of the carbohydrates, all organisms were grown in agar stab cultures of carbohydrate media. The basal medium consisted of Proteose Paptone, 5 gm; NaCl 5 gm; carbohydrate, 5 gm; agar, 3 gm; and phenol red, 0.02h gm per liter. Carbohydrates employed were: adonitol, aesculin, arabinese, cellobiose, dextrin, dulcitol, fructose, galactese, glucose, inulin, lactose, maltose, mannitol, mannose, melebiese, melezitose, raffinose, ribose, rhamnose, salicin, sorbitol, sorbose, soluble starch, sucrose, trehalose and xylese. Only new culture tubes were employed to avoid mistakes as to presence or absence of growth in the non-peptone containing media. In addition, only those tubes were selected which gave 100 per cent trans- nussion when filled with distilled water. These were compared to a tube selected as a standard in the Cence Photometer employing the ‘blue filter, as described above. This was necessary since it was lloped to detect both cases of slow carbohydrate utilization and cases .cm :H \r ;.r 0: Aomn... D on)... up .002, ummH sHoapmn HHom may: Mam mremv mpsmH .o.o.e.< .HGurxHSHHm JCHHH 00.0.9.4 mmqwmmoumm u . 4 chm> 9s mmmH IHhm .em poem; nephew chmbaxmccmm mo :+H pe>Hca . Hmcopmz human we thmsobHCD spworm ca oHcQH: mo thm-m>HcD oJWomoD COHfiOn) MA HH 3 mmmH .mehoms ocHs m Amvaon aw HommHm bmHmmm .m.m.D COHpoomcH mmmH .mHmnooo oquom AmvaommH HmmmHm mobmmm HHom moGMmm xmoalm .H.m.x.z noHpeHomH COHHmooH mohsom nopmmemman Loudthpcoo COHpmcmeoQ popmeoo< mm;mpmm HmcmepO human pcmmopH m p cH new: mmpszso .dgm EaH . thOUQOEokao Mo mmohfiom «emscHszooV HH mqmeg mmma momwhh henna pHsmanB Nmmmm adopmmm wdepwcw wcsHoom N hnHuuo>Hna memHefiH mmmm mmm-m momlimmmlimsmii ”Rum mHmnm «one . .mJIm .enum .m,-x cmmanoda . o . t m o. a . . - . J 1 a I l NmmH .pHoppmo HHom mums mmauu .omum .HH : .4 m bamaavm mmma «mmMCchum HHom mama cHomepom admfipmo «UQmHmH mmm -. mmma ISmHnocmuhom HHom mew: ommSOHgoconhcz _ H; sHmsH / . ...r.. .0 .L . Mr... . 2 RMMH ammmnm mpg. « Hfloq mSJT. m «m mPSrH - \l Admsuoa mmma «noHcaa HHom mama cmvmmo deHm:: mmsmo 2m: ammH ocmecm pm mmq pm>Hm amfinsm HHH ocmecm HHo: JMmH pcmHmem Go:fioo mam mmnasm HH vcmawcm HHHx encamcHeu :mma Unmfiwcm aw meg uvam cwenzm H owchm :oHumHomH COHpmooq meadow poummemo>cH popanppcoo sedumcwamoa dopmmooa ,1 .3 ,u . o a o r rlmo o+ea HmcHquO Newpm pcmmmnm one :H pom: mmhspHdo .mmm EdempomnoEonno Ho Apeschccov HH mnm poshfiam pom> mmmH massamo .er HHom ween 9H: humaumo mmma .QHopmcdmpH HHom mam: :Hmpmcsmpe oz npmmcm m¢w npmmcm mmmH eemHmcm HHom apmoem :smoem man gpmocm mom nammcm He: .H.m.m.z new: .g.m.m.z mmmH chnomHHmo pops: nm>Hm mmcxmz mam: .H.m.m.z Mme: .H.m.m.z AQHmH .o.e.o.zv mmimm 83$ :33; _ Mw acmaumo _ mmmH .soacse HHom mnmm soHss: mmmH pcmamcm pmpHHa papa; cmfipsm pm .m.?«£ mmma UcmeQm .HmuHHb hops; Cmcfldm mm .m..~_.....2 HmHz cm> N.m .w.z mangsu «HanomHHmo UmpmcHawpcoo HmHz cm> HmHz cw> H.m .w.: :Hmmempnqu mmmH ansUHw> HHom mcmm chpmcmpaoHH GOHpmHomH QOHumooH meadow popmmemm>zH popannpcoo GOHpmGmemQ empamoo< til we opma HwCHmHAO hospm pcmmmhm 0:» CH pond mmhdeSU .maw mepmpownothno mo mooasom Aeoschqoov HH mqmee _ 27 _ TABLE III Viable Chromobacterium spp. cells per ml. of 70 per cent transmission saline suspensions inoculum as determined by Nutrient Agar plate counts Count peg Strain ml. (x10 ) 121133 OH—I ILES : Frazer's Hill . 10.30 Cambridge Univ. 3.00 I\I.R.R.L. 8—1085 7.20 Sealey 6.70 Univ. Nancy 7.30 Univ. Michigan 5.30 Valley l.hO PSYCHROPHILES: Eng. III 3.50 H-20 6.00 H-27 5.70 H-29 6.50 NoRoRoLo 1‘71 6050 Smith DA 7.90 -28.. Twenty-five per cent of the psychrophiles also produced varying degrees- of acid from glucose in the lower half of each tube, with an alkaline reaction above. The remaining 75 per cent produced an alkaline reac- tion throughout the media. The average final pH for the psychrOphilic cultures was 7.55 (range 7.20 to 8.00). An alkaline reaction was shown by 66 per cent, an acid reaction by 7 per cent, and no reaction by 27 per cent of the cultures in maltose. No correlation between psychrOphiles and mesophiles could be seen; but the pH average in this medium for the former was 7.75 (range 7.20 to 8.35), and for the latter it was 8.h5 (almost uniformly). -0ne hundred per cent of the cultures showed only an alkaline reac— tion in both lactose and mannose media. The average pH was 8.30 for the psychrophiles and U.h5 for the mesophiles. In sucrose, an acid reaction was produced by 15 per cent, an alka- line reaction by h5 per cent, and no reaction by h0 per cent of the cultures. The average final pH for the psychrophilic cultures was 7.35, and that for the meSOphilic cultures was U.h5 (range 6.10 to 8.h5). In the semi-solid carbohydrate agar, all psychrophiles showed an alkaline surface with all carbohydrates within hd hours incubation. No acid formation was evident. The mesophiles showed varying degrees of acid production with fructose, galactose, glucose, glycerol, mannose, sorbose, sucrose, trehalose, and xylose, often accompanied by an alka— line surface reaction. All except strains Lewitus and Shahan in mannose showed acid in glucose, fructose, mannose and trehalose. This group showed eitqer no reaction, or merely an alkaline surface in arabinose, adonitol, aesculin, cellobiose, dexrin, dulcitol, inositol, inulin, lactose, maltose, nannitol, nelebiose, melezitose, raffinese, rhamnose, salicin, sorbitol, and sorbose. Carbohydrate utilization in the peptone-free media of Ayres, Rupp and Johnson, and of Elrod and Braun is shown in Table IV. A plus sign indicates an increase in turbidity during the seven day incubation period, and a negative Sign indicates a lack of increase. General reactions for the psychropniles and meSOphiles are indicated at the head of each proup. Only cultures in each group which did not behave as their type are indicated in tea renainder of the table (i.e. the exceptions). Growth of the meSOphiles in fructose, galaetose, glucose, élycerol, nannitol, and xylose was not as decisive as might be desired. In most cases the turoidity of different organisms in various of these carbo- hydrates barely reacmad 10 per cent transmission. 0n the other hand, the differentially useful a‘abinose, inositol, maltose, salicin, sorbitol, sucrose, trehalose, and xylose gave large turbidity increases with the psychropniles and aisolutely none with the nesophiles. Lactose was utilized slowly, increased turbidities being first evident in most cases only after 5 days incubation. Hung-so, en 3,7’ifliil‘3 Production Detectable hydroaen cyanide was produced within hb thFS of incu- bation, often within 2h hours, by all mesophiles except strains Frazer's Hill and Univ. Michican. None was detected Tron the psychrophiles. The negative reactiOns with strains Frazer's hill and Univ. Michigan recurred upon repetition. - 3o _ \\\\\\\\L\k \‘k‘k‘k‘k‘k \‘k‘k \\\\k\\\\\ seamen nm .Um popes .me. m.m .o.2 H.m .u.2 chpmcopgoHH aHmpmcuom HH ecmecm H vcmamcm JOHHH .o.o.a.< «meoHpmmoxm __-__J. \\-\\a\\\ «monBD cwanon thmhmbHca hoammm mobmmm Namw .o.9.o.z nephew oqu nohHm H045 .0.o.a.< mmm .o.o.9.« umGOHpmmoxm n u n x u x u u u u u x a. ..... u u - x - x x x u "monHoame mmwe UHqumommz Gov. Q I. knew. .L.w nu Taco no es Jaw. Ea Otwwmimnp mac Wis Jnmmme HOWWWEWUMJ 1.8 J W o.A T.o TaTLn x 8.0 T.T.o T. T.J a O.w n T. e 1.0 Q. s o o u Ice Tia. U J 8 Q.Q.T. o o.w o o e o G. o n t.I.I.e I.I. 1.2 I.J e o 0.0 o o s 0 S o o o 1.I. I.T. 1.1.1.J 1.1. u I.u I. T.s 1.8 s a. a s O a s S 1.0 w u I. O 0 O o O O O 1. u 0 e 0 e a I. a S a a 0 S u T.T.T.T.T.T. s o s s o e s a s e s a a s a a e a mefiHo maonooad mmUHhmeoom mmUHhmcoomw mmUHhmnoommocoE nooddw :thm Ian A . 0 "LE‘!’“' mHoos menu meopaom :H .mam ssaeosompoeoueo an cOHpmNHHHps mpmteanonpme >H mqmH mumee :oHpmuHHHus on u I “COvauHHHps u.\ «map . Cale 2 mHl© is ac :pmmcm _ mm .3 “8m; Sm: «mCOHquoxm x- xxxxu- u--\ .\.x\. xxuxxxxxx £523Ee§pgdfiggh SV 8 .1.an . .. 0 l8. S 1.0 JV ea ”Wammw mmmM ammmmm mammmmmmm .L.0 QUSOOW Jets... QJGGTGTTL quo 0900.. a.“ 1.1.1.81. 1.2m.J 900.000 so 80000.1. _L qqflvvunufflf UT: T... Ivor-#880; asoessqou um wmmwwm a: u m.m..: 9% .amaa 8m 0 9 m 9 0 3 0 mmeHo mHonoon wmeHnegoow mmanmeoomm mmvHamnoommocoz ToosHm IhHom IHQ.II_ r rlili mHees mesa mcopdme :H .mnm EsHumpompoEonno an GOHpmuHHHps mpmnohnobamo HemscHscouv sH mamas _ 33 - lbthylene Blue Thiocyanate Reduction Letnylene blue thiocyanate was einpletely reduced by all strains within 13 minutes. Nitrate Reduction and hruonia Prouuction Only strains Veer, K.1.R.L. b-lJZO, §.R.R.L. 3-1005 and Ind. Univ. snowed no nitrate utilization in Eitrate Peptone Broth at hB hours. By 7 days, only N.R.R.L. strains 8-1020 and B-lOBS had failed to reduce nitrate in this medium. At h8 hours, most mesophiles showed no nitrate reduction in Dimmick Nitrate Solution while most psychrophiles did do so. After 7 days, only strains N.R.R.L. B-1085, Valley, mentekab, Lake Garden, and Lewitus were negative. Ammonia was produced by all strains in both media within h8 hours. Gelatin Destruction Gelatin stab cultures snowed significant liquefaction by all meso— philes after 72 hours incubation. All psychrophiles showed little or no liquefaction at this time. By 1h days, the meSOphiles had all liquefied at least two-thirds of the medium. Only a few psychrophiles (15 per cent) had produced any degree of liquefaction within 1h days, and in no case did it exceed one—quarter of the medium. All mesophiles showed a zone of gelatin destruction on Frazier Plates after 72 hours incubation, while no psychrophiles did so. After 7 days, only nine mesophiles (lb per cent) showed no zone of gela- tin destruction. _Bh- "”|)Y Tryjtophine Utilization ail “HJP Reactions Indol and acetylmethylcarbiaol were not Corned by any of the cultures, and tn: MR reaction was negative in all cases. Metility and Hydrogen Sulfidr Production Iotiliby of all strains was afyarent near the surface of the medium witnin 2h hours. It was also observed microscopically in all cases 0 Hydrogen sulfide had not been proouced by any strain in either medium after 1h days incubation. Urea Hydrolysis Urea was not hydrolyzed by any strain. Stains All cultures were gram negative. The mesophiles averaged 0.75 x 2 microns in size and the psycnrophiles 1.0 x 3.5 microns. Mith Lgffler's alkaline methylene blue stain, the mesophiles (+- usually showed bipolar staining, while he ps"chropnilos often showed netachromatic granules. Pigment All mesophiles were constantly dark violet pigmented on Nutrient Adar (Difco), Nutrient Gelatin (Difco) and glycerinated potatoes. The nsyehrophiles, on the other hand, ranged in color from blackish violet to crean Jith or without scattered traces of bluish pignentation. .‘1 3. . : . .. Lenperature Requireients Tne temperature range ini optimum temperature for each strain examined are shown in Table V. The average results of plate counts with broth cultures of 10 strains diluted to each 10 per cent transmission from 10 to 90 per cent are snown in Table VI. These data can as used to calibrate Figure l. The positive readings for all psychrophiles and then those for all meSOphiles at each 10 per cent transmission fron 10 to 90 per cent after no hours incubation were averaged to yield data for the curves in Figure l. . *3 _ 36 _ TABLE V Temperature optima and ranges for Chronobacterium spp. strains examined . T““‘ Range Optimum ‘train (Go) (90) RESOPHILES: A.T.C.C. 353 8-36 28-36 A.T.C.C. 6357 8—36 32-36 A.T.C.C. 7&61 12-36 28-32 A.T.C.C. 12t72 12-uu 26—36 Birch 6-36 26-36 Brown 8-36 23-36 Cambridge 6-36 28-36 Frazer's Hill 6-h8 32-36 Inst. Pasteur 532 8-36 2o-36 Lake Garden lZ-hh 28-36 MEt. Water Bd. 12-36 32~36 N.C.T.C. 7917 h—36 28-36 N.R.R.L. 8-1085 12—36 28-32 Reeves 16-h8 26-36 Sealey l2-hh 28-36 Univ. Michigan lZ-hh 32-36 Univ. Nancy 16—36 32-36 Univ. Pennsylvania 8-36 32 Valley 5-36 2a—36 PSYCHRCFHILES: A.T.C.C. 1110b 0-36 2h AoToCeCo 121473 0‘36 21.]. Berlin 16 0—36 20 Berlin 18 0-32 2h Berlin BO 0-32 2h Berlin V—l 0-32 2h Berlin V-8 0-32 2h Berlin 35/2 O~32 2h Corpe h-A 0-32 2h Creuss-Callaghan Strain 16 0—32 2h England I 0736 2h England II 0—32 2h England III 0-28 2h English Garden 0—32 2h Futa Pass 0-28 2h H-h 0-32 2h H-ll 0-32 2h 11-20 0—32 21; H-ZS 0-32 2h _ 37 _ TABLE V (continued) Strain Range Optimum (00) (°c) H-27 0-36 2).; H—29 0-32 2).; H—3O 0—32 21; H—31 0—28 20—21; H¥33 0-32 20—2h H—3h 0—28 2L; H935 0-32 2h H—36 0—25 2).; H—39 0.32 2L; H—SB 0-32 2h Herrenchiemsee 0-32 2h Bernstein 0—36 2h Ind. Univ. X 0—32 2h Inst. Past. S2227 0—32 2h Lichtenstein 0-32 2h ASS. 2.1 0-32 20—2h M.G. 2.2 0—32 2).; 1.1.3.8. 25 0—32 21; MJ‘LB. 27 0—36 2).; lhnich 0-32 2h h'..'l.R.L. 3-1020 0-32 20-21; N.R.R.L. use 0-36 20—28 N.H.R.L. to? 0—32 2h N.R.R.L. 1470 0—36 21; N.h.R.L. u71 0—36 2h Sheath DA 0-32 2h Sheath GA 0-32 2h Sneath NC 0-32 21; Sheath RU O~36 2h Traunstein O~32 2h Ulm 0-36 2h Veer h—32 2h 6-1 0-36 2h 6-h 0-36 -2h 6—5 0-36 2h 6-10 0-36 21; 6-13 0-36 21; 6—15 0—32 2h 6-18 0—32 2h 6-20 0-32 2h 6-21 0—36 21; 6-22 0-32 20 zoadnaoz. neg—o... 0v ¢Uhs¢ 0.00 O... 0 lock 0* xo< go a. u¢3h¢¢ucluh 0* to at on an on Out on o. a. O . o . ‘11 . O o . ON On 0' 00 Oh 8 (ALIOIOUM. snum as con muons fioo awaits-30>»; -I 09 E 34.2.33: . I i3. . 0.50; ‘- .1. {LA at? I \x) \O I TABLE VI Average viable cell counts at each 10 per cent transmission from 10 to 90 per cent of five psychrophilic and five nesophilic Chromobacterium spp. strains grown in broth culture, determined by nutrient asar plate counts Percent Count per ml. (x108) Transmission hesophiles Peychrophiles 10 .32 .57 2o .75 .99 30 1.03 1.58 to 1.86 1.88 50 3.20 2.68 60 5.73 L.18 70 7.00 é.hh 80 15.77 12.80 90 22.87 16.15 J [Jilix I. .II A l};;. -h0- DISCUSSION Carbohydrate utilization in peptone—free media, acid production in peptone containing media, hydrogen cyanide production, temperature range, gelatin digestion, and pigmentation are useful differential Characters in this genus. Carbohydrate Utilization Ayparently the mesophilic and psychrophilic groups can be dis- tinguished by the production of acid by the mesophiles from fructose, glucose, mannose and trehalose in solid peptone containing media, while the psychrophiles fail to do so. Failure of the psychrophiles to utilize carbohydrates in agar solidified peptone media may well be due to their reluctance or fail- ure to grow under the nicroaeronhilic or practically anaerobic conditions in the depth of the medium. The stron,ly alkaline reaction produced by tnese strains at the aerobic surface, however, precludes any formation of an acid reaction here. These reactions are summarized as follows: TABLE VII Acid production from glucose, fructose, mannose and trehalose by mesophilic and psychrophilic Chromobacterium spp. in peptone containing media Glucose Fructose Mannose Trehalose Mésophiles / / /(-) / Psychrophiles — - - _ / 3 acid; (-) 3 acid rarely produced; - a no acid In addition, utilization in peptone—free media of adonitol, arabinose, inositol, lactose, maltose, rhamnose, salicin, sorbitol, and sucrose can also be used as distinguishing characteristics. As can be seen from Table IV, the mesophilic organisms are able to utilize fructose, galactose, glucose, glycerol, mannitol, ribose and trehalose as sole carbon sources. Fermentation of mannose and xylose in the semi-solid peptone medium would indicate these two carbo- hydrates can be fermented but cannot be used as the sole source of carbon. This is strikingly p rallel to the findings of Liu (1952) with Eseudomonas aeruginosa. Liu found P. aeruginosa able to utilize the same carbohydrates as sole carbon sources, and to be unable to utilize adonitol, dulcitol, inositol, inulin, lactose, maltose, raffinese, salicin, sorbitol and sucrose. Additionally, Liu found 2. aeruginosa able to ferment arabinose and rhamnose, but unable to use them as a sole carbon source. Elrod and Braun (l9h2) disagree in respect to arabinose, and Salvin and Lewis (l9h6) agree with respect to rhamnose ‘with.P. aeruginosa. The meSOphiles in the Genus Chromobacterium are perhaps related to PSeudomonas aeruginosa. Similar studies with other gram negative bacilli would.be highly valuable for comparative purposes in this respect. If si4giificant differences were to be found between other gram negative organisms and both the violet mesophiles and E} aeruginosa, the meso- .philes are likely related to f. aeruginosa. Such a statement to this effect at present, however, would be premature. The violet pigmented psychrophiles, on the other hand, are as much different from P. aeruginosa as are the violet pigmented mesophiles. ’) A lack of sufficient conpirative studies make“ A b it impossible to liken these organisms to any other species or genus at this tine. Tic occasional fermentation of raffinose and inulin by the psychrophilic cultures is of particular interest. Adams, Richtmyer and hudsen (19L3) snoxed that the ease of enzymatic hydrolysis of sucrose, raffinese, stachyose and inulin by bakers' yeast and brewers' yeast inve'tase are in the proportion lOO : 23: 6.8 : 0.036 and 100 : 12.5 : 3.1 : 0.006 respectively. Accordingly, one might deduce that all cultures utilizing raffinose are capable of utilizing sucrose. Such was the case. In aidition, stachyose would have been utilized by some number betsoen ll and 16 of the cultures studied here. Signifi- cant, however, is the fact that utilization of these carbohydrates is apparently more a matter of degree than of possession or lack of a particular enzyme by the organism. Since the enzyme would appear to be present in all psychrophiles as "sucrase", its function in the cases of inulin and raffinose is relatively unimportant. Unfortunately no similar correlation can be found between either adonitol or rhamnose and the other carbohydrates utilized by the meso- philcs. The use of tnese carbohydrates in peptone-free media for separat- ing the two groups of violet chromogens are summarized in Table VIII. Occasional discrepancies in carbohydrate utilization shown in Tables IV and VI may be due to the use of chiefly old labora- -tory'stock cultures for representatives of the meSOphilic group. ‘Attempts by the author to isolate mesophiles by both.Corpe's Rice Grain ikathod and by direct agar plating of both water and soil samples were -2! _u3- TABLE VIII Utilization of 11 differentially useful carbohydrates by meso- philic and psychrOphilic Chromobacterium spp. in peptone free media Q) m o 3 r4 r4 r4 0 m a) a) v o o O O O C C. O m m to co 7‘? +9 .0 +3 "-4 H 5 o o o 0 "—1 H «4 o ,0 .3 H +3 $4 +3 £1 ,0 r: m 0H e w E‘; 0 s a a 2: O 0 a is; £3 £3 .3 (0 if? {—4 U) '3 I5 0') mesophiles - - - - (%) - (-) — - _ I ‘k FSychrophiles / / / (f) / - / / . utilization; (/) - utilization uncommon; - a no utilization; (-) a no utilization uncommon unsuccessful. These discrepancies are of minor importance only in the cases of sucrose and trehalose, however; and those for trenalose are corrected in the peptone containing media. Linardos and Clevcrdon (1955) reported that Chromobacterium spp. require organic nitrogen and carbon (as amino acids) for growth. Growth, per se, in the peptone free media employed here indicates the error of this statement. Hydrogen Cyanide Production Hydrogen cyanide production by mesophilic cultures, with two exceptions (strains Frazer's Hill and Univ. Michigan), is useful for distinguishing the two groups present. These two exceptions might possibly be eliminated by alteration of the cultural conditions, but such was not attempted. Gelatin Destruction Two modes of differentiation by gelatinase activity are possible. While practically all (86 per cent) the strains showed the presence of the enzyme, the relative activities are of differential value. This is summarized as follows: TABLE IX Gelatin destruction by Chromobacterium spp. at 72 hours determined in Nutrient Gelatin stab and on Frazier Plates Nutrient Gelatin Frazier Plate mesophiles / / Peychrophiles (;) - / - destruction; (X) - destruction rare; - - no destruction -US- Pigmentation All meSOphiles produced the pigment, violacein, out the psychro— philes were variable in this respect. This is of value in that achromogenic or only partially pigmented strains can be recognized as members of the psychropnilic group at once. Tempe ‘ature One of the best criteria for the differentiation of the two groups proved to be their temperature ranges. One group, the mesophiles, was found to grow in the range h to h8OC, with optima between 28 and 3600. Tue other group, the psychrOphiles, grew from O to 36°C with an opti- mum at ZhOC. The two groups are best distinguished by incubation at o, 12, 28, 32, 36, hO and thC (Figure 1). Only the exceptional mesophile grew above 3600, however, According y, the upper limit for diagnostic work should be confined to that level. These temperatures would be of value in comparative studies in which members of both groups were present, and in which comparable inocula of all strains were employed. Then, these cultures growing best at 8 and 1200 and poorest at 28, 32 and 36°C in hb hours or ess would be the psychrophiles. Tnose growing best at 2C, 32 and 3600 and poorest at b and 12°C would be the meso- philes. High turbidity caused by a minimum of 250 million cells per cc. after h8 hours incubation at the intermediate temperatures of 16, 20 and 2hoC (i.e. room temperature and slightly below) make tiese temper- atures valueless in differential work unless a photolometer or similar 1 ‘ . — u- . r v‘ ,‘A ~ 1 ,‘vv ._ I ,.J ”-2 — ‘V ‘b'v— -'o - ;~r\-~I\‘\ would be breatlJ iniluenoed, and even iavesoeu in Jaiue, o, arireoiiw unequal inocula. \ for the occasional QGuCrz' -uation, therefore, where only one or a 'V - .“ .‘1H ’1 ‘ y-s -" .v ,- . ‘r- ‘ 2 . “'3. ‘ h I) f.": ,0 ‘ r,' l r ‘ r - v H fOu seepineno are anal...b infideli;ud, it and 3x t are thd oei erat ires . of Choice. The diiforential value of the‘e two inundation temperature: J u) ‘ x. r.'_ -.‘1' V.an as Lollons: TABLE X \ ul fier3.ti at on of mesophilic and pep chropwi Lnionoozctrriu" 33p. by ten.erature of incuba ion I ha hours incubation at: 12°C 35°“ Lbsorhilos _ f t“\\r u. -,." . V / - .LuJCnrOiJLLll‘jb r where / represents "good growth" (viable cell count in excess of 103 million/cc) and - represents "poor growth" (viable cell count less than 32 million/CC). _ g7 - Tie biochemical characteristics found useful in this stiiy are summarized in tabular form in Table XI. Inadequate descriptions of early nancd species renders impossible 4 r r I- -;V. \~ .‘\ ‘ “‘ u 1‘ r‘ ~ y- ’ ~: ‘ - . ‘r" "‘ “.‘ ‘.‘ . | ‘. , V "r“v- ‘ the attacgnenc oi and one nane to either OJG mesoplilic o: tn: psych- . ' ‘ , a ‘4 4.. ' ¢, , ,. 7‘ - 3,, 73..” ,‘L‘ \ _fi’w r’f ,, - ,, 133 ilic group {LUA certainoy. leccrdinelJ, oneaun (19,3-3o) piOposeo . H ! t r a fi' ‘v-r“!‘,.' ‘IV".‘| '3 r. fl 7 \‘ "’e‘. . . ,\ 3‘- ‘, 2 . j » this tie ni-e Udrdflogl.tulll. lioiaceun (sunrotei) aflfbunélnl oe con- served for the type species by virtue of its wide acceptance and that this name be applied to the mesophilic strains. He has deposited strain Mbntekab in the National Collection of Type Cultures in England and in the American Type Culture Collection in dashington, D. 0., to serve as a type for this Species. In addition, Sheath preposed the name Chromdbacteriun lividum (Vogco) Holland for the psychrnphilic group, saying tiat it is the "first permissible." While this author agrees with the first proposal, it is felt that the name Bacillus m-mbranaceus amethystinum Eisenberg, now Chromobacteriun amethystinum (Chester) Holland should also be conserved and be applied to the psychrophilic strains.- However, until sue} names are officially accepted, it is strongly 1 suggested that cultures be referred to only as being nemoers of either the psychrophilic or mesophilic group. "i r.!, “coflpommb oanmflpm> u m “ceasommp ebfleemmc I .o>flpmwmc hampmn "AIV m0>HpHmom hacks“ "AXV I «soapommp m>HpHmom I \ Co W I I AIV I - - 4. - 4, - 114.114. 1-- message 7:) TV x 4. .. 4. 4. 4. 1-47.1134; guess 7 co.. «W “a c rkl.ocninim%"v rev-a no i N. Swim-fl...» fl. w... :v. ._ e em. 0. e. we m mammeemmwew mmmm. Z QJ go.“ N TS .CWQJQUlq U. 00 a in we mnnnrmmmmwu ewes J nu veg-mg an ”M. uommmeeeewm mews Au m-a. Apopn mwn+ m. T. a he a a m .22 .o n .- t.u o m m. fi w m. «Heme $3 88nd flew: a. spaced J o meopmmm m. u .mem ssflhmpomnoeoumw owaflcmomes use oeaflnmohnokmm em» you unflwb Hmflpcmemmmfle Mo mzofluommh HmoflEanOHb UmNHhImEEdm HN mqm<8 f r? '1'?“ -LL-) .LbLLub :11 . . ,‘ ‘1 '~‘_:AI’_ "- r n _ if (A, n _w"~ fl .-,- H Alina ;.., nit.-tmuer, N. h., ufld Judson, C. o. 1913. ooze enzfnes present in 1:1,11J zurifiod invertase pre;:ara ioxm 5 A ontxiiitioI to the stud; of 1-1cto-3rIHOs1H-ses, aiictosii m: ;llCOolQaScu . - . ‘ .1. .r. / and mannosidgses. J. A“. Chen. ooc., £5313 9—1360. Anderbaud, ., Sansin, X., Ceccalii, J., and "erveille, P. 195L. Isole.nentl'1n Chronoocct rium violaceum a partir do esions hepa- h.” tiques oos-zrvees chez un singe Carcopithecus cephus; etude ct pouvoir pathogene. Ann. Inst. Pasteur, 37 hl3-uli. Ayers, S. H., Rupp, P., a: i Jo‘inoon, J. T. 191;. A study of alkali- forning bacter 1a in mi--k. t. 5. Dept. Agric. 321.732. ‘rfi ‘- Sannton, J. h. 1913 U‘bir 1131 aceus und Kemdranaccus .me _Jstinum. Centralbl. f. Baht., Orig. Abt. l. 71: 129-lh6. Seijerinck, M. N. 1891.- Die leo 0:13 tciiic:1te einer p Qienthacterie. Botanische Zeitung, L9: 706—711. Rei}3rinck, M. 5.1892. La biologie d'une bac .L be ArchiV3s h.;rlandaisos aes Sciences Exactes et 230 TI”'onzini, 3. 1591. 33.ra un nu ovo batterio colorato. Annuario ,I..L H iclla oocieta dei Ni .‘r listi in hodena, 1h fasc., Ser. 2, lL9-158. Black, H. 5., and J ahan, J. 1938. Bacillus violaceus infection in a iiuman being, J. Am. had. Assoc., 110: 1270-127l. ‘3owhill, T. 1399. Kinual of Bicteriological Technique and special Bacteriology. Oliver and BoJ d. Edinburgh. jBreaudat, L. 1906. Sur un microbe nouveiu producteur d'acetone. Ann. de l'Inst. Pasteur, 20: 87h-879. Breed, :1. So, Ifdl‘ray, E. C0 D0, and IIitC‘ien-s, A. PO 19118. Bergey's 'Manual of eterminative Bacteriology. Sixth Edition. Williams and Vilh ins 00., Baltimore. {Ruester, F. D. 1910. A Lanual Determinative Bacteriology. The rhcmillan (30., 1134'! Yep {. (fiaolkBVitCho 1922. Seen in: Breed, R. 8., et al. l9h8 Bergeyts Itinual of Determinative BacterioloiJ. Oljuxssen, H. 1890. Ueber einen indigoblauen farbstoff erzeugenden Igacillus aus wasser. Centralbl. f. Bakt., 1: 13-17. .PI; 1 .II ’1' J. ClaJson, B. J. and Young, C. C. 1913. Preliminary report on the pro- duction of hydrocyanic acid by bacteria. J. Biol Cnem., 15: D19. Cohn F. 1872. Unt» 5" n. Beitrage zur Biologie der fflanzen, E s " Corpe, a. A. 1951. A study of the wide spread distribution of Ch” bacterium SpOCiSS in soil by a s'mgle Technique. J. of Bact. 315-7317. J \- A" Crease-Callaghan, C., and Corman, M. J. 1935. On the cnaracteristics of Bacterium violaceum (Schroeter) and some allied species of violet bacteria. Sci. Proc. Royal Dublin Soc., 21: 213-221. Davis, J. G. 1939. Chromobacterium iodinum (n. sp.). Zentralbl. f. Difco Manual of Dehydrated Culture Media and Reagents for 1953. Micro- biological and Clinical Laboratory Procedures. 9th edition. Difco Laboratories. Detroit. Dinmick, I. 1917. Thosphorus deficiency in relation to the nitrate reduction test. Canad. J. Research, Sect. 0., 25: 271-273. Eisenberg, J. 1891 Bakteriologische Diagnostik Hilfstabellen zum Cebrauche Beim Praktikum. Arbeiten. 3 Aufl. L. Voss. Hamburg and Leipzig. Elrod, R. P. and Braun, A. C. 19h2. Peeudomonas aeruginosa: its role as a plant pathogen. J. Dact., hh: 6333h5. ,ElzarirVOlcani, B. 19hO. Studies on the microflora of the Dead Sea. Teesis, Hebrew Universi y, Jerusalem. TFlugge, C. 1886. Die Kikroorganismen. Vonel, Leipzig. jFrankland, G. 0., and Frankland, P. F. 389. Uber einige typische rnikroorganismen in wasser und in boden. Zeitschr. f. Hyg., Q: 373- DOG. ,Fraziery W} C. 1926. A method for he detection of changes in gelatin (iue to bacteria. J. Inf. Dis., 32: 302-309. iynyiucheau, H. A. 1907. Fatal infection by Chromobacterium violaceum. C. 11. Soc. Biol., Paris. 1: 278. Cited by Sneath, P. H. A. gt_§l. 1953. Lancet, 265: 276-277. Germnnno, P. 1892. Der membranaceus amethystinug mobilis. Centralbl. r. Bakt., 153: 516—519. ‘— 011nmu1, J. P. 1952. Studies on certain species of bacteria assigned to tkne genus Chromobacterium. J. of Bact., 6S: h8-S2. Iv ill. I‘ll." I I {$31.1 Gilnan, J. P. 1953. Personal communication. Godfrin, P. 193M. Contri‘ution a 1'etude des bacteries bleues et violettes. Thesis, Nancy. Grin s, H. 1927. An aerobic capsulateu bacterium chromogenic on sugar media. Centralbl. f. Bakt., Abt. II’.Z§: 367-368. Grimes, M. 1931. A study of two new species of bacteria belonging to the genus Cnromcbacteriun. Sci. free. Royal Dublin Soc. 19: 331-3Qh, Guignard, K. L. 1905. Sur 1'existenca, dans le sureau noir, d'un compose Tournissant de 1'acide cyanhydrique. C. R. de 1'Academie des Sciences, 1h1: 16-20. Guianard, M. L. 1906. Le haricot a acide cyanhydrique, Phaseolus luna- tus L. C. R. de l'Academie des Sciences, 1&2: 5&5-553. Hans, R. J. 1953. A taxonomical and physiological study of the genus Chromobacteriun dergonzini. Thesis. Kayne University. Detroit. Harrison, F. C., and Barlow, B. 1905. A new chromogenic slime producing organism. Centralbl. f. Bakt., Abt. II, 15: 517-539. Jordan, M., and Edwin, D. 1890. Exyerimental investigations by the State Board of Health of lbssachusetts. Purification of Sewage. p. 633. Cited by Godfrin, F. 193D. Contribution a l'etude des bacteries bleues et violettes. Tease, Nancy. Kraal. 1899. Bacteriologisehes laboratorium. Pra¢., sammlung. Cited by Godfrin, P. l93b. Contrilution a l'etude des bacteries bleues et violettes. These, Nancy. jLasseur, Ph., and Giabicani, R. l9h2-hh. Parallelisme entre B. mesentericus niser Lunt et 3. lactis Garini. Travaux du Lab. de *i) ‘nicrObiol., Fac. Pharm. d3 Nancy, Fasc. X111, 16 —173. it; jbasseur, Ph.,iDupaix—Lasseur, A., and Mblcion, J. 19h2-hha. Arti ice Ivermettant de cultiver sur pemme de terre certaines bacteries qui, (an general, s'y development tres dificilement. Travaux du Lab. de Iiicrobiol., Sac. Pharm. de Nancy, Ease. XIII, 187-188. lipaseur, Ph., Dupaix-Lasseur, A., and Mblcion, J. 19h2-bbb. Characteres anstigeniques de Chrggebacterium chocolatum (Knutsen) forme Violette est forme orangee. Travaux du Lab. de Microbiol., Fae. Pharm. de Nancy, Laennaur, Ph., Dupaix-Lasseur, A., and Melcion, J. l9h2-hhc. Observa- ‘tions sur les deux formes d'agglutionation chez Chromobacterium choc: cflgrtum (Knutsen). Travaux du Lab. dc Microbiol, Fac. Farm de Nancy, Fasc 0 XIII, 313"3170 Lehmann, K. 3., and fieumann, E. C. 1596.it1as und (buxmi im der Daktericlogie. Teile I und II. J. d. Le}. .ann, Hunich. Lehmann, K. B., and Neunann, H. C. 1912. Atlas 1nd Grundriss der Bakteriologie. Teile I und II. J. C. Lem; ann, Munich. Lesslar, J. E. 1927. Fatal infection by Clru”0 acte riura violaceln. kpo IllSl. ‘1'?ng 113.03. 5‘. IA. 8. r). 2:)- Viuej I)"; 51163 T11 11, P. H. A. Ct a1. Lancet, 265: 270-277. Linardos, C. 8., and Cleverdon, R. C. 1955. Sane nutritional require- wants of some Chromobacterium spp. Bact. Proc. 9. 9. Liu, P. 1952. Utilization of carbohydrates by Pseudononas aerurinosa. .fl' {(3 F '1 ‘:___ Jo 0131(3qu ’ >11: 773-7d...0 Lustir, A. 1393. Di agnsstik d3? ..LLcr‘en les Wasscrs. 2 Auflage. Fischer, Jena. T.bce, 3. 1887. Sur quelquas bacteries des eaux de boisson. Ann. d'Hyg. Ibblique et 13 “bd. legals. 17: 3Eh-35”. Kace, E. 1913. Traite Practiqle de Bactari clogia. Tone II. 3rd edi- tion. J. B. Bailliers et Nils, Paris. Manual of methods for the Pure’ Cultvr-c Siui 1v Bacteria. 19h7. Society of American Bacteriologists, Cene.a, H. Y. ‘uartin, P., 1931.1ata1 infection by Chromobacterium violaceu.. Rey. Inst” ltd. RC3. F. 3. S. p. 68. Cited by Sneath, P. H. A. et al. Lancet, 265: 277° Maschek. 1867. Baxterioloxiscne unter.3*c1ungen der Lei=i eritzer trink- 3" wasser Jahresbericht der Komm. aber R ;a133Lu1e zu Leitr 3 its. Boehnen. Cited by Godfrin, P. 1934. Go tributiona 1'etude des vacterie" bleues et violettes. These, Nancy. Migula, A. 1900. System der Bakte rien. Zweiter Band. dustav y 3cher, Jena. fatal infection by Chromobacteriun violaceum. Minnett, 1:. P. 19 . 1 do (£11111. ‘9. hhv Cited 02,7 811031.111, P. H. A. et :11. 1?:‘3. .7 l Brit. Guiana Lie Lancet, 265: 2 Navy, F. C. 1953. Per anal conmunicatiun. Plagfe and Proskauer, B. 1887. Bericht Uber die untersuchung des Berliner leit'ngswassers in der Zeit vom 1. Juni 1885 bis 1. Aoril lbbo. Zeitfchl. f. Hyg., a: bfl-}65. Salvin, 3. 3., and Lewis, M. L. 19h6. External otitis, xith additional studies on the genus Pseudouonas. J. oi 3.3t. 51: 1495—506. ili’ifi Schat tenberg, J. J., and Harris, U. P. l9k2. Chromobacterium violaceus var. manilae a. a pathogenic microorganism. J. fo Bact.,_hh: 09—52 . ,u .1 .. , . ,. . Scnroter, J. 1b72. Ueber einige uurch bacteriengeoildete pigments. B: itrage sur Biolo ie der PTlanzen, Heft? a, 109-126. a ‘ n _ , w , , scnrotar, J. 1b36. Cohn's hryptogamne rlora von SchleSien, Pilze- Ers te Band, 157. Cited by Godfrin, P. 193k. Contribution a 1'etude des bacteries bleues et ‘iolettes. T‘cse, Nancy. chrote r, J. 1889. Cain's Kryptogamen Flora von Schlesien, Pilze. Dri te Band. Cit ted by Godfrin, P. 193M. Contribution a 1'etude des bac erics blsuss et violettes. These, Nancy. Sinpel, W. L., Medina, G. and Atwood, M. 3. 195‘- J. An. Vet. Lbd. a- 2‘. 15 3' Juan. l 1.10 'lx 7"l'_..o r Snith, A. J. 1887. A new chromogenic bacillus---Baci11us coe; uleus. The Xeiical ers, 51: 758-759. 1'“;— Smith, N. R. 19b6. Aerobic mesophi lic spore-forming bacteria. U. 3. Dept. of Agriculture. misc. Publ. No. 559. In Sneatfi, 9. H. A., J11c< ian, J. P. 3., Singh, R. 3., and Edwards, D. 19; . Fatal iuiection by Chro nobactaciun violaceum. Lancet, 265: 276-277. Sneath, P. H. A. 1953-56. Personal communication. death, P. H. A. 1956. In .ress. Soule, 3.3 H. 1939. A study of two strains of B. violaceus isolated from human-sci L3. Amer. J. of Path., 15: 592-596. lbirey, S. 1900. Cited by Godfrin, P. 193k. Co ltribution a 1'etude des bacteries ble aes at violettes. These, Nancy. Thonas. 1930. Etude comparative de Bacillus mesentericus fuscus Flugge et Bacillus mesentericus Niger Lunt. These Pharmacie, Nancy. Cited by Godfrin, P. ’193h. Contribution a l'etude des bacteries bleues et violettes. These, Nancy. Tobie, J. C. 1939. Bull. Assoc. des Diplomas de Hie obiol. Fae. Pharm., o o ‘2': ' h’"7 W . t '. ‘ . o o A A \ r" Toni, J. 3. and Trev1sin, V. 1009. Sy110ge Sccizoayce tum 3X saccar «0 Sylloge Funborum, vol. 3, p. 933- van der Sleen. 39L. N,te s l'exaznen blot rrologique qualitatif de l'eau Haarleu fie ritiez rs Lo- J33. Arch. T eyler, Serie II, t. IV, 8 Dartie, p. 121. 1 u. S“; _- VOL es, c. 1C93. Uc ber einiL 3 ii Nasser vorkomnende pigment-b3? mt rie . '3 '5 \ U21 T14-rllnlo lo aJ'qSit.’ l-+:U031"315 '6'.- ‘ 1“ T 1’ I} r. .I‘ n" 1 I.1 t“ \ d ' “V v“ "‘ 1“" “‘v ‘4" .‘ “ uielde1c, u. 1) o. 11tude d'un Dacllle lscls d'dn (us 1'T1131 1€nbd136: C.‘ . '. ‘ -' ‘ fi"\ v‘: ~v-\, ‘: v ‘. I . w.- saCillus Violaceus Uv no son LiLJeJc: 1a Jlulacelne ristallises. "'1; 'Nr‘.\ ‘1,“ IT... 1 ' f‘t . fi‘. ‘ 1. a 1L 1vo‘v UL 113.113. ’ Q FdQQOdl {3' Hard, H. N. 1390. A violet baCLllus from the Thames. Cen'ralol. f. __ ~. ~ } ‘ ‘9 ‘1", ‘ L ‘ 31;t., Sand L, Refezatc, u: 902. be: also: hazd, H. I. 1C9b. SOL: 1 1 r 1 ’u (T 15’ _ r L ' Tnanes Jacterlal. Ann. o1 satang, 12: 207. Jinslow, C. E. A., ct a1. 1920. Tue amilies and Le nera of tne bacteria / ,. . .. ' A N A ." .— ‘w ~ ‘ A 1‘ ‘ n J. ’ r‘ K \Einal Io: rt o1 L‘s UO‘S ittcc CT the JUClCJJ c1 .meric1n Java11013“ gists.) J. of Bact., 5: 191—229. - wooley,. . G. 1905. Bacillus violaceus manilae. Johns Hopkins Hospital C111.,1(>: 39—93. Zopf, J. 1885. Die Spaltpilze. 2 Aufl. E. Trewcndt. Breslau. Zobell , C. E. 1932. Factors influencinL the reduction of nitrates and nitr i U83 by bacteria in semisolid me dia. J. Bact.,'£g: 273. 1,; ‘W‘ :' "‘r ‘. '1 V‘— «4-4,: Audrcsbes oi LJOdsratiLb inhfzau oatuis ‘1 “‘4 .'|~ Ltfi‘v T : R‘: 1 ' Ir” ?.'-',r ‘. [3" " 4 '2 iorams, 3:. “£3443, immunwlog/ Div_s lun, Arm“ 13113“ 33;; cc . '11 .3, v. j , AWWT'J' ~ ‘r‘l‘ . 'l INK”.2 .) ', fl?” .. ,3.’ Y 1’) ¢1l~-I‘!‘JEJ~t-43 QUIT d 01’ " W-Lt ‘ ‘w;-i :‘0‘ luv a‘mj"k -Ca* V‘QVA f"; , I. 1 )3. ., .;) VKjAl .LL , A "1 u. L. J! . T ‘ T7 T» .F‘ L t 4' f ‘1 H} "J’ “ ’I. ( Ifi" F, :-,—-‘ ,. '- . ‘ ~n~ I." -', c (‘0 I. O .1. , .LLAU JJ— Uut L L1; 414.;- l-’V—s L134, vr)l‘§ . , ALL': jLL'ln‘uAASQ s.) ULTJQQe L") t) a -: r..-‘,\ Ar ‘,i-, :1 31"]..11'1‘1Jd 1195.0.) ‘JVL. .lL‘I'IJ' Jul‘“an, N. 1., fiLtropolitan qulf Sfari, Lennon, England Urrjc, Jr. Milliia L., Larartnent of Biolcgf, "estcrn Kc nLuckr State College, Boxling fireen, Aentucky tiynes Jillian 0., FeimitnLa ion Section, Yortacrn Utilization Re- I 3 search Branch, Agricultur 1 Re sea rcll 3L rvicc, United States Tafartnant of “5113313130 Fe Jria Illinois Kluyver, Prof. A. J., LaboratLrium voor IIiLrouiolo is, PL01M-L 1e K00 L3L1331, Ni:afe Laan 5, De.Lft, Hollan LcClang, Dr. L. 8., nosartmant 3f Bacteriol31y, Ind diana Uni Veg3itg, filoamin¢t3n, Ingiana P... ‘LJ 0 :3 f" c (D :1, (D . ,' ‘1 ." r 1 I _I v ‘.‘ ‘_ A 15- C ‘1 q '.. V ‘ a ‘ ‘ O r 4“, .1 iuilLaai, Prof. J. u., LaoLraLolre »3 LiCfi‘DiulbbLe, -w _‘ “ ‘ v" \‘4 ‘ ‘ .-~ 1’ ‘Yr‘ '1'," “" "3 Phafmdbic 48 Adnbj, LinL,, glance . 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