S’I’UDIES ON A RED PIGMENT PRODUCED BY PSEUDOMONAS AERUGINOSA Thesis for file Degree of DE. D. MECEIGAN STATE WWERSETY Frederick Just Post 1958 FREE!“ P \\ HWWW \iiiiiijii 12930 WU ‘ 00/ This is to certify that the thesis entitled STUDIES on A RED inHTYT yxwruuflu I"? l’ li'JU UK ll GNU} 2“. i": f’fU £11101"HK J4. presented by Frederick dust Post has been accepted towards fulfillment of the requirements for A— degree in __I‘.’1 i CID. b 1.0 10 53y Majbr professor Date 14 Lily 1958 0-169 LIBRARY Michigan State University '~s-‘ -_ PLACE It RETURN BOXto movothb checkoutfrom your record. TO AVOID FINESMumonorbdomdlto due. :1“— DATE DUE ’ DATE DUE DATE DUE we?“ __ L_- IS!- flit—l at) MSU chn Afflnnatlw Action/Equal Opportunity lnctltwon Wm: STUDIES ON A RED PIGMENT PRODUCED - BY PSEUDOMONAS AERUGINOSA By Frederick Just Post AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree or DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1958 Approved W WW \ Frederick J. Post 1 An organism has been isolated from soil which produces a water soluble red pigment. The organism has been identified as a variant of Pseudomonas aeruginosa var. erythrogenes (Gessard). The wine red pigment was later termed pyorubrin and it has been reported occasionally by various authors since that time. The cultural requirements for the production of the red pigment have been determined and it has been found that a medium composed as follows gives the best results: DL-alanine 1.0% glycerol 0.5%, K2HPC4 0.01% pH 7.0 A procedure has been demonstrated for isolating the red pigment from synthetic media. Because of the fact that the pigment is soluble only in water, methyl alcohol,phenol and acetic acid, the procedure linvolves precipitating the pigment on a powdered cellulose column and washing with various solvents. This treatment is followed by chromatographic separation from various contaminants on powdered cellulose columns using methyl alcohol and 1N NH¢0H as solvents. In characterizing the pigment it was found that it had E8 at ph 8.0 of +0.215 volts. It was also found that the pigment could act as an electron acceptor in the succinic dehydrogenase system of yeast. It has further been noted that the pigment has four distinct absorption peaks at 254, 281, 390, and 515 mu at pH 8.0. Frederick J. Post 2 It was discovered that the pigment, as isolated from culture, was altered in strong base (1N NaOH; very poorly if at all in NHQOH) in such a way that it now became a very ef- fective indicator. the color of the original pigment changed slightly toward a violet color below pH 5.0 but remained red at all pH's above 4.0. The altered pigment retained the acid characteristics but now became orange-yellow above pH 4.0. The possibility that some group is removed from the molecule by the action of the base is discussed, and absorp- tionSpectra for all of the pigments derived from the one isolated are included. The theory that the red pigment is a derivative of the phenazine nucleus is advanced on the grounds of the similar- ities of absorption spectra with the phenazine pigments, its chemical reactions, and finally by consideration of the fact that the organisms, to which these isolated belong, character— istically produce phenazine pigments. in organism obtained from the National Collection of Type Cultures, London, and identified as 33, aeruginosa var. erythrogenes on the basis of pyocyanine production, also produced a red pigment identical with that obtained in the present studies. The identification of the organisms, by other means, used in this study is therefore verified. STUDIES ON A RED PIGMENT PRODUCED BY PSEUDOMONAS AERUGINOSA BY Frederick Just Post A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1958 r/’ r," 6' / Ar. . 0 If) ’ ’éf‘isl Vita Frederick Just Poet Candidate for the degree of Doctor of PhiloSOphy Oral Examination, 13 Kay 1958, 5 PM, Room 325 Giltner Hall Dissertation: Studies on a red pigment produced by Pseudomonas aeruginosa Outline of studies Major subject: Microbiology Minor subject: Biochemistry Biographical items Born, 20 February 1929 Undergraduate studies, University of California 1946-52, B.S. in Public Health Sanitation Graduate studies, Michigan State College 1952-53, M.S. in Bacteriology Experience: Bacteriology Laboratory Technician at the University of California Sanitary Engi- neering Research Station 1950-52, Grad- uate Research Assistant at Michigan State College 1955, Bacteriologist United States Army Medical Service Corps 1953-55, Grad- uate Assistant Michigan state University 1955-58 * member of the Society of American Bacteriologists, the Society of the Sigma Xi, the American Association for the Advancement of Science, and the Albertus Magnus Guild ACKNOWLEDGMENT The author wishes to eXpress his appreciation to Dr. W. L. Mallmann whose guidance aided appreciably in the progress of these studies; to Dr. H. L. Sadoff for his useful suggestions; and to all the others who were so helpful and patient. ad majorem Dei gloriam TABLE OF CONTENTS Introduction Historical I. II. III. IV. V. Identification of the Organism under Study. SXperimental Results Conclusions Pigmentation and Carbon-Nitrogen Source. EIperimental Results Discussion Pigmentation and mineral requirements Emperimental Results Discussion Isolation and Characterization The Pigment in Culture Isolation Characterization General Summary Figures Literature Cited 37 38 43 49 51 51 60 74 78 78 80 89 92 94 102 113 117 150 INTRODUCTION Ever since sédillot (1850) first reported the peculiar occurrence of blue tinted bandages, the pigments of the group of organisms belonging to the genus Pseudomonas (Borgey's Manual 1957) have excited great interest and a phenomenal amount of research. The pigment observed by Sedillot was later isolated and described, long before the organism itself was isolated. The genus embraces organisms producing colors ranging through the spectrum from blue to red. The pigment which one associates with the genus in general, is the so-called ”bac- teriofluorescein", a greenish fluorescent pigment usually con- sidered characteristic. The second most frequently encounter- ed pigment, blue pyocyanine, is the preperty of one species and is usually considered the identifying characteristic when it occurs. The variability encountered among the organisms of this genus is oftentimes extremely eanperating to workers attempt- ing to identify isolates or classify its members. A common example of such variability is as follows: A pure culture of Pseudomonas aeruginosa may produce, 1. blue pyocyanine, 2. green bacteriofluorescein (henceforth called fluorescein), 3. a black melanine—like pigment, and 4. a red pigment, all of which are water soluble. Progeny of this culture may ex- hibit all of these, any combination of them or, in fact, no pigment at all. The alteration of characteristics may remain as a permanent feature and pigmentation as a means of iden- tification is thus limited. Biochemical and morphological characteristics are equally disjointed, resulting in confusion at times. One must examine as many variables as possible in order to arrive at any comprehensive idea of a species. Sver since Gessard (1882) first isolated and described Bacillus pyocyaneus (Pseudomonas aeruginosa) there has been a running argument as to whether this organism produces a red pigment as a separate entity. everyone who has worked on the subject of pigments in this organism has granted that a red- dish hue deveIOps in older cultures, but have offered a vari- ety of explanations as to its source. Only a few authors have truly recognized a separate red pigment and reported it as such. One of the first problems encountered with this red pig- ment (when it has been reported) is its complete insolubility in all common organic solvents and the consequent difficulty in isolating and identifying it. Further, it has usually been reported to occur simultaneously with pyocyanine and/or fluo-» rescein. .Few attempts have been made to study the pigment directly due to these facts. During the course of a problem in industrial waste dis- posal, the author isolated an organian which produced a sin- gle and unmistakable red water soluble pigment. It was later identified as a Pg, aeruginosa culture in spite of the fact that no pyocyanine was produced. The purpose of this work may be considered fourfold; l. to show that a red pigment does exist as a separate entity in strains of £2, aeruginosa, 2. to determine the nutritional requirements for the production of the pigment, 3. to develop means of isolating the pigment, and finally, 4. to find some chemical and spectroscOpic characteristics of the pigment. HISTORICAL A. General One may reasonably assume that wound infections showing blue pus had been.observed many years prior to the first re- port by Sédillot (1850), since his report was concerned with the transference of the agent of this condition. Fordos (1860) succeeded in isolating the prominent blue coloring material by the use of chloroform, and three years later (1863) recog- nized a second yellow pigment from stained bandages. This in- vestigator gave the name pyocyanine ("blue pus") t0 the blue pigment, and pyoxanthcse to the yellow pigment. Interestingly, the pigment of the blue bandages was isolated and studied twenty years before the causative organism was isolated. Lflcke (1866) probably was the first to associate the blue pus coloration with "rod shaped vibrios'. The organism may have received its name about this time, for at the time of Schroeter's publication (1875), the name Bacillus aeruginosum had been assigned to a pigment producing bacterium and Bergey's Manual (1957) authorized this species name for the organism, later called Bacillus pyocyaneus (Gessard), on the grounds of priority. Gessard (1882) was the first to isolate the organism responsible for blue pus, which he called Bacillus pyocyaneus on the basis of the identity of the pigment with that iso- lated by Fordos. The initial work by Gessard was his disser- 4 tation ("to demonstrate the parasitic origin of the phenom- enon", 1.6., blue pus) and his attachment to the study of this organism lasted until his death in 1925. On the basis of prior usage this organism is now called Pseudomonas aeruginosa. Ledderhose (1888) and Charrin (1889) studied the path- ogenicity of this organism for animals, and header 33 al. (1925) studied the animal pathogenicity of a red strain of the organism. Elrod and Braun (1941) suggested that a plant path- ogen Phytomonas polycolor, pathogenic for laboratory animals, was identical with 33. aeruginosa. These authors (1942) also showed that 22, aeruginosa could act as a plant pathogen. Many other examples of pathogenicity could be mentioned but a more complete list of references on this tepic may be ob- tained from the excellent article by Kietzmann (1955). Emmerich and Law (1899) observed and isolated an "enzyme: which they called pyocyanase, and demonstrated its toxicity for other organisms. One might say this was one of the first antibiotics isolated. Hosoya (1928) had similar results. This antibiotic action has been shown to be due to substances other than an enzyme (Schoental 1941, Hays 22 31. 1945, wells 1952) and will be discussed more fully later. Gessard (1882) found two pigments, blue and yellow, in cultures of P3, aeruginosa. Ernst (1887) reported the "cha- meleon phenomenon" which was a multiple color change along a a scratch created by drawing a needle through a heavily grow- ing agar culture. This was also reported by Jordan (1899a). Ernst eXplained this to be the result of the production of a leuco base which became colored on exposure to air. Gessard (1890b) now claimed only two different pigments, a blue pyo- cyanine becoming yellow pyoxanthose, and a green fluorescent type. Later (1891) he established four races of fig, aeruginosa on the basis of these two pigments formed on various media, namely: race A forming pyocyanine and fluorescein; P forming only pyocyanine; F fluorescein only;and S forming no pigment. Jordan (1899a) assigned the term variety and Greek letters to these same groups. Thumm (1895) felt that 33. aeruginosa formed only one pigment which assumed various colors and shades, depending on the amount of ammonia produced in the culture. Gessard (1898) found yet another pigment- a black one formed on tyrosine substrates. Charrin and do Nittis (1898) reported the simul- taneous production of black, blue, green, and yellow pigments by Pg. aeruginosa. Jordan (1899a) thought the black pigment was an oxidation product of the fluorescent pigment and the green was a stage in the process of pyocyanine oxidation to the black pigment. Krause (1900) found six different types of pigments, depending on the solvent used to isolate them. Gessard (1917) reported yet another pigment, this time a red one. Turfitt (1957b) felt that there was more than one pigment and Fraser and Mulcock (1956) demonstrated four separate pigments on the basis of solubilities and indicator properties. Razidha (1898) reported that 23. aeruginosa and B. fluorescens (now 22- fluorescens) were one and the same species although (1899) he felt all the fluorescent bacteria may be divided into two groups, one like Pg. aeruginosa and the other like 23, fluorescens. Aoki (1926) reported that the organisms were so variable one could not tell them apart. He suggested that they may be variations of the same organism. He included here also Pg, putida. Sandiford (1957) thought much along the same lines, arriving at the conclusion that one is justified in including the two organisms together since it was so difficult, if not impossible in some cases, to tell than apart. Beginning with Gessard's (1891) studies, it became ap- parent that gg. aeruginosa could be induced to lose complete- ly the ability to form any pigment at all. This was also demonstrated by Hasserzug (1887), Kruse and Pasquale (1894), and Remlinger (1898), among others. This point will be discuss- ed. further under Pigments since it raises many problems. B. Pigments 1. Pyocyanine Of all the pigments produced by 23. aeruginosa, pyocy- anine has been the most studied by far, due, mainly, to its ease of isolation. It has given a great deal of information on oxidation-reduction reactions and of electron transport. The discovery by Fordos (1860, 1865) that the pigment of blue pus could be extracted from water solution by chloroform led to his further study of its prOperties. once the chloro- form layer had removed the blue pigment from water, the or- ganic layer was removed. He found that the addition of acid 7 to the ruiloroform.1ayer caused the pigment to become red and at the same time no longer soluble in chloroform and it then returned to the water layer. Barium carbonate (or any base) restored the blue color. A yellow pigment which he called pyoxanthose remained in the tailoroform.after the acid pyo- cyanine was removed. - His work showed that pyocyanine was soluble in alcohol and .cfloroform and less soluble in ether. Pyoxanthose was slightly soluble in water and easily so in alcohol, ether, choloroform, carbon disulfide, and benzene. Gessard (1882) observed that pyoxanthose was red in acid and violet in al- run" He felt that it resulted from the oxidation of pyocyanine. Jakowski (1895) observed that pyoxanthose in ammoniacal solution was yellow by transmitted light and gave a green fluorescence. He reported this substance to be soluble in water and alcohol and insoluble in ;.chloroform, ether, and amyl alcohol, contrary to Fordos, if this was the same pigment. Thumm (1895) thought the original pigment was a yellow (yel- low-orange in concentrated solution) with a blue fluorescence and alkali determined whether it would be a dark green or a light green. Boland (1899) observed that a « chloroform solution of pyocyanine, upon standing for a day, changed to green and then to yellow, lending support to the idea that pyoxanthose (also called pyoxanthine) arose from pyocyanine and not vice versa. Jordan (1899a) felt that pyoxanthose arose from pyocyanine after passing through green and black intermediates. 0n the ,x. ¢ ‘v\ . other hand, de Seixas Palma (1907) felt that pyoxanthine be- came the green color of pyocyanine and then became red and finally brown in the last stage. Ernst (1887), as already mentioned, theorized that pig- ment formation resulted from leuco compounds formed by the organism which became colored on exposure to the air. has- serzug (1887) reported that oxygen was the most important single requirement for pigmentation. Further, he observed that pigment was produced during the second of two stages; (1) growth, followed by (II) pigment formation. This was borne out by Harris (1950). Christomanos (1902) also sup- ported the leuco base theory. Sullivan (1905) has an excel- lent list of references up to 1905. Ledderhose (1888) thought pyocyanine to be an aromatic closely related to the anthracenes. It was not until the concerted efforts of Trade and Strack (1924, 1925, 1928, 1929) that a probable chemical structure was suggested. Michaelis (1951) finally established the presently recog- nized structure: 0 (If: k / ,// t 1) He CH3 acid form basic form The genus Pseudomonas represents the only known group of organisms producing phenazine derivatives, which is the nucleus of the illustrated structures. The structure on the left exists in acid solution and is red. When in basic sol- hnion the oxide group on the number one carbon atom ionizes and in this form is blue. In the blue form it is soluble in chloroform and the unionmed form is not. The molecule is rather easily reduced and from the blue form (under alkaline conditions only) proceeds by one step to leucOpyocyanine. Under acid conditions (and only acid conditions) the reduction proceeds in two distinct steps through a very stable green semiquinone form (half reduced) to leucOpyocyanine. Elema (1951), Michaelis (1951) and Friedheim and Mich- aelis (1951) finally brought some light to understanding of the multitude of pigments observed in fig. aeruginosa cult- ures, though not everything has been explained as yet.sred, blacks, browns, etc. an excellent reference on this aSpect is that of Bracken (1955). Pyocyanine is demethylated very easily in the presence of air under alkaline conditions or by 8302 to the yellow relative l-hydroxy-phenazine. This compound has been report- ed to be produced in older cultures. Between pH 1 and pH 11 this compound is lemon yellow and above and below these pH's it is red. The partially reduced semiquinone is green. This demethylated pyocyanine is probably the pyoxanthine (-ose) of previous authors. The spectral absorption curves of pyocyanine and pyo- xanthine may be found in the works of Cluzet 23 31. (1921), Lasseur and Girardet (1926), Vellinger (1955a,b) and Hugo 10 and Turner (1957). The function of pyocyanine and its related pigments has been a subject of considerable debate and is by no means definitely settled yet. The oxidation-reduction potential of pyocyanine lies between methylene blue and indigo trisul- fonate and therefore can accept electrons rather easily (Elema 1951). Stheeman (1927) actually had prOposed this. Friedheim (1951) and Ehrisman (1954) also thought that it was involved in respiration, and it has been used by many, along with its quaternary relatives (safranine, etc.) to study respiration. Harris (1950), however, felt that it was merely a waste product on the grounds that little or no pyo- cyanine was formed until maximum growth, in terms of numbers. had been reached. A short review of this subject has been prepared by Campbell gtflgl. (1957), who reported that pyo- cyanine can complex magnesium.and specifically inhibit those enzymes requiring Mg in their activity. 2. Fluorescein Fluorescein is the most universally occurring pigment formed in the genus Pseudomonas and is one of the key char- acteristics of the genus. According to Turfitt (1957b) this pigment was first observed by Kunz in 1888 when he removed the pyocyanine and found a green fluorescent material which was soluble only in water and alcohol. Babes (1889) observed the same results but added that the culture was red-brown in diffuse light and emerald green in refracted light. An acid solution fluoresced (in contrast to the reports by most 11 investigators) while a basic solution did not. The coloring matter was of two parts; a) soluble in alcohol and green like cholorOphyll, and b) insoluble in choloroform, benzene, 032, other, petrol, and amyl alcohol. The solution was red-orange by diffused light and blue-green by diffracted light. Acid and base changed the color. He considered it a separate pig- ment from the pyoxanthose of Fordos. Sullivan (1905) reported the green pigment insoluble in choloroform but soluble in alcohol and ether. Boland (1899) reported a reddish-brown pigment accompanying pyocyanine, extractable in alkali but insoluble in ordinary solvents. header 35,31. (1925) reported that a red pigment, pyorubrin (to be discussed in more detail later) occurred to some degree in all Pg. geruginosa. Little work was done in the intervening years due to the difficulty in obtaining a sufficiently pure product for study. Giral (1956) studied the preperties of two pigments 130- lated from.§g, fluorescens. One was an unidentified pigment which was reddish in concentrated solution, yellow in dilute solution and with a blue fluorescence in either case. The other was a yellow green pigment with a blue-green fluores- cence in water. The absorption spectrum resembled the fla- vines and the pteridines in some respects. He felt that the pyorubrin of header 2: 31, (1925) was nothing more than con- centrated fluorescent pigment. Turfitt (1957b) isolated the fluorescent pigments of several species of Pseudomonas by a process of electrodial- 12 ysis. He found that the pigment was amorphous and greenish- brown, soluble only in water, phenol, acetic acid, and aque- ous alcohol. Concentrated solutions were yellow in acid, red in alkali with a brilliant green fluorescence. Neutral solu- tion fluoresced blue. He found no evidence of a leuco pre- cursor, in contrast to Jordan (1899b) and header at 31. (1925). He assigned an.empirical formula of 043702N for the compound. The absorption spectra of the fluorescent pigments of several species were included in his work and he concluded that they were nearly identical. Turfreijer 33 31. (1958) criticized Turfitt's work on the grounds that his samples were not pure. This work gave an empirical formula for the fluorescent pigment as 03284103N7. It absorbed strongly at a wave length of 420 mm in alkali and 560 in acid. Oxidation gave a yellow-red fluo- rescence and the molecule lost a fragment of 05H505. The nucleus of the remaining pigment contained one oxygen atom and a heterocyclic system involving two nitrogens. In later work Turfreijer (1941) isolated four separate fluorescent pigments using a combination of charcoal absorption and pre- cipitation with phosphctungstic acid. Chodat (1951) separated blue fluorescing and yellow fluorescing pigments. Absorption spectra led to the same con- clusion that Giral had reached, flavine and pteridine resemb- lance. Rivera 23,21. (1956) gave some chromatographic Rf values for various pseudomonad pigments on paper. Bonde‘gt,gl. (1957) isolated a green fluorescent pigment from £3, calci- 15 pgecipitans and found an empirical formula of C14H31N53015Na4.5, He found that the green pigments of yOung cultures migrated slowly . on paper chromatograms developed by an isobutanol- HCl—HBO mixture, while that of older cultures migrated much faster and separated into several spots. 1 In spite of the work done so far, no agreement on the structure or relationships of all the fluorescent pigments has been reached. Hugo and Turner (1957) suggested that the green fluorescent pigment was related to or identical with 1-hydroxyphenazine since its spectrum was identical with the one he isolated from Pg, lemonnieri and also with those re- ported by Turfitt (1957b) . Absorption spectra of various fluorescent green pigments were reported by Cluzet 33 31. (1921), Giral (1956). Turfitt (1957a,b), Turfeijer gt_al.(1958), Chodat (1951), Hugo and Turner (1957) and Bonds 23 31. (1957). 5. Black Pigment A black pigment was first reported by Gessard (1898) and was shown by the same author to be an oxidation product of tyrosine under the action of an enzyme, tyrosinase. The pigment itself was melanine (black) formed through an inter— mediate red or reddish-brown pigment. Gessard (1901) gave the organism a variety status and it was known as 823. pyg- cyaneus var. melanogenes. Burkholder and Starr (1948) found about one half of the cultures they tested gave a reaction indicating the presence of tyrosinase. Little work has been done on this pigment (as a pigment of fig. aeruginosa) since 14 1901. However, much has been done on the chemistry of melanine formation outside bacteria. The red intermediate has been called d0pachrome and is a highly unstable substance spontaneously oxidizing to mel- anine. See McElroy and Glass (1950) for more information on this tOpic. The spectrum of this compound and others of sim- ilar nature was reported by Bu'lock 33 a1. (1950). It might be of interest to note that tyrosinase is one of the cOpper containing enzymes. 4. Red Pigment Mlle A. Raphael in 1897 (see Lasseur 91 3;. 1937) isolat- ed a strain of fig, aeruginosa from a disease process, which produced a red pigment as well as pyocyanine. Gessard stud- ied this organism, but it was not until 1917 that he report- ed and gave it a variety statusfigg. aeruginosa var. 23132- rogenes. His previous studies (1890a) showed that this organ- ism. produced a "greenish-yellow pigment which turned red with time". The organism isolated by Mlle Raphagl produced mostly the greenish-yellow pigment which turned red and only a small amount of pyocyanine. He reported that it produced a weak fluorescence when he first received the culture but that this power was soon lost. His description indicates that the greenish-yellow color was the reduced form of the red since shaking in air caused the culture to become"a beautiful vivid red of variable wine or current shade". Pyocyanine was produced in very small amounts but not in all media. 15 hamelle (1918) worked with a culture isolated by Lasseur from.adenoid pus which produced a bright red pigment. Com- parison with the culture of Gessard showed it to be identical. He also verified the observation that oxygen was required for pigment formation and added that the loss of power to form the red pigment paralleled that of pyocyanine which occurred here, again in small amounts. Rochaix and Bansillon (1925) showed that while the power to form both pyocyanine and the red pigment could be lost, they were able to restore only the pyocyanine function. Gessard (1919c, 1922) felt that the only difference be- tweena so-called normal culture and the red strains was the ratio of red pigment to pyocyanine. Leonard (1924) and Meader 33131. (1925) agreed that all Pg. aeruginosa produced the pigment to some degree and named the pigment pyorubrin, since they isolated their cultures from the urine of an infected rat. A short description of the pigment follows; it is form- ed from a leuco base and is not an acid-base indicator as is pyocyanine, the precursor is greenish in the culture, oxida- tion produces a leuco compound which cannot be reduced to red again, reduction of the red gives a leuco base and the red can be restored upon oxidation, no solvent could be found to dissolve it but alkaline ng caused an orange precipitate in 24 hours. It was reported to be non-fluorescent although this was apparently decided without the aid of an ultra- violet lamp. Lasseur t l. (1926,1957) studied the red variety and 16 suggested that it did not sufficiently define the species whereas pyocyanine did. Don and van der Ends (1948) thought that while the red pigmented forms were rather rare, the pig- ment did differ from the red-brown observed in other forms. They isolated from the urine of a patient with cystitis a culture which formed a red pigment rather weakly in serum or glucose broth. Glycerine improved pigment formation especially on solid media. Colonies formed a metallic effect. Chernomordik (1956) studied the red pigment of five cul— tures of fig. aeruginosa, all of which showed marked antag- onism to other bacteria. Glycerine enhanced pigment formation. The pigment was insoluble in all organic solvents tested and it was unaffected by acid or alkali. Oxidizing agents did not affect it but reducing agents gave a brownish-yellow color and H203 restored the red color. He also noticed that upon standing the solution lost its red colon,except on the surface, and became brown. The red could be restored by shak- ing in air. He disagreed with Vellinger (1955b), who thought that pyorubrin was nothing more than pyoxanthose (yellow) in the presence of alkali (red), since acid had no effect on the color. Schwarz and Lazarus (1947), Ringen and Drake (1952),and King st 31, (1954) reported the isolation of pyorubrin pro- ducing strains. Cluzet El 9);. (1921) reported that the eryth- rogenic pigment begins absorbing light at 4500 or 4700 A° and absorbs completely in the ultraviolet range. Bonde _e__t 9_1_. (1957) isolated a red pigment from another Species which was 17 soluble in acidified choloroform, had a blue fluorescence and an absorption spectrum similar to the above. Babes (1889), Gessard (1890a), and Boland (1899) report- ed red-brown pigments which may or may not be pyorubrin. Hefferan (1904), in a comprehensive study of bacilli forming red pigments, included several cultures which could very easily have been pyorubrin producing strains, at least from the descriptions. Nogier 93 31. (1915) isolated gs. aeruginosa from.a lesion. It formed a red pigment "the color of vinegar" which was not the normally found red-brown pigment. Rahn (1916) included a red pigmented Pseudomonas which liquified gelatin but was not otherwise described. Legroux and Genevray (1955) described a culture of £2. aeruginosa which produced a yellow or rose pigment on gelatin or Dorset's medium. Rivera 33 El: (1956) studied the chromatographic prOperties of otherwise unidentified red pigmented strains of Pseudo- ,Qgggg. Giral (1956) and Turfitt (1957b) suggested that the red pyorubrin was nothing more than the green fluorescent pigment in concentrated solution. The problem of this pigment has still not been resolved. 5. Achromogenesis The variation of pigmentation in.g§, aeruginosa is "subject to certain caprices", as Aubel and Colin (1915) Stated, but the lack of any pigment at all may be a serious problmm, especially where the organism occurs as a pathogen. Kruse and Pasquale (1894), Hemlinger (1898) Lomry and Gillet (1929), and Gaby and Free (1955), among others, have isolated 18 non-pigmented forms of £3, aeruginosa from disease processes. The last named authors reported 4% of all the isolations in their laboratory fell into the non-pigmented group. The fact that achromogenesis could occur was observed very early by hasserzug (1887), Phisalix and Charrin (1892), Charrin and Phisalix (1892a,b), Blanc (1925), and Rochaix and Bansillon (1925) who reported that they could induce tempo- rary or permanent loss of ability to form pigment. Gessard (1891) and Jordan (1899a) both had a category for achromogens in their division of Pg, aeruginosa. Beginning in 1919 (a,b) Gessard started an intensive study of achromogenic forms (1920a,b,c;l922) and suggested calling them Bacillus pyg- gyanoides because of their resemblance to Pg. aeruginosa on the grounds that only if it produced pyocyanine could it be called 22. aeruginosa. Domingo and Cullell (1952) reported that the results of Gessard's methods of classification were too variable to use. A variant might gain ascendancy and impose its characteristics on.the rest. Lasseur and Thiry (1915) showed that selection Of the prOper medium could induce pigmentation in organisms not otherwise producing any. Lomry and Gillet (1929) and Legroux and Genevray (1955) demonstrated the practicality of this observation by inducing pigment formation in cultures of P3, aeruginosa which had never formed any before. More Will be said about this aspect under Nutrition. 19 6. Other Pigments Several other pseudomonad species produce pigments re- lated structurally to the phenazine nucleus of pyocyanine and for continuity will be briefly mentioned here. Tobie (1945) suggested that the Pseudomonas be classi— fied on the basis of production of phenazine pigments. Four of these have been isolated so far besides pyocyanine. Pg, chlororaphis produces a green crystal within its culture medium which is the semiquinone of oxychlororaphin. This pigment, which is called chlororaphin, is structurally as follows (Braken 1955): ’0 N \\NH2 N ./J Birkofer (1947) has isolated this pigment from cultures of Pg, aeruginosa which produced pyocyanine as well. This indicates the very close relationship between these organisms. Clemo and icIlwain (1958) and Clemo and Daglish (1950) have succeeded in identifying the pigment of Pg. (Chromo- Iggcterium) iodinium (Bergey 1957) as the di-N-oxide of 2,5- dihydroxyphenazine. OH 0 Kluyver (1956) and Haynes 33 g;. (1956) N§§§ '\\ isolated a new species, Pg. aureofaciens which produces several pigments, one of E ‘ which is the greenish-yellow or yellow 0 CH phenazine l-carboxylic acid. Note the similarity (structure on next page) of this pigment to that of Pg. chlororaphis. 2O ‘,0 Pg. sypcyanea has been reported /, N H to form a phenazine pigment (Liu 1957) i also. QM A few other pigments occur which phenazine-l—carboxylic acid may be related to those of the above group: Pg, nigrificans reported by Fatznelson and white (1950) to form its black pigment under cultural requirements similar to those of pyocyanine; Pg. viscosa found by China (1955) forming greenish crystals in the medium; Pivnick (1955) named Pg, rubescens, found in cutting oils, which formed a pinkish to yellow-orange pigment; and finally, Pg. lemonnieri forming a blue pigment (unlike pyocyanine) isolated by Hugo and Turner (1956, 1957). This is by no means a complete list of pigment formers among the pseudomonads but it fairly represents the multitude of colors produced by this genus. It will require a great deal more research to establish the true natural relation- ship between all of these organisms, indeed if there be any. It would certainly seem that all of the phenazine producers are related but it remains to be seen just exactly how. C. Identification -he problem of identification of Pg. aeruginosa has been complicated by the insistence of classifying on the basis of pyocyanine formation. Gessard (1919a, 192Ca, 192;) insisted that the production of pyocyanine wasa must in order to call the organism.Pg, aeruginosa. Tobie (1945) felt that classi- 21 fication on color alone is irrational and ”swan (1956) echoed the warning of Charrin and Pnisalix (1892a) when he stated that division based on a single characteristic is un- desirable but may be necessary for want of adequate knowledge, however, it should cease to be used when sufficient knowledge is available. Tobie suggested classifying the Pseudomonas on the basis of production of the phenazine nucleus. Special means of identification began with Gessard (1891) who divided the Species into four races based on pigment production in certain media after he discovered (1890a,b) that substrate had great effect on the type of pigment pro- duced. Later (1919b, 1920b) he develOped his classification into an elaborate system whereby pigment in bouillon de- mmibed the race and pigment in peptone decribed the variety. He also used glycerol-peptone~agar to authenticate the Species by its color production. Dissatisfaction in this method was eXpressed by Domingo and Cullell (1952) and evidence accumulated through the years that many cultures lost the power to produce pigment and that many organisms known to be Pg; aeruginosa did not form any pigments. Several selective media have been developed but none has gained wide acceptance. Vitiello (1954a) incorporated 0.75% tannio acid into an agar medium which he claimed was selective for Pg, aeruginosa without hindering colony shape or pigment formation. Bhat (1956) used formate as a sole carbon source for Pg. fluorescens. Ringen and Drake (1952) used 9 mg per 100 m1 of pyocyanine as a selective agent and 22 reported that growth of almost all other organisms was in- hibited. Haynes (1951) develOping the observation made by Charrin and Phisalix (1892a,b) that 23. aeruginosa could grow pro- fusely at 43 C, prepared a three step procedure designed to identify Ps. aeruginosa whether it formed a pigment or not, and to distinguish between this organism nudge. fluorescens. Bis tests included, growth at 42.5 C, oxidation of potassium gluconate and formation of heavy, stringy slime after several weeks growth at room temperature in potassium gluconate. Gaby and Free (1955) deve10ped a method of identification based on colonial morphology, odor, gram.reaction, acid from glucose only, gelatin liquefaction, and a negative urease test. 2g, aeruginosa represents an organism which is highly variable in morphology and in chemical activity. Bergey's Manual (1957) gives a series of characteristics to be used in.identification, however, each point is variable in some :respect as reported by various authors. About the only Completely acceptable character is that the organism.is gram negative. A. Flagellation has been variously reported. Nicolle and Bey (1896), Burckhardt (1917), Petschenko and Wroblewski (1928), Christie (194s), and Bergey's Manual (1957) all re- ported one polar flagellum, whereas Fuhrmann (1907) and Bartholemew (1949) reported 1-3 polar flagella. Edson and Carpenter (1912) reported polar flagella, in some cases 1, 23 others 3-6 and some tufts, while Aoki (1926) reported some 2, and.some 5-4 but seldom 5. As forgg, fluorescens the situa- tion is the same. Generally, the majority or gs. aeruginosa will be found to have one polar flagellum, and occasionally nmre, while 23, fluorescens has a polar tuft, usually, and ,nmy occasionally be found with 1-2 polar flagella. B. Gelatin liquefaction is generally considered to occur :rather rapidly with gs, aeruginosa although there are excep- ‘tions (Aoki 1926, Sandiford 1937, Salvin and Lewis 1946, I?ote1 1955, and Wetmore and Gochenour 1956). C. Colonial morphology on agar is extremely variable. Igang (1915) found that gs, aeruginosa and fig. fluorescens czould not be distinguished on colonial morphology. Gaby (1946, 1955) surveyed.the problem.and arrived at three (:olonial types characteristic of the species. Gainer and Ide ( 1951) and Williamson (1956) discovered colonial variants of I>s. aeruginosa composed of different cellular types, each of which had its own characteristic biochemical properties. D. It is generally considered that Ps. aeruginosa «coagulates, reduces, and peptonizes litmus milk with an al- kaline reaction. 23. fluorescens usually is Just alkaline and possibly reduction may occur. (Petschenko and Wroblewski 1928, Sandiford 1937, Seleen and stark 1945, Salvin and Lewis 1948, and Munoz 23 31. 1949). E. Indol is reported ascncurring, especially in the older literature (Jordan 1899a, Sherwood gt 31,1926). Sandiford (1957) showed that most reports of indol production 24 : ,‘Xlllilfllfrcvt' I . resulted from the addition of an acid reagent to cultures containing pyocyanine which naturally turned red-the positive indol test color. His work amply demonstrated that fig. 32525- igggg should be considered indol negative. F. Nitrates are generally reduced to nitrites and in many cases to nitrogen gas. However, these reactions do not occur in all strains. Jordan (1899a), Acklin (1925), Sherwood (1926), sandiford (1957), seleen and stark (1943), Gaby (1946), Salvin and Lewis (1946), and Burckholder and Starr (1948) should be consulted for a further discussion of this subject. G. Blood is hemolyzed by most strains of fig. aeruginosa but not all (Petschenko and Wroblewski 1928), and has been used in identification (Gaby 1946, Potel 1955 and Christie 1948); however, Liu (1957) has shown that too many other Pseudomonas species also hemolyze blood which limits its usefulness in identifying fig, aeruginosa. H. That g3, aeruginosa has a very distinctive odor was reported very early. Babes (1889) said that the odor was similar to that of linden or lime flowers or basswood. Nicolle and Bey (1896) refermd to it as a flower odor. stettenheimer (1915) and Cmelianski (1925) also refermfl to it as a linden flower odor. Gessard (1919c) thought the odor distinctive enough to be able to definitely identify 23, aeruginosa.1n 1924 he published a paper on the substrates producing the odor and found that trypt0phane was the most efficient. Lomry and Gillet (1929) and Chernomordik (1959) both refenbd 25 to use of the specific odor of this organism for identifica- tion. It has been refermfl to as trimethyl amine with a grape- like odor by Gaby and Free (1955). I. Hydrogen sulfide production, while not included as one of the characteristics in Bergey's Lanual, ap- parently occurs in less than half of the isolates. Acklin (1925), Clara (1954), Salvin and Lewis (1946), and Wetmore and Gochenour (1956) all reported negative results for this compound. Jakowski (1895) and Sullivan (1906) reported Has. Edson and Carpenter (1912) found about one fourth of their cultures were positive. Ransmeir and stekol (1942) reported small traces of H25 from.cysteine in synthetic medium, Chernomordik (1959) divided the species into two groups with H23 production as the chief dividing characteristic. Sherwood 91 §_l_. (1926) and Munoz _e_t_ 21- (1949) found that about half of their cultures produced H28. J. The fermentation reactions of fig. aeruginosa represent the most variable of all the biochemical reactions. Just about every conceivable result has been reported at some time. One may consult the works of stein gt 31. (1942), Munoz gt_gl. (1949) and Liu (1952) for references not listed here. Kendall and Farmer (1912) maintained that gs. aeruginosa does not utilize carbohydrate but this was effectively re- futed by Sein 33 21. (1942) who used potentiometric methods to show that carbohydrate was used even though no acid could be demonstrated. 1'. Edson and Carpenter (1912) boiled their cultures to re- move any amuonia which might mask acid production. Aubel and Colins (1913) showed that addition of glucose reduced the amount of ammonia produced and they thought this due to neutra- lization of acid. gears and Gourly (1928) showed that acid would be produced if the peptone concentration was 1% or 1688. Clara (1954) showed that acid could be produced in glucose, galactose, fructose, mannose, arabinose, xylose, mannitol, glycerol, but not in rhamnose, sucrose, maltose, lactose, raf- finose, saliCin, or starch when the carbohydrate was used as the sole source of carbon and the the nitrogen source was an inorganic ammonium salt. Brom cresol purple was the indicator. Elrod and Braun (1942) modified the medium somewhat and found similar results except no acid in mannitol or glycerol. Salvin and Lewis (1946) used phenol red as the indicator because of its greater sensitivity to small amounts of acid and the results were as Clara found, except fructose, mannitol, and trehalose were negative. All strains fermented glycerol, and seleen and stark reported identical results on this substrate. Gainor and Ide (1951) demonstrated that colonial variants frequently had Opposite fermentation reactions, e.g., on arabinose one variant produced acid while the other was strongly basic. Williamson (1956) reported similar results. Liu (1952) used a synthetic medium and found results similar to those of Clara but, although acid was formed from arabinose, xylcse, and mannose, these could not be used as a sole source of carbon by the organism. He also thought it of particular 27 importance that the time of incubation be reported. His theory was that protein was preferentially attacked and carbohydrate was not used until after the protein had been used, at which time the ammonia content was high, thus masking any acid pro- duced. Wetmore and Gochenour (1956) compared the reactions of several species of pseudomonads on synthetic carbohydrate media and found differences between them, fig, aeruginosa and fig. fluorescens were very similar, however. Simon (1956) distinguished between these two organisms and other pseudo- monads on the basis that only fig, aeruginosa could ferment D-arabinose in infusion broth. It is generally considered that in standard carbohydrate test media the two closely related organisms fig. aeruginosa and fig, fluorescens are highly variable and usually consid- ered inactive on all but glucose which may or may not be fermented (Bergey 1957). For this particular group of organisms much more information may be obtained if the nitrogen source is limited. fig, aeruginosa is a highly aerobic organism and was in- cluded by Gordon and McLeod (1928) among these organisms giving a reaction indicating the presence of cytochrome ox- idase. hovacs (1956) and Gaby and Uadley (1957) have adapted this test to the identification of fig, aeruginosa.All 436 strains tested by hovacs gave a positive reaction with tetra methyl-p-phenylene diamine dihydrochloride whereas Gaby and Hadley distinguished between several closely related species with p-amino dimethyl aniline oxalate. 28 Another test which has been little used as yet is the one for HON production. 3merson.gg,g;. (1915) and Patty (1921) reported its presence in fig. aeruginosa. Sherwood gt 31, (1926) found that all of 22 strains gave a positive HCN test. This question was re-examined by Lorck (1948) who found that not all these organisms produce HCN but suggested the use of picrate paper to test for its production. Emmerich and wa (1899) reported the presence of the so- called enzyme pyocyanase which was shown by Schoental (1941) to actually be a mixture of pyocyanine, l-hydroxyphenazine, and an unknown colorless antibiotic substance. Hays g} 31. (1945) and Wells (1952) isolated and identified a series of ‘ antibiotic substances from.fig, aeruginosa and found them structurally related to kynurenic acid, a tryptOphane deriv- ative. Young (1947) described the relationship of pigment production and antibiotic activity. He found that if the medium was acid and kept that way, neither pigment nor anti- biotic substances were formed. Tobie (1946) suggested the possibility of using this pr0perty to aid in identification. This was used by Potel (1955) who found fair correlation between antibiosis and animal pathogenicity. Two cultural characteristics have been reported which should be mentioned. First, crystals in the the cultural medium have been mentioned with the pigments, but there have been a few reports of colorless crystals from time to time. Dorset (1896) and Edson and Carpenter (1912) found crystals due to the precipitation of calcium and magnesium.phosphates 29 in the strongly basic culture. Beebe and Buxton (1904) found crystals due to fat formation. Second, the so-called metallic spots or plaques are often reported (Legroux and Genevray 1955, and Linz 1958) and are explained by Don and van den Ende (1948) as associated with bacteriOphage carried by this organism. Consult this last named refernce for more infor- mation. Jamieson (1942) suggested observing cultures under ultraviolet light to determine any fluorescence before pig- ment is visible. To sum up this section is the example of Gaby (1955) who studied twelve species of Pseudomonas,including fig. aerug- lgggg, and found that standard biochemical and morphological procedures could not distinguish between many of them. D. Nutrition and Pigmentation 1. General Pseudomonads are generally considered to be quite non- Specific in their choice of carbon and nitrogen sources and do not require the addition of any extraneous nutritional factors. In fact they get along on so little that Braun and Cahn-Bronner (1921) considered fig, aeruginosa an autotrOph since it could grow onan ammonium carbonate medium. Turfitt (1957a) observed the same results on the same substrate. Robinson (1952) felt that ammonium carbonate initiated growth but did not support it. The reaction occurred through the intermediate carbamate ion. Englesberg and Stanier (1949) 30 , l ..._ rho observed invisible growth with fig. fluorescens when no added carbon source was present. This prompted them to say "..... ‘fig. fluorescens, a typical bacterial autotrOph...". For more information on the early studies of pseudomonad nutrition, see the thesis by Turfitt (1957a). 2. Pigment Nutrition Lepierre (1895) claimed that fluorescence was the result of the presence of xanthine, creatine and soluble albumin in meat extract. He stated that heating under pressure destroyed the fluorescent producing capacity. Georgia and Poe (1952) diaproved the latter statement but did note that peptones had a great deal to do with pigment formation. some produced pig- ment while others did not. This effect was also noted by Maggio (1946). Goldsworthy and still (1956) and Lourie (1945) noted the suppressing effects of meat extracts on the pigment production of Bacillus prodigiosus and fig. aeruginosa respec- tively. Georgia and Fee (1952) showed that for maximum pigment formation the initial pH of the medium should be between pH 6.5 and 7.7. higher or lower pH's retarded pigment formation and lowereu the amount produced. This was also noted by Vitiello (1954b) and Young (1947). Hueppe (1880) cultured fluorescent bacteria on ammonium tartrate, KgHPO,, MgSO., and 08012. Gessard (1892) observed that 0.00625% K2HP04 resulted in pyocyanine, about 0,0125% resulted in both pyocyanine and fluorescein, and 0.15% gave only fluorescein. Fe thought that excess nitrogen favored 31 . . Fl.‘ .3. pyocyanine aid excess phosphate favored fluorescein. :33 also thought that lecithin was required. Lepierre (1895) disputed the effects of phosphate. Jordan (l899a,b) decided that for the fluorescent pigment both phosphate and sulfate were re- mflred and the cation was not important. Pyocyanine did not need either. Sullivan (1905) agreed with Jordan that phosphate and sulfate were required for fluorescein. sullivan (1905) found that alkali and high phosphate favor fluorescein and that I acid and low phosphate favor pyocyanine. \ Benecke (1907) found 0.0001 mg of potassium was required for growth and a slightly higher level for pigment. Potassium could not be replaced by Li, Na, or NH, while Rb and Cs could replace it but at a much higher concentration. Fe found that phosphate, magnesium, and sulfate were required. Goris and Liot (1921) reported that pyocyanine was produced on plain 2% agar while if large amounts of phosphate and Mgso, were added, only fluorescein was produced. Friedlein (1928) found that K,Na, and Cl ions were dis— pensible in an ammonium lactate medium and that Mg and 30, ions only enhanced growth but were not required. Georgia and Poe (1951) observed that MgSO, gives the best fluorescence and although some is produced in its absence, it is probably due to contaminants. Phosphate was also required. They found (19:52) that 0.1% Keane. and 0.1% 91330., when added to peptones which did not produce the.fluorescent pigment, now produced it. 32 Robinson (1952) found that phosphate and magnesium were required and that there was no growth without them. He also reported that potassium and sulfate were not required for growth or pigmentation. Hoffa (1954) found that phosphate and Mg aided pigment formation. Jamieson (1942) felt that 80,, POg, and Mg ions were required for pigment formation. Warring and Workman (1942) used organic complexing techniques to re- move iron and found that 0.08 ppm Fe was required for full growth of fig. aeruginosa. King 23 gfi. (1948) and Burton.g§_gi. (1948) studied the mineral requirements of fluorescein and pyocyanine, reSpec- tively. They found that the ions Mg, 30,, K, P04 and Fe were required for production of both pigments, but that the con- centrations determined which one would be formed. For instance, fluorescein was produced in large amounts at a low iron content while at a high iron content pyocyanine was the only pigment produced. It was also necessary that the Mg and 80% ion concentrations be low for fluorescein and high ( about 1%) for maximum pyocyanine production. Excess iron (above 0.005%) inhibits pyocyanine formation as does excess phos- phate ( over 0.05%) while fluorescein is not at all inhibited by 0.1% phosphate. The effect of low iron concentrations on fluorescein production has been verified by Totter and Mosely (1955) who found the most fluorescein produced when no iron was added. Hellinger (1951) agreed on the requirement for Mg, 30,, and.P0, ions for pyocyanine production and added NH, and and glycerol to the list. He disputed the requirement for K 35 . .Illrlr 11“le it!!!“ I I and Fe ions but added CaCOs to enhance pigment formation due to keeping the pH near neutrality. He also thought that in- creasing the MgSO4.7H20 content from 0.025% to 0.2% inhibited pyocyanine. He felt that Burton's (1948) medium was the best presented so far; 0.04% KeHPO., 2% MgSO,.7H20, 0.001% Fe30,. 7H20, 1% glycerol, 0.6% glycine and 0.6% L-leucine. This medi- um. gave Optimum production of pyocyanine. Grossowicz gg'gi. (1957), using stationary cultures, found Mg ion was absolutely essential and could not be replaced by Co or Fe ions. Frouin and Lebedet (1912) found that sodium vanadate completely suppressed pigment formation and that a series of rare earth metal salts could replace magnesium to some degree. Cataliotti (1955) found that zinc salts suppressed pigment formation and Kharasch gt 31, (1956) demonstrated that copper at l-50,000 inhibits pigment formation. King g£_gl, (1954), however, mentioned that 100 ppm CuCl increased the production of pyorubrin. It had been noted for many years that pigment formation depended on the carbon and nitrogen content of the medium as well as the salts Just mentioned. More references on this point may be found in Sullivan's (1905) publication and Turfitt's (1957a) thesis. Hueppe (1880) used ammonium tartnates in his synthetic medium.and arnaud and Charrin (1891) and Sullivan (1905) found asparagine well suited. Lepierre (1895) thought that dibasic acids with two methylene groups were required. This point was refuted by Jordan (l899a). 34 9'.- F.- ‘III 3 till.‘ I 9].! . Aubel and Colin (1915) found that ammonium salts of fat- ty acids and mineral acids gave good production of pyocyanine, except ammonium nitrate which gave only fluorescent pigment. Asparagine was the most favorable and glycine did not work at all. Goris and Liot (1922) found that on mineral agar many amino acids could induce pigment formation, and (1923) they found the ammonium salts of the amino acids gave best pigmenta- tion. Glycine did not give any pyocyanine. Glycerol, glucose, fructose, and mannitol gave good pigmentation. Liot (1925) concluded that an ammonium salt is necessary for pigmentation and its nature is unimportant. Carra (1924) found that alanine produced the greatestpigmentation, and Cicconi (1942) found that alanine overcame inhibition of pigmentation caused by rice bran. Burton gg‘gfi. (1947) found that a medium composed of gly- cine (or DL-alanine) and L-leucine and glycerol gave pigment production comparable to that on glycerol peptone agar. Glycine, alanine, valine, and tyrosine were the only amino acids individually to give pyocyanine but leucine enhanced their effects. Fee and White (1955) found higher alcohols, disaccharides, and polysaccharides favor fluorescence while pentoses and hexoses prevent it formation. King g3 gfi. (1954) developed a medium for the demonstra- tion of pyocyanine-pyorubrin and one for fluorescein. The former had Mgso, added and the latter had KgHPO, added. Glycerol was the carbohydrate. Peptone we used for pyocyanine and proteose-peptone for fluorescein. 35 Blackwood and Neish (1957) found,through tracer studies, that glycerol and dihydrcxyacetone were the best carbohydrate sources for pyocyanine production and that alanine, leucine, isoleucine and glycine were incorporated into pyocyanine most readily. Grossowicz g} 2;. (1957) found that glutamic acid, gamma amino butyric acid, L-hydroxyproline, and alanine were most active in producing pyocyanine by active resting cells. 0f the carbon sources, those of the tricarboxylic acid cycle were the most active. 36 I IDENTIFICATION OF THE ORGANISM UNDER STUDY The organism of this study was one originally isolated from a sample of soil obtained during studies on the dispos- al of nitro toluene wastes. At the time it produced a wine red, water soluble pigment only on certain media. The ques- tion of why it produced its pigment only on certain media led to its identification and directly to the matter of the pigment itself. This section will be concerned with identifi- cation. The standard biochemical and morphological tests indicat- ed the genus Pseudomonas. However, the lack of characteristic pigments presented a problem in determining the species. or all the species included in this genus, it most closely re- sembled fig, aeruginosa, however, the pigment was not typical and no pyocyanine could be isolated. Several authors have registered doubt as to the significance of the pigments in identification due to the so frequent occurrence of non-pig- mented forms (Gaby 1946) and cultures which lost the power they once had to form pigments. With this in mind, four basic methods of identification were chosen (Gessard 1920b, Haynes 1951, Gaby and Free 1955, and Wetmore and Gochenour 1956) besides the standard proce— mmes (Bergey's Manual 1957, Manual of Methods for Pure Cul- ture Study of Bacteria 1955). Several tests mentioned in the literature supplement the above criteria. 37 Experimental Observations of characteristics reported in Bergey (1957) were made using media and reagents recommended by the Manual of Methods for Pure Culture Study of Bacteria, unless other- wise mentioned. gfiggigjggg_The Burke Modification was used with 18 hour old cultures. Flagella Stain This was according to the technique of Leifson (1951) with the time of staining at 15, 15, and 17 minutes. Motility This was determined by hanging dr0p prior to flagella staining. Gelatin Liqgefaction Plain 12% gelatin and nutrient gelatin were used. Tubes were incubated at 57 and .20 C for up to two months. Plates ggg_51ants These were prepared from.Nutrient Agar (Dif- co), Tryptone glucose extract (Difco), Dextrose Agar (Dif- co), and pigment medium modified from King 33 gl, (1954). This was composed of 2% BactOpeptone, 1.5% Bactoagar, 1% glycerol, 1% KeSO,, and 0.14% M8012 at pH 7.2. figggg This was Nutrient Broth (Difco) or 1-2% Tryptone (Difco). Litmus gig; Prepared from.Difco Litmus Milk. Potato Slants These were prepared by slicing a white potato and cutting a cylinder out with a cork borer of the ap- prOpriate diameter. The bores were then cut in half on a slant and soaked overnight in water after which they were washed and used, or soaked for one hour in a 6% glycerol- saline solution (Simon 1956). Small wads of cotton were 38 placed in the bottoms of culture tubes and saturated with water or 6% glycerol-saline. A small slot was out down the side of the slanted potato cylinder, to allow expan- sion of the liquid in the cotton, and it was placed in the tube. They were autoclaved at 15 lb for 12 minutes. A heavy inoculum was used. Ammonia Production of ammonia was determined in Dunhams so; lution using Nesslers reagent. nggfi Dunhams solution with Pringsheim reagent and 2% tryp- tone broth with an oxalic acid soaked paper above the culture. A pink coloration indicated indol. Tubes were observed up to 20 days. Nitrate Reduction.Peptone broth plus 0.1% KN03 and a gas in- sert for N2 detection was used. Tubes were read at 7, 24, and 48 hours. Hemolysis Plates of 5% sheep red blood cells in blood agar base were observed at 24 and 48 hours. Carbohydrates A 1% carbohydrate-purple broth or -phenol red broth base was used. The carbohydrateeused.were glucose, lactose, sucrose, xylose, sorbitol, mannitol, and malt- ose. Observations were made up to 20 days. Citrate gg_gglg Carbon source Simone Citrate (Difco) was used. Methyl figg and Voggs-Proskauer‘figggg Difco MR-VP medium.with the reagents recommended in the Difco Manual (1955) §3g£gg,Hydrolzsis Dextrose Starch Agar (Difco) and Lugols Iodine, tested periodically over two weeks. Egg The method of HCN detection suggested by Lorck (1948) 39 was used. Nutrient Agar plates were heavily seeded with the culture. Paper strips saturated with picric acid, with several drops of N82003 on it, were placed across the top of the inside of the dish. Plates were incubated at room (24 C) temperature for two days. Nutrient broth cultures had a strip placed Just below the cotton plug and above the medium. A red color indicated HCN production. Gessard (1920b) develOped a scheme or code for the iden- ‘ufication of fig. aeruginosa on the basis of the type of pig- ment produced on certain media. To verify the species he used 2.5% glycerol- 2% peptone-agar. To determine the race he l“w used 0.5% bouillon and for the variety he used 2% peptone (tryptone). In bouillon he determined the presence of both pyocyanine and fluorescein (A), or pyocyanine alone (P), or fluorescein alone (F), or none (8). In peptone each of the above could produce pyocyanine (Pe), red pigment (E), black pigment (M), or none (0). An organism.oould then be classi— fied or coded, SE, APe, SO, etc. The peptone used was tryptone (Difco) as recommended by Simon (1956). The medium was incubated at 57 C for 4 days and then at room temperature for 6 days to allow full pigment development. I Haynes (1951) suggested the criteria of growth at 41 0, oxidation of potassium gluconate, and the formation of slime, oyster, reverse whirl and transitory film when incubated at room temperature for up to two weeks. Slants of dextrose agar were streaked and incubated in 40 a water bath at 41:1 C and observed at 18, 24, and 48 hours. An equimolar amount of sodium gluconate was substituted for the potassium salt and distributed 100 ml to a 500 ml flask, inoculated and shaken on a rotary shaker, as prescribed by Haynes, for up to two weeks at 57 C. The cepper reagent used to test for reducing sugar was that of Shaffer and Hermann (1921) as suggested by Haynes. Ten ml of the reagent was added to one ml of culture and brought to a boil for ten min- utes, and then cooled in running water. Red copper oxide in- dicated the presence of a keto or similar group,presulab1y 2-keto-g1uconate. After samples were positive, or after 14 days, the flasks were set at room temperature. Typical fig. aeruginosa would form a slime thick enough to draw upward on a needle about 6-12 inches before snapping. The cultures also showed the phenomenon of “reverse swirl". Rotate the flask in a circle on a flat surface and then stop. The slime moves in a circular direction, slowing down, and reversing direction for a few centimeters. These cultures also show a heavy film adhering to the side of the flask when shaken and an "oyster" -a heavy mass of slime in the center of the liquid that moves as a unit. Gaby and Free (1955) described six tests which should determine the identity of non-pigmented fig, aeruginosa: 1. One of the three colonial variants, 2. Grape-like trimethyl amine odor, 5. Gram negative, motile with a polar flagellum and forming a pellicle in broth, 4. Acid from glucose (in peptone broth) but none from lactose or sucrose, 5. Gelatin 41 liquefaction and, 6. Negative urease test. 111:: these were determined on media already described, except urease which was determined in Difco Urea Broth. Wetmore and Gochenour (1956) used a series of reactions, some of which overlap with the proceeding sections. Only those techniques which differ from those already described will be mentioned. Carbohydrates were studied (in contrast to the studies reported on page 59) in a synthetic medium in which the nitro- gen source was the ammonium ion. The medium wss composed of 0.2% (NH,)2HPO,, 0.02% MgSO,-7H20, 0.02% NaCl, 0.02% K2HP04, and 0.002% phenol red as the indicator. At the suggestion of Simon (1956) the indicator was added in a water solution since alcohol could be fermented. The medium was dispensed 2.5 ml per 4 mm diameter tube, plugged and autoclaved. All carbohydrates showing acid were repeated using filter steri- lized carbohydrates added separately to the sterile broth base. Carbohydrates used were L-arabinose, D-arabinose, xylose, glucose, galactose, fructose, lactose, sucrose, maltose, tre- halose, raffinose, inulin, glycerol, mannitol, dulcitol, sor- Intol, adonitol, and salicin. Tubes were inoculated with cul- tures grown on nutrient agar slants, removed with a needle to keep added nitrogen and carbon sources at a minimum. They were incubated at 57 C and observed for 40 days. Sterile dis- tilled water was added to the tubes periodically to keep the water level up. Growth at various temperatures was determined by inoculating dextrose agar slants and incubating at 42, 57, 42 20, and 5 C. In order to test tolerance to sodium chloride, this salt was added to veal infusion agar in concentrations from 0-6%. The various cultures were steaked on quadruplicate plates at each concentration. They were incubated at 57 C for 48 hours. Acid tolerance was determined by streaking on Sebourauds dextrose agar at pH 5.6 and incubating for 48 hours. Some of wetmore and Gochenour's tests were omiumd as not suf- ficiently definitive. miscellaneous tests included the oxidase reaction used by this author and also reported by Kovacs (1956) and Gaby and Hadley (1957). The test consisted of several drOps of p-amino dimethyl aniline hydrochloride placed on nutrient agar colonies. The observation of a color change from pink to maroon to violet black indicated the presence of oxidese. The test for antibiosis was'roughly" qualitative since no prescribed procedure exists. A single colony was started in the center of an agar plate and allowed to develop for 72 hours. Cultures of Escherichia coli, Serratia marcescens, Staphylococcus aureus , Salmonella typhosa, and a beta strep- tococcus were streaked radially outward from near the colony. The plate was incubated for another 24 hours and inhibition of growth noted. Results The organism which is the object of this study, was ob- tained by streaking a soil sample on Tryptone Glucose Extract Agar (TGE, Difco). Two separate colonies which showed the red pigment were picked and restreaked on TGE. From each of these, 43 two colonies, differing slightly in colonial form, were pick- ed. Four cultures were used henceforth designated 16-1, 16-2, 17-1, and 17-2. These four isolates were carried as separate organisms for comparative purposes. To compare with these, nine known cultures of £3, aeruginosa were used. Five of these (l,2,4,5,6) were typical pyocyanine forming strains and one (number 3) formed no pigment in most of the studies. These six strains were isolated by the author from a series or cases of external otitis. One culture (7) was a laboratory contaminant. The remaining two cultures (8 and 9) were strains of 23, aeruginosa var. erythrggenes obtained as # 5083 and 6749 respectively,from the National Collection of Type Cultures, London, England. The last culture to be used (10) was a single culture of fig. fluorescens A3.12 obtained from Dr. H.L. Sadoff of this department. All tests with this culture were incubated at 30 C. The results of the physiological tests are summarized in Table I and while these are the indicated results, a few supplementary remarks must be made about certain of them as well as a few things not included in the table. The flagella stains indicated two types of flagellation. Cultures 3,4 and 10 fall into a group which showed polar flagella, predominately monotrichous, but some multitrichous, with morethan two waves. Culture 3 is the non-pigmented culture, 4 is a typical pyocyanine producer, and 10 is 23, fluorescens. The rest or the cultures fell into a group which showed exclusively polar monotrichous flagella with 44 "‘l D’._ ' In“. r»... TABLE I PHYSIOLOGICAL REACTIONS OF THE PSEUDOMONAS CULTURES Characteristic Strain Number 16 16 17 17 1 2 3 4 5 6 7 8 9 10 -l -2 -l —2 Gram Reaction .......... - - _ - Gelatin Liquefaction + + + + + + + + + - + + + + Litmus Milk coagulation + + + - - + - - - - - - 4 - peptonization + + + + + + + + + - + + + + reduction + + + + + + + + + - + + + 4 basic reaction + + + + + + + + + + + + + + Indol .......... - - - - Nitrate Reduction + + t + + + + + + - + + + + N2 produced + + + + + + - - + + + + Hemolysis, beta + + + + + + + + + - + + + + Hes ---------- - - - - Citrate Growth + + + + + + + + + + + + + + Methyl Red ---------- - — - - Voges-Proskauer .......... - - - - Starch HydrOlYS1s ---------- C C- - - HCN + + + + - + + - - + - + Temperature Tolerance 41 C + + + + + + + + + - + + + + 37 C + + + + + + + + + i + + + + 20 C + + + + + + 4 4 + + + + + + _ 5 C ---------- O '- - - Gluconate Oxidation + + + + + + + + + + + + + + Slime Production + + + + + + + + + - + + + + reverse swirl + + + + + + + + + - + + + + film formation + + + + + + + + + + + + + + oyster formation + + + + + + + + + — + + + + Odor-trimethyl amine + + + + + + + + + - + + + + Urea Splitting - - - - - - - - - (con't next page) 45 TABLE I (CON'T) PHYSIOLOGICAL REACTIONS OF THE PSEUDOMONAS CULTURES Characteristic Str ber l6 I6 17 17 1 2 3 4 5 6 7 8 9 10 -l -2 -1 —2 Ammonia + + + + + + + + + + + + + + Acid Tolerance pH 5.4 + + + + + + + + + - + + + + Oxidase Test + + + + + + + + + + + + + + Antibiotic + + + + + + - + + 4 4 + + + Carbohydrates-Stand- ard media glucose + + + + + i t + + + + lactose ------- - - - - sucrose ------- - - - .- xylose ....... - - - - sorbitol ------- - - - - mannitol ------- - - - - maltose ------- - - - - Carbohydrates-Syn- thetic media L-arabinose + + + + + + + + + + + + + + D-arabinose - + - - + + - - - - . - xylose + + + + + + + + + + + + + 4 glucose + + + + + + + + 4 + + + + + galactose + + + + + + + + + + + + + + fructose + + + + + + + + + + + + + + lactose .......... - - - - sucrose ---------- - - .. .. maltose - - - ....... - - - - trehalose + + + + + + + + + + + + + + raffinose ---------- - - - - inulin .......... - - - - glycerol + + + + + + + + + + + + + + mannitol + + + + + 4 + + + - + + + + dulcitol ---------- - - - - sorbitol ......... - - - - - adonitol .......... - - - - salicin ---------- - - - - Motility + + + + + + + + + + + + + + NaCl.Tolerance 4% + + + + + + + + + + + + + + 5 + + + + + + + + + x + + + + 6 + 1 + + + + + - + - + + + + 7 ---------- 3 - - - ..a N more than two waves. Note that all the cultures are essen- tmlly identical in terms of flagella. In 24 hour old broth cultures, 1 through 7 formed a group with heavy turbidity, moderate slimy pellicle, scanty viscid sediment with a putrefactive odor. Cultures 8 and 9 differed only in that the sediment was not viscid and the pellicle was very heavy. Culture 10 had a ring but no pellicle, scanty turbidity, moderate granular sediment and a putrid odor. Cultures 16-1, 16-2 and 17-1 differed from 8 and 9 only in the remarkable lack of turbidity. Culture 17-2 was more like the first group. The colonial characteristics on dextrose agar fell into two of the three colonial patterns described by Gaby and Free (1953). Cultures 1,2,3,4,6 and 7 are large and Spreading with smooth, convex translucent centers; effuse, flat, wavy transparent periphery and irregular lobulated edges, while 5,8,9,l6-l,16-2,l7-1 and 17—2 were round or slightly irreg- ular, slightly raised, umbilicule (or umbonate) finely (or coarsely) granular simulating a rough colony. 0n slants of pigment medium only 16-1, 16-2,l7-l, 17-2, 8, and 9 were compared. In 24 hours the first five looked identical, red at the slant tip with the rest yellow green. Culture 8 differed in one respect, it did not show the silvery aplotches associated with the unknowns in this medi tmn Culture 9 formed an almost brown-red pigment. 0n plain potato slants, all the cultures showed only pinkish growth while on glycerinated potato 1,2,4 and 6 47 showed emerald green to blue pigment while 3,8, l6-l,16-2, 17-1, and 17-2 showed pinkish growth exactly as on plain potato. Culture 9 was deep brown and culture 10 did not grow. Gelatin liquefaction in cultures 8,16-l,16-2,17-l and 17-2 was peculiar in that a large crater was created in the medium.but no liquid. The hole was just as large as it would have been if there was liquefication, A very small amount of liquid finally formed at the bottom of the crater after 3 weeks for culture 8 and 2 months for culture 16-2. Culture 4 took 20 days to show liquid and culture 10 showed none at two months. On temperature tolerance, all cultures grew in 18 hours at 41 0 except 10 which did not grow in 48 hours. At 37 c culture 10 showed very slight growth in 48 hours. At 20 C culture 10 grew rapidly and the rest slowly. At 5 C none grew in 5 days. The results given for salt tolerance are the compilations of quadruplicate plates. A + means growth on 3 or 4 plates, * growth on two plates, 3 growth on one plate, and - no growth. When grown on D-arabinose as a sole carbon source, the only cultures showing acid were 2,6,8, and 17-1. The last one turned acid at 37 days and the former ones at 60 days in filter sterilized media. In addition (in contrast to Simon 1956) fig, viscosum-acid in 37 days, §§._geniou1atum-l7 days, 25. fluorescens 11251 -37 days, 33. £3251 4973 -17 days and fig. fluorescens -37 days, all fermented D-arabinose as a sole carbon source. This test, as one of identification,was not further considered. 48 Gessard determined a code from the pigments produced in various media as follows: Bouillon (A) both pyocyanine and fluorescein, (P) pyocyanine alone, (F) fluorescein alone, and (3) none; Peptone (tryptone). (Pe) pyocyanine, (E) red pig- ment, (M) black pigment, and (0) none. To be a £3, aeruginosa to qualify for this coding at all, pyocyanine had to be pro- duced on gycerol-peptone-agar. The results on his media are: Strain Glycerol- Bouillon-code Tryptone-code Code Number tryptone agar 1 pyocyanine no color 3 pyocyanine Pe SPe 2 pyocyanine pyocyanine P pyocyanine Pe PPe 3 yellowish no color 8 no color 0 OS 4 pyocyanine no color 3 no color 0 OS 6 pyocyanine no color 3 pyocyanine Pe are 8 fluorescein no color 3 pyocyanine Pe SPe 9 brown-red pyocyanine P pyocyanine Pe PPe brown-red brown-red lo fluorescein fluorescein F fluorescein F0 l6-l fluorescein no color 3 yellow-red % 8! 16-2 do. do. do. SE 17-1 do. do. do. SE 17-2 do. do. do. 8E The tryptone tubes of the last four cultures showed a red band on t0p of the yellow which extended throughout the tube upon shaking. Note that only 1,2,4 and 6 can legitimat- ely be considered to be £3. aeruginosa by this scheme. All cultures except number seven showed some antibiotic activity against the gram.positive organisms (beta strepto- coccus in particular) and against one of the gram negative organisms (S, typhosa) but none against g. coli or g, marces- cens . Conclusions From Gessard's media these unknown organisms cannot be classified as 23. aeruginosa since they did not produce 49 pyocyanine. The classical classification as represented in Bergey's Manual strongly suggested.§§. aeruginosa but it is not definite either since there is no pyocyanine. According to the criteria of Haynes (1951) and Gaby and Free (1953), however, the organisms appear to be definitely £3, aeruginosa and by comparison with the reports of red pigmented fig. aeruginosa in the literature, it does indeed appear to be this organism. To recapitulate, the chief bases for calling this organ- ism.§§, aeruginosa; 1. From Haynes (1951)-growth at 41 C, ox- idation of gluconate and the subsequent formation of slime, reverse swirl, film and oyster on this same substrate; 2. From Gaby and Free (1951)-colonial morphology, trimethyl amine odor, polar flagella, negative urease, pellicle in broth, and acid from glucose; 3. General agreement with Bergey's Manual description; 4. Antibiotic activity; 5. Excellent comparison with the fermentation and other reactions with strains of hetmore and Gochenour (1956); A strongly positive oxidase test; and finally 7. The excellent correlation with known 33, aeruginosa including Gessard's variety erythrogenes. The author is convinced that the organism is 23. aerugi- £222. (var. erythrogenes) forming the red pigment reported by Meader 9.13. g}, (1925). SO II PIGMENTATION AND CARBON-NITROGEN SOURCE It was noticed early that pigmentation depended on the ‘ medium used to culture the organism. For instance, on Difco TGE agar lot J393948, the organisms showed the characteristic red pigment, while on lot 3412238 the pigment was poorly de- Wflnped and often not produced at all. A study was therefore undertaken to determine the most suitable nitrogen and carbon sources for pigmentation. For convenience of presentation the succeeding section is divided into two parts: A, studies on complex media, and 8, studies on chemically defined media deve10ped as a result of the studies on complex media. Experimental A. Unless otherwise indicated, the following general procedures were followed for these tests. The pH of all media was adjusted to pH 7.0 prior to autoclaving. sterilization vwas by autoclaving at 15 1b pressure for 12 minutes, and the tubes were slanted prior to use. The particular culture used in these studies was that designated 17-1. It was prepared by streaking a Nutrient agar (Difco) slant and incubating for 24 hours at 37 C. Inoculations were made with a 100p of cul- ture from the agar slant by heavily streaking the entire sur- face of the test medium. All tests were done in duplicate. All media were incubated at 37 C and observed for periods up to one week,or longer where indicated. 51 “‘ 1. A series of experiments was inaugurated to determine whether either the tryptone or the glucose of TGE agar could be replaced by other peptones or carbohydrates. The base medi- um in the one case was 0.3% beef extract, 0.1% glucose, 1.5% agar, and in the other case 0.3% beef extract, 0.5% tryptone, and 1.5% agar. Peptones studied included; Albimi peptone M, proteose peptone g 3 (ppj3), tryptose (Difco) and trypticase soy (BBL). Carbohydrates studied were galactose, fructose, lactose, sucrose, maltose, inulin, mannitol, and sorbitol. Controls were complete,dehydrated TGE lot 5393948 and lab- oratory prepared TGE agar (prepared from the individual ingredients). 2. Laboratory prepared TGE agar was utilized to deter- mine what effect, if any, 0.01% asparagine, 0.05%g1ycerol, and 0.01% FeSO.-7H20 and 0.01% mgso.-7H.o had when added in various combinations. 3. A 0.5% Albimi peptone K, 0.1% glucose and 1.5% agar medium was compared with the same medium with 0.1% glycerol added in place of the glucose, and also with peptone N and agar alone. / 4. knowing that nutrient agar did not support the pro- duction of pigment, it was mixed with TGE agar in ratios of 1-1, 3-1, and 7-1. The total amount of added material was kept at the 2.4% specified in the TGE agar formulation. To a duplicate set of tubes was added 0.001% glucose, the approx- imate amount lost during the dilution of the TGE agar. 5. Various combinations of glucose and tryptone plus 52 1.5% agar were studied- glucose to tryptone C.5-0.1%, 0.25- 0.1%, 0.25-c.2%, c.1-c.2%, and 0.05-0.27. 6. The effect of autoclaving was studied using Difco TGE agar slants. The slants were autoclaved for 0,5,10,13,15,17, 20,25 or 30 minutes, streaked and observed for relative amounts of pigment formed at periods of 1,2,3,4 and 9 days. Similar tubes were steamed at 100 C for 0,5,10,20,25,30, and 45 min- utes and 1,2,3,and 4 hours. Sterile medium was prepared as follows: TGE agar medium (dehydrated) was made up to volume in water and filtered through thatman 31 filter paper, which removed the suspended agar. The medium was then sterilized by filtering through a sterile Seitz filter. The medium was then.added to agar already sterilized by autoclaving. The resultant suSpension was heated to dissolve the agar and then.autoc1aved, heated with steam, or used as controls. 7. To test the method of carbohydrate sterilization, several peptones were used as nitrogen sources at 0.5% con- centrations. To this was added 1.5% agar and one half the Preper amount of distilled water. Glycerol or glucose was added to water in an amount so that when a measured portion or the solution was added to the base medium, the volume was correct and the carbohydrate was 0.1%. Three types of steri- IUzation were studied (autoclaving was done at 15 1b for 12 minutes); a) base autoclaved-carbon source filter sterilized through a Seitz filter,4 b) base autoclaved-carbon source autoclaved separately, c) base and carbon source autoclaved together. 53 These experiments also yielded information on the suit- ability of these peptones for pigmentation. 8. The effect of the salts, reported for pyocyanine by Burton.gj,gl. (1948), is described in detail in the next section; however, it was found that; 1% Mg30.°7320, 0.01% KeHPO., 0.0001% FeSO‘-7H20, 0.5% pp§3 and 0.1% glycerol gave optimum pigment production and was used henceforth. I 9. Optimum glycerol concentration was determined by vary- ing the content of this compound in the last mentioned medium between the values 0.01 and 10%. 10. Casamino Acids (Difco) was substituted (1%) for the ppg3 and glycerol was added in 0.1% and 1% concentrations to separate sets of tubes. 11. The amino acids contained in Casamino Acids were divided into four groups and madeup according to the percent- ages reported for casein by Hawk 23 31. (1954). Group I Group III tyrosine 6.3% arginine 4.1% phenylalanine 5.0 histidine 3.1 threonine 4.9 lysine 8.2 serine 4.3 glutamic acid 22.4 leucine 9.2 aspartic acid 7.1 isoleucine 6.1 valine 7.2 glycine 2.7 alanine 3.0 Group IV Group II cystine 0.3 methionine 3.4 proline 11.3 tryptOphane 1.2 Each group was added to 25 m1 of distilled water so that an equal volume of each would yield an amount approximately equivalent to 1% Casamino Acids. Glycerol was 0.3% and agar 1.5%. 54 A preliminary test of the complete medium was made to deter- mine whether minerals were needed. The various groups were now added to 0.3% glycerol and 1.5% agar and the preper mineral concentrations in such a manner that each group could be observed alone and in all combinations with the others. 12. Each of the amino acids in a desired group was pre- pared in 1% concentration with 0.3% glycerol, 1.5% agar and the mineral ingredients. B. Since agar prohibited quantitative studies of the pigment and added unknown salts and organic material (Difco Bulletin i81, 1925), a new method of obtaining pigment in an exact synthetic medium was deve10ped. The test medium (without agar) containing the amino acid or carbohydrate under study was prepared in 40 m1 amounts and added to oversized 22x200 mm test tubes at 20 ml per tube. The tube was then sealed at the tOp by placing a square piece of aluminum foil over the top and pressing it firmly down and around the sides. The edge was lifted slightly to allow the liquid to eXpand when autoclaved without blowing the foil off. Immediately after autoclaving, the foil was again pressed against the sides effectively sealing the contents from the air and keeping the outside of the tube sterile for several centimeters down from the tep. Sterile Petri dishes were placed on the table and the contents of one tube placed in a single Petri dish and the contents identified on the outside cover. 55 Lillipore membrane filter disks (47 mm diameter, white or black) were previously sterilized by autoclaving at 15 1b for 15 minutes in stacks of ten (pad, separator, filter disk, pad, etc.) in a petri dish. D1 sks were removed with sterile flamed forceps and drOp- ped on the surface of the liquid medium, one disk to each plate- The disk thus floated, on a medium of the type used in these studies, would remain afloat for periods up to ten days or longer. That liquid seeped up through the disk could easily be seen especially with the black filter disks. Careful observation of the white filter disks verified this. The organism was prepared by washing off a 24 hour old nutrient agar slant and removing the liquid. This suspension was centrifuged, washed with sterile saline, centrifuged again and suspended in a quantity of saline. These steps were all done aseptically in a screw cap test tube which also served as the storage vial of the suspension. When required, a snap ension of this was made to match a llcFarland Turbidity #3 Standard by diluting with saline. A11 plates thus received a unif‘tarm inoculum. The culture was placed onto a filter disk floating on the medium by running 0.05 ml from a one m1 pipette onto the disk and then touching whatever remained on the tip of the pipette ‘0 the disk. The drOp on the disk could then be streaked over the Surface of the disk by using an Open 100p bent at a 45° “313 at the tip. The wire was touched gently to the drOp and moved toward the side. The disk slid across the liquid- 56 and came to rest against the side of the Petri dish. Gentle sliding of the needle while the disk pushed on the side of the :11 sh allowed the culture to be spread along a line. Be- peated streaks at slightly different angles covered the sur- face rather uniformly. For cptimum effects several mm around the edge of the filter disk should be left free of growth to prevent the bacteria from getting into the medium too soon. A growing culture on one of these disks will eventually sink it (4—5 days), but a strictly aerobic growth will have been inaugurated. ' After the plates had been incubated at 37 C for an appro- priate time (5 days in all but one experiment), they were ‘ removed and the duplicate plates combined and washed into a 50 m1 volumetric flask and made up to volume. After this was shaken well, 10 ml was removed with a 50 m1 syringe and plac- ed in a centrifuge tube. A11 tubes to be used were then cen- trifuged at 2500 rpm in an International Centrifuge with a 1.5-3 cm radius head for one hour. A 50 m1 syringe was used to remove all but a small portion containing the packed cells. 1“ Fae diluted if need be, or placed directly into a cuvette f0? use in a Bausch and Lomb Spectronic 20 colorimeter. Pre- 11“ii-nary studies indicated a strong absorption peak in cul- ture media at 525 mu and 365 mu (Figure l). The former was Chosen as more indicative of the visible color. Dilutions were made and the readings were directly preportional to the °°n°entration (relative) (Figure 2). Relative concentrations in the 50 m1 sample were calculat- 5‘7 ed according to the following formula: Optical density the dilution relative reading it 100 x denominator - concentration With the new technique the following experiments were formulated: l. The studies of the individual amino acids were repeat- ed entirely, using 1% aminoacid (except 0.6% tyrosine) in a base or: LigSO"7HgO 0.5% glycerol 0.3 KgHPO. 0.03 pH 7.0 The salt mixture was altered slightly for this experiment. The relative pigment concentration was determined at 525 mu on the Optical density scale (absorption). An entire absorption curve was determined for the tubes of alanine, tyrosine,1ysine, serine , histidine, phenylalanine, glutamine, arginine, valine, leucine and isoleucine because of the visual observation that there were differences 01110118~ the colors of the pigments (Figures :5 and 4). 2 . A comparison was made between the pigment producing P°"°I‘8 of alanine, leucine, isoleucine, and valine alone (at 0°89; Concentration) and in combination (one set at 0.2% each and a second set at 0.4% each). These were compared to Casamino A“1‘18 (0.8% and 1.6% concentrations); amino acid Groups I and III (0.8% and 1.6% concentrations); agarless (filtered) TGE 1°t 6393948 without added salts; agarless (filtered) TGE lot 5393948 without added salts but with 0.3% glycerol added; and finally, proteose peptone ”Z3 at 0.8% and 1.6% concentrations 58 without added salts but with 0.3% glycerol. The basal medium was 1% MgSO.-7H20, 0.01% KeHPO. and 0.3% glycerol at pH 7.0. :5. Quantitative effect of variation in the alanine con- centration on pigment production was determined using the last named basal medium with alanine concentrations of 0,0.1, 0.5, 1.0, 1-5, and 2.0%. 4 . The effect on pigment formation of 0.3% concentrations of Various carbohydrates. The carbohydrates used are listed as follows: Tricarboxylic acid cycle intermediates; sodium citrate, sodium succinate, sodium malate, and sodium fumarate, Sugars; xylose, arabinose, ribose, galactose, glucose, fructose, trehalose, Alcohols; ethyl, butyl, mannitol, glycerol (control) Acids; sodium lactate, sodium acetate, sodium butyrate, and sodium gluconate. The base medium was as follows: alanine 1.0 yi830‘ .7320 1.0 0.0 KgHPO‘ 1 pH 7.0 5° Finally the effect of varying the glycerol content in the base medium. The experiment was conducted in two sections. The first consisted of concentrations of o, 0.01, 0.03, 0.06, 0.09. 0-1. and 0.273 glycerol incubated for five days. The second was °°mP°8ed of concentrations of 0, 0.1, 0.2, 0.5. 005a 1.0, 2-0» 5-0: and 10 % glycerol incubated for ten days. 59 Results The organism on TGE agar slants behaved as follows: growth occurred during which a yellow,or in most cases greenish, pig- ment was produced (it has never been observed after the red appears). This disappeared and was replaced by the wine red pigment. since only one lot of TGE agar produced good pigmentation and. another lot of the same medium produced it poorly or not at all, an attempt was made to reconstruct TGE agar from lab- oratory ingredients and at the same time substitute various peptones, and carbohydrates into the medium. Unfortunately, the only one of these which produced pigment was the lot which was already known to produce it. Addition to the laboratory prepared IGE agar of compounds known to stimulate pigmentation in other organisms, uniformly failed to produce pigment, with the exception that 0.01% asPaI'E=lgine and 0.05% glycerol when added together produced a very slight pink after 16 days. Albimi peptone I25. plus agar and glucose produced a very slight red pigment in 2 days which remained constant for 9 days. Peptone if. plus agar and glycerol showed extremely strong “‘1 Pigment formation beginning the first day. Peptone 1v: plus 5851‘ Showed no pigmentation. This suggested that media other than 'IGE agar lot $393948 contained something inhibiting pig- ment formation. TGE agar was diluted with Nutrient agar at l-l, 1-3, and 1'7: keeping the total weight of the added material at 24 8m 60 per liter. To insure adequate source of carbon for pigment formation, 0.001 gm of dextrose per liter was added to a dupli- cate set. No pigment was formed in any of the tubes indicat- ing that what ever it was in the nutrient agar, a 1 to 1 ratio was sufficient to supress pigment. It couldn't be the lack of carbon since sufficient was added. It could be the peptone except that peptone M gave good results. One ingredient present here, not present in the peptone N studies, was meat extract, a highly variable substance and reported to inhibit pigment formation in several other species. With the meat extract inhibition idea in mind, a series of experiments was set up to test the effect of its absence in laboratory prepared TGE (E left out) agar. success was immediate. Pigmentation was poor but it was produced. The combination of the highest concentration of tryptone (0.5%) and the lowest of glucose (0.1%) produced a great deal of red pig- ment. In the others, even though the glucose concentration was raised, the tryptone concentration was lower and pigmentation less. It had been noted earlier with TGE agar lot §393948 that autoclaving for too long materially reduced the amount of pig- ment formed during the first few days of incubation. The low amount of pigment produced above might have been the result of over autoclaving. This experiment was in two parts. The first determined the effect of autoclaving and steaming on TGE agar and the second dealt with comparisons of filtering and autoclaving the carbohydrate. The results of this exper- iment may be found in Tables II and III. 61 TABLE II EFFECTS ON PIGMENT FORMATION OF AUTOCLAVING TGE AGAR AT 15 LB Time in Days of observation ___ minutes 2 4 9 O 44 444 4444 4444 4444 5 44 444 444 444 4444 10 44 >44 >44 >44 444 13 4 44 44 44 +44 15 4 4 4 4 444 17 4 4 4 4 444 20 4 4 4 4 444 25 4 4 4 4 444 30 4 4 4 4 444 +444 wine red 4 slight pink TABLE III EFFECT ON PIGKENT FORMATION CF oTEAMING TGE AGAR Time ‘ Days of Observation p__ l 3 4 5 0 minutes 4 4 44 4444 4444 5 ” + 4 +4 (4444 (+444 10 " 4 4 44 444 444 20 ” 4 4 4 f T 25 " + + 4 oLIGHT 3O " 4 4 4 GRADATION 45 n + + 4 l i 1 hour + 4 4 (444 (444 2 " + + 4 >-44 >'44 3 " + + 4 44 44 4 ' 4 4 4 4 4 4444 wine red 4 slight pink It? is apparent that autoclaving or steaming excessively affects: pigment formation mainly in the early days of incub- ation. iIf incubated long enough, they are fairly equal. Since the media all formed about the same amount of pigment over a period. of time, the simplest method and shortest time of sterilizing which would insure sterility were chosen. Twelve 62 minutes of autoclaving was the combination chosen for use. When results on the methods of sterilizing the carbohydrates were compiled, it became apparent that results depended a great deal on the carbohydrate and/or the peptone. Table IV contains the results of these tests. TABLE IV EFFECT OF CARBOHYDRATE STERILIZATION ON PIGMENT FORMATION Nitrogen Treat- Days 0:? observation source ment 4 glucose egceroI l 2 Z 1L 2 5 1. peptone a t 4 4 4 44 444 b 1 4 4 4 44 444 c 1 4 4 - i 444 2. tryptone a - 4 4 1 1 )4 b -_ 4 4 - 4 4 O - 1 3 - 4 4 firyptose a - 4 4 - i 74 b - 1 4 - g 4 c - 4 4 - i 4 4. prO‘beOse a - 1 4 4 444 4444 Rep t one b - 4 >4 1 44 >4444 r23 0 - L 4 - 44 4444 . PhYtone a - t g - 4 4 b - 1 g - 4 4 ¥ 0 - t a - 4 4 a) - filter sterilized carbohydrate b) - autoclaved separately c) - autoclaved with base 4444 wine red 4 slightly pink 111 all cases the amount of pigment produced with glycerol far GIceeded that from glucose regardless of the peptone. PrOtBOse peptone #3 produced by far the most pigment, and while autoclaVing seemed to retard pigmentation somewhat, it was not enough to warrant filter sterilizing. It might be noted from “bl-8 IV that autoclaving affected the pigment forming power to some extent in all cases but it was most apparent before 63 the second day. From these experiments, then, the medium chosen for further studies was 0.5% ppifs, 0.2% glycerol and 1.5% agar sterilized by autoclaving at 15 lb for 12 minutes. ‘Ttle addition of 1% MgSO..7H20, 0.01% K3HPO‘ and 0.0001% FeSO‘ .71130 to the medium Just mentioned enhanced pigment for- mation and increased the reproducibility from tube to tube ( 538 next section for details of salt concentration effects). The cptimum nitrogen source, carbon source and salt composition have now been determined. Before continuing with the nitrogen studies it would be well to consider the Optimum Glycerol content to be used with the above medium. An explor- story set of tubes containing 17‘; Casamino Acids in place of the protease peptone ,73 and 0.1 and 1.0% glycerol. The results of these experiments will be found in Tables v and VI. ‘The cptimum concentration of glycerol (Table V) was 10%. Th" I>igment was not formed at this concentration, however, ‘Hrtidl after three days, a limitation in time which was shared TABLE V EFFECT OF GLYCEROL CONCENTRATION ON PIGMENT FORMATION De 3 of observation % glycerol — I 2 :5 '7 .01 4 4 4 44 .05 4 4 4 44 ,1 4 4 (44 44 .5 <4 4 44 444 1.0 (4 4 4 >444 3.0 <4 4 4 (4444 5°C t 1 t 4444 1000 1 a 3 )4444 4444 wine red 4 slight pink 64 by 1,3 and 5% glycerol concentrations. Furthermore, the amount of glycerol used was prohibitive. By the third day 0.5% glycerol was the strongest red, a very little more than 0.1%, and a great deal more than 1%. It was felt that concentrations over 0 .573 caused inhibition of pigment formation in the early days of incubation, and it would be advantageous to sacrifice quantity for speed. Since it was not determine whether or not 0-555 was at the very edge of inhibition, a concentration between 0.1 and 0.570 was chosen - 0.5%. TABLE VI EFFECT ON PIGLENTATION OF 1% CASAIJINO ACIDS IN PLACE OF PROTEOSE PEPTONE 173 Days of observation 1 s % glycerol 2 d 7 0,1 4 44 44 44 1.0 1 4 44 44 4444 wine red 4 slight pink As indicated in Table VI, Casamino Acids 1% could be sub8t1tuted equally well for 0.5% proteose peptone 43. The seVen‘ day readings of both glycerol concentrations were equal to 0 .528 glycerol using 0.5% ppy3. since Casamino acids could be substituted for the ppfi3, it should be possible to take the individual amino acids which make up casein (of which Casamino Acids is a completely acid hydrolysed product) and determine which of these was respon- sibis for the pigment. It has already been described how the medium for this section was prepared, and the division of the amino acids into groups. The groups were Prepared 3””de 65 to the amino acid concentrations taken from Hawk §_t_ §_l_. (1954). The groups were combined in a preliminary trial to give a com- plete synthetic 1,6 Casamino Acids medium. One set of tubes had no minerals added while the other had minerals in the concen- trations already mentioned. The purpose here was to gain evi- dence <3f?the need for these particular minerals. Pigment pro- duction in the former set was weak while it was very heavy in the latter. The minerals were required, as seen here. {Pile groups were combined in a nanner to show all possible combinations, and the results can be seen in Table VII TABLE VII EFFECT ON PIGMENTATION 0F AMINO ACID GROUPS I, II, III, and IV, ALONE AND IN COMBINATION Grou Da 3 of observation Combinagions ‘ I *2 5 I - 44 444 II — - - III - 3 444 IV - _ - I,II - 44 444 I,III "' +4 4+4 I,IV - 44 444 II,III - 44 444 II,IV - - a III,IV - - 444 444+ wine red 4 slight pink Groups II (proline) and IV (tryptOphane, cystine, and methionine) apparently did not donate anything to pigmentation and were no longer studied. 01‘ the two remaining groups, I was the earliest to develOp pigment whereas III developed later b at arrived at the same apparent concentration of pigment. G POups I and III both produced a yellow fluorescence under an 66 ultraviolet lamp. It was very intense with I and only slight inIII- The next step was to break each group down into its indi- vidual amino acids. This was done by raising the concentration of each to 1% (except 0.6% tyrosine). The medium formulation used was as follows: amino acid 1.0% (0.6fityrosine) 1418304, .VHBO 1.0 glycerol 0.5 KeHPO. 0.01 FeSO..7HgO c.00c1 pH 7.0 Red pigment can be supported by all amino acids except glycine (growth did cccur) (Table VIII). L-Alanine, however, TABLE VIII EFFECT OF AMINO ACIDS ON PIGMENT FORMATION 1==================F=E .mino Acid Days of observatiog Group I l 2 5 9 glycine - - - - L-alanine 444 4444 4444 4444 valine - 4 44 4+ leucine - — - 44+; 1$Oleu31ne - t 4444 444+ threonine - 4 44 4; phenyl alanine — 3 RB RB serine - t 4444 444+ tyrosine - RB BB RB Group III arginine 4 4444 4444 4444 histidine 4 4 4444 RB lysine - - 444 444 glutamate 4 44 4444 4444 aspartate 4 44 44 ill 4444 wine red 4 slight pink RB red-brown 67 was by far the most potent pigment substrate, producing almost full pigmentation in one day. subsequent tests indicated that DL-a1anine could just as effectively replace the L-isomer. In order to study the pigment quantitatively, it was nec- essary to remove the agar and work with a solution. The pig- ment is not soluble in any organic solvents except methyl alcohol, phenol, acetic acid, etc., so that it was essential that the pigment be in free, albeit water, solution. work had not progressed very far with this organism when it was dis- covered that pigmentformation would not occur except in the presence of large amounts of oxygen. Broth cultures would not produce pigment even when aerated or shaken. The only effective method was to grow them on a solid surface. Agar was ideal but it interfered with isolation and quantitative measurements. As discussed under the experimental procedure, a method was devised in which a Millipore filter was floated on the surface of the medium under study and then the culture was streaked over the surface (or swabbed on the filter with a OOttOn swab saturated with the culture). After a period of time (5-10 days) the liquid was collected and made up to a certain volume, centrifuged and read in the spectroPhotometer at 525 mu. With alanine it was three days before the organism would produce appreciable pigment but once started there was 5 great deal produced. The studies of the individual amino acids were repeated US1DE the new technique and a slightly different basal medium. The I‘elative pigment concentration was determined according 68 to the formula given in the EXperimental. The results are presented in Table IX. Accompanying this is a list of the vis— ual appearance of each tube prior to reading in the spectro- photometer. TABLE IX QUANTITATIVE STUDY 03 RED PIGMENT FORMATION ON VARIOUS AMINO ACIDS Amino acid Relative Description Concentration DL-alanine 225 dark wine red DL-valine 150 do. but lighter L—leucine 144 do. but more violet DL-isoleucine 125 do. but lighter L-tyrosine 81 brownish L-glutamine 28 reddish brown L-histidine 25 brownish DL-phenylalanine 24 brownish-red L-arginine 19 pink DL-serine 14 very faint pink L-lysine 12 very faint pink DL-threonine 0 -- L-asparagine 0 -- L—glutamate 0 «- L-aspartate 0 -- Pigmentation fell into two distinct groups visually and this was verified by the absorption curves (Figures 3 and 4). Histidine, phenylalanine, and tyrosine fell into one group forming mostly a red—brown to brown pigment, while the rest fell into another group producing the typical wine red color. Glutamine seemed to exhibit characteristics of both groups. DL-alanine, l-leucine, DL-isoleucine and DL-valine were the be“ Pigment substrates with DL-alanine by far the best. 013701119 “33 not used since it failed to produce pigment in the tests with S011d media. One rather obvious difference between the results reported 69 here and those in Table VIII is the fact that threonine, gluta- mate and aspartate failed to produce any pigment and that growth was slight on these substrates. It was thought that some idea should be gained of the relative efficiency of the various media and substrates used up to this point. Alanine, leucine, isoleucine, and valine were prepared alone and in combination and compared with media previously shown to produce pigment. Results are recorded in Table x, TABLE X RELATIVE mOUNT Oi" PIGMENT PRODUCED 0N VARIOUS NITROGEN SUBSTRATES =— Nitrogen Relative Nitrogen Relative Substrate Concen- substrate Concen- tration tration 0.8% alanine 1'75 0.8% Group I 128 0 .8543 valine 148 1.6% " 180 0.8% isoleucine 73 0.8% Group III '75 0 .876 leucine 66 1.6% " 52 0.2% each of above 103 TGE (agarless) 18 0.4713 each of above 158 TGE lus glycerol 44 _l. 6% Casamino Acids 105 0.85 ppgs 18 As seen from the above table, all the synthetic media gave better results than complex media, with the exception of Cas- amino Acids. Alanine again demonstrated its superiority- 0.8% was surpassed only by 1.6% of Group I amino acids. Leucine, 180leucine and Group III amino acids were relatively the same in Pigment producing powers. The more concentrated Group III actnailly appeared to reduce the amount of pigment formed. TGE and ppfi‘?) were about the same and as eacpected, glycerol increas- Od the yield of pigment from TGE. '70 since alanine was the best source of pigment it was felt necessary to determine the importance of its concentration over a wide range. Concentrations from 0 to 2% were chosen using the filter technique and incubated for 10 days. The results may be found in Table XI and Figure 5. The results clearly indicated that an alanine concentra- tion of at least 1% is required for maximum pigment formation and that over this amount does not lead to any further signif- icant rise in pigment production. TABLE. XI QUANTITATIVE EFFECT OF ALANINE CONCENTRATION ON PIGMENT FORMATION % Alanine Relative Pigment Concentration 0 0 0.1 5 0.5 160 1.0 2'75 1.5 290 2.0 280 TABLE XII QUANTITATIVE EFFECT ON PIGBal'iNT FORMATION OF VARIOUS CARBON COMPOUNDS \ AT 0. 373 CONCENTRATIONS \——_k C Omp ound Relative Compound Relative Concentratio Concentration Tram. 96 ethyl alcohol 29 snominate 20 butyl alcohol 42 galate 4 glycerol (control) 118 umrate 4 mannitol . 65 xYIOSG 40 acetate . 0 ell‘etbinose 28 lactate 25 l‘1bose 31 butyrate 42 glucose (10 days) 39 gluconate 129 fIli'uctose '73 ‘Sglactose 90 71 The next experiment,in order, was a survey to determine if any carbohydrate or carbon compound could serve as a sub- stitute for glycerol. All compounds tested were at the same percentage concentration, and acids were employed as the sodium salts. Readings were made at 5 days except glucose and acetate which were held 10 days (zero at 5 days). Results will be found in Table XII. The various carbon sources tested did not fall into any real pattern as far as producing pigment was concerned. The TCA cycle intermediates were relatively non-reactive with the exception of citrate. 0f the carbohydrates, only galactose showed any efficiency, with fructose showing a fair degree. Glycerol was the only alcohol which showed much pigment, but this was a great deal. Among the acids, only gluconate showed great production, but this was about 10 more units than glycerol Showed. why one compound shows more activity than another undoubtedly is dependent on its route of metabolism, but just What this route is is beyond the scepe of this paper. Despite the fact that glucose was the carbon source of the medium on which the pigment was first observed, it is a relatively poor source. This might be expected, for such results as indicated in Table IV show that glycerol always pr°du€=ed relatively more pigment. The slowness of pigment formation from glucose, however, was unexpected. No pigment was Observed in five days, but there was some in ten days. Just “by this should be so is unknown. The last eXperiment to be of concern in this section was '72 the determination of cptimum glycerol content. The eXperiments were conducted as before, with the filter plates. The experi- ments were in two parts. From 0 to 0.275 glycerol incubated for 5 days was one part. From 0.1 to 10% glycerol incubated for 10 days was the other part. Results will be found in Table XIII and Figures 5 and 7. TABLE XIII QUANTITATIVE EFFECT OF GLYCEROL CONCENTRATION ON PIGNENT FORFATION t Glycerol , Relative Concentration eye 0 days 0.0 1.5 .01 46 .03 60 .06 110 .09 195 .1 182 280 .2 180 280 .5 280 .5 270 1.0 300 2.0 285 5.0 290 10.0 340 As seen in Table XIII and Figures 5 and ’7, the glycerol contenlt is not of great importance above 0.1%, which is the borderline. Below this concentration the amount of pigment PPOQuced drops off sharply toward zero. The small amount of Pigment recorded at zero glycerol concentration is undoutedly due to the fact that the organism can utilize the carbon chain of alanine to some extent as a substitute for glycerol. This is partially borne out by the fact that lactate can result in pigment but is a poor substrate (Table XII). '75 Discussion crom the experimental evidence presented it is apparent that the red pigment formed by this culture of £3. aeruginosa is highly susceptible to beef extract as an inhibitor; more likely it is something contained in this substance. that this might be is unknown but the next section deals with one pos- sibility. It would seem that this inhibitory power of beef extract is not the only factor affecting pigment production. Table IV illustrates the point that various peptones either inhibit or lack something, since with the same carbon source, the amount of pigment varies with the peptone. The next section deals with a possibility here also. One may also see in this same table that the carbon source greatly affects the amount of pigment. This is further demonstrated in Table XII where various carbon sources range from 0 to 129 units on an otherwise Optimum substrate. The explanation of this likely lies in the pathways and rates of assimilation of these com- pounds and is not within the 800138 of this thesis. Cnce it was discovered that Casamino Acids could replace the more complex ppg‘o, work progressed rapidly to a synthetic medium containing agar. Agar itself, however, was an unknown Quantity and because of it no quantitative work could be done, nor could the pigment be isolated. The develOpment of the technique whereby Itfiillipore filters “mid be floated on the surface of a liquid long enough to allow the organism to produce large amounts of pigment resulted in the quantitative determination Of pigment on substrates '74 develOped. Furthermore, it presented an Opportunity to deter- mine the relative efficiency of the various substrates and arrive at a more scientific determination of Optimum conditions for pigment formation. It has been found that alanine above a 1% concentration and glycerol above a 0.1% concentration are the Optimum carbon and nitrogen sources. Glycerol may be slightly less effective than an equal concentration of sodium gluconate, but it was chosen because there was so little actual difference between them and because so much work had already been done with glycerol. For the sake of comparison, it was retained. Also, it was more readily available. As already mentioned, the pigment formed on the various amino acids fell into two groups (Figures 5 and 4). The ring compounds produced a red-brown pigment while the others pro- duced the typical wine red pigment, although glutamine seemed to form both pigments. It is suspected that the ring compounds produced melanine-like pigments under the action of tyrosinase, since these compounds are the prime sources of these pigments and this enzyme has so Often been reported in these organisms. It is important to note that, from visual observation, some of the wine red pigment apparently occurs in this group, but the absorption spectra Of the two pigments overlap and it is impossible to distinguish one from the other, especially when the brown pigment is in high concentration. The compounds producing the wine red pigment, however, all bear some relationship. Whether this relationship is true rather than apparent must be determined by further research. that follows thmis speculation on this apparent relationship. The following are the structures of the amino acids producing the most pigment (in descending order-Table IX); H\ k CH-CHNBg-COCH alanine n ans-CH2. JD CH-CEh’I—ig-COGH valine H CHa-CHg‘ A? CH-CHNHg-COCH leucine CH3 en‘s-Chg“ 2,0H) CRNHg-COCH isoleucine OH, H The basic or important group lies to the right of the VertICle line. Note that extending the carbon chain tends to decrease the amount of pigment formed, and that the location of the methyl group in leucine or isoleucine alters the amount of Pigment somewhat. Any other groups attached to the basic alanine structure reduces the amount of pigment to about 1/10 th 01' the above amino acids. Serine, for instance, substitutes an hydroxyl group for one of the hydrogens on the beta carbon of alanine but produces only one tenth the pigment that alanine ROSS. The same holds true for arginine and lysine which are elitensions of the valine structure. That these amino acids must be in free or very short chains may be one eXplaination ‘hy pigment may be formed on some media (peptones) and not others of the same source (casein). 76 In this section the determination of the carbon and nitrogen sources for a synthetic medium for production of the wine red pigment produced by this organism has been demon- strated. The apparent "antagonism" of beef extract to pigmenta- tion and the relative efficiency of various carbon and nitro— gen sources in producing the pigment have also been demonstrat- ed. Einally, some thoughts have been presented on the signif- icance of the structure of the amino acids which produce the Pigment. The carbon and nitrogen sources and the concentrations sufficient to produce growth and abundant pigment were finally chosen to be: DL-alanine l.% glycerol 0.5 salts- see next section. ‘77 III PIGMENTATION AND MINERAL REQUIREMENTS The previous section dealt with the determination of the composition of a synthetic medium in terms of carbon and nitro- gen source. This section deals with the minerals and the con- centrations required to produce the red pigment under study. Up to the point in the last section where it was discov- ered that ppf5 could be used as a source of nitrogen, no salts had been added with any regularity. The one attempt (page 52) did not produce any pigment except when the salts were absent. with the advent of the ppfiS-glycerol medium, Burton's paper (1948) came to the attention of the author, and it was thought that pyocyanine and the red pigment might be enhanced by the same minerals. Again in this section the research must be divided into two sections; the study of mineral requirements on A. complex media, and B.-synthetic media using the filter technique. Experimental A. General considerations are the same as given in Section II A except that glassware was cleaned in aqua regia where required. 1. The first eXperiment is the same as that reported on page 52 where 0.01% MgSO..VHgO, 0.01% 36304.7H20, 0.05% glycerol and 0.01% separagine were added to laboratory prepared TGE. 78 2. The following mineral salts at various concentrations were studied for their effect on pigmentation: K2HPO‘, thO. -Hgoo M6012.6320, K250... and F680..7H20. The basal medium was composed of 0.5% ppfifi, 0.1% glycerol and 1.5:? agar. NO other salts than the one under study were added to the medium. 5. A series of experiments was performed to determine these mineral effects on a 1% alanine, 0.5% glycerol and 1.55.") agar base. Different concentrations of minerals were added to the medium alone and in combinations. Results were recorded from visual observations. B. The hillipore filter technique was next develOped (page 55) and the pigment concentration was determined spectro- photometrically and plotted on graph paper, giving typical curves for each mineral component. Plates showing no pigment at five days were kept until 10 days. These were uniformly neéiative at this time also. 1. All studies were conducted on a base medium composed 0f 1% alanine and 0.5% glycerol at a p}! of 7.0. The various mineral salts under study were added in the concentration ranges desired and altered in accordance with the information reiuired. The Optimum concentrations or wgso..7H.O and 21.211130... were determined as well as the effect of substituting various ions in place of these. For the latter determinations, salts were added at concentrations so that the ion under study was e(“livalent in concentration to that found in the ideally constituted medium. The other ion that necessarily went along with the ion under study was also controlled in a separate '79 plate. For example: MgSO. ions were studied using Lig012 and Naeso‘, each in a separate determination. In order to control the effects of the Na and Cl ions which were necessarily added, NaCl in an equivalent concentration was used. i'ineral ions which might inhibit pigmentation were also studied-Mn, Zn, Cu, and ASO‘. 2. In order to ascertain more fully the effect of initial PH on pigmentation an eXperiment was designed to study a range of pH from 6 to 9. The initial pH was adjusted in a base medium containing 17;. alanine, 0.3,; glycerol, 1,8 Ng30..'7H20 and 0.01% K2HPO... - KOH or £12250. was used to adjust the pH in order to “01d adding any mineral icns not already present. After five days 1Ileubation, the resultant pH was determined and the rel- ative Pigment concentration measured in the spectrophotometer. . The DH‘ 8 were determined using a Beckman model G pH meter. Results The results on laboratory prepared TGE have already been reFOI‘ted in section II. No pigment resulted when 0.01% MgSO. '73‘0 or 0.01% F630..'7H20 was added. In the tube where no minerals at all were included, there was a slight pink formed, but it was very faint. The addition of minerals to pp,,3 when it was discovered that they would support pigmentation, gave the results indicat- ed in Table XIV (page 81). This table represents concentrations ram-82'Lng from zero upward for each mineral studied. It is of intelZ‘est to note, that pigmentation occurred even in tubes w hich had no added minerals, although the addition of certain 80 TABLE XIV VISUAL DETERMINATION OF RED PIGIVZENT AS A RESULT OF MINERAL SALT CONCENTRATION ON A PROTEOSE PEPTONE (ES-GLYCEROL AGAR BASE Mineral Concentration Concentration Optimum concen- range range of tration range studied pigmentation for pigmentation heiiPO‘ 0'005% 0-0.01% 0.001% M330. .7HgO 0-2.0 0-2.0 2.0 MgClg.6HgO 0-5.0 O-3.0 0.5 K280. O-3.0 0-5.0 1.0 amounts of each enhanced its formation. This is undoubtedly the result of salts contained in the agar and the ppff3. The Difco Bulletin #81, 1925 indicated that practically all the minerals represented above occur in agar to some extent. From the Difco Manual (1955), ppffls is estimated to contain an equiv- slant of 0.13% KgHPO‘, 0.0067. Lasso. and 0.000175. F630,. As a result of the data of this eXperiment, a mineral base of 1;, wzgso..7H.0, 0.017; Kgnrc4H and 0.000173 Peso..7neo was used in all work with ppffié. That this composition Was not ideal can be seen in the fact that pigment formation on the PPJf3 medium with the salts added was not always constant, oc- '°8.81onally producing more and sometimes less. These eXperiments were repeated when it was found that DI-"8.lanine could replace Ppn‘s, and the results were identical “1th the exception that more pigment was found to be produced “an r‘eso. was left out of the medium. It is noteworthy that the amount of pigment varied considerably from eXperiment to eJtperiment indicating that agar was still adding ions which affected the results. 81 Jit; was with the develOpment of the hillipore filter technique that the exact concentrations of the ions could be ascertained. Table IN and Figures 6 and 7 contain the results of two separate eXperiments on the concentration of Ngso...'7HgO required for Optimum pigment formation. The first considered concentrations from 0.1 to 10% incubated for 10 days, while the second considered concentrations from 0.01 to 0.5% incubated for 5 days. This medium contained 0.01% KgHPO‘ as well- TABLE IV EFFECT OF MgSO...’7HgO CONCENTRATION ON THE PRODUCTION OF RED PIGMENT =================:======================= % MgSO..7HgO relative pigment 02222f££§£122§_. 5 days 10 daz§_ 0.0 O 0.01 0 0.05 4.5 0.05 46 0.09 75 0.1 155 520 0.2 165 0.5 170 322 1.0 260 2.0 1'70 5.0 0 10.0 0 It can be seen that IthO..7HgO is required in a concen- trati on of at least 0.05% for any pigment formation to take Place at all and the optimum lay at 0.5%. Below 0.03;; a slight amount of growth did occur, but it was week. No growth or p1Binaritation was shown at 0, 5 and 10% concentrations. The importance of the concentration of phosphate is 11- lustrated in Table XVI and Figure 6. The medium used con- tained 1.0% Mg.)0.,.'72120. 82 TABLE XVI EFFECT OF KgHPO. CONCENTRATION ON THE PRODUCTION OF RED PIGMENT % KgHPO. Relative Pigment Concentration 5.0 0 0.005 26 0.01 240 0.02 590 0.06 440 0.04 360 0.05 156 0.06 108 0.07 50 0.08 0 0.1 0 The effect of phosphate is dramatic. Within a concentration range of 0.005 to 0.07% the pigment rises to a maximum, produc- ing more than ten times the amount of pigment formed at the extremes of the range. It becomes apparent that phosphate added as a contaminant becomes extremely important in the production of this pigment, and must be rigidly controlled. The previous results of these studies (Table XV) indicate that a ph0$phate concentration of 0.5% is also inhibitory. Occasionally tubes containing phosphates above 0.07% will show a precipitate with MgSO. and if this removes enough phosphate a small amount of red pigment will be produced. The results indicated are those without precipitate. Ferrous sulfate septahydrate was used next to determine the effects of iron on pigment formation. Results are given in Table XVII (page 84). The base medium here included 1% MgSO..7HaO and 0.01% KgHPO.. The amount of iron required for full growth by these organisms is reported to be about 0.000,04% (0.08 ppm Fe) 83 TABLE XVII EE‘ECT OF FBSO¢.7H20 CONCENTRATION ON THE PRODUCTION OF RED PIGMENT % F680..7320 Relative Pigment Concentrgtigg 0.0 175 0.00001 200 0.00005 190 0.0001 215 0.0005 225 0.0005 210 0.005 200 0.01 74 0.05 0 0.1 ' 0 BeSO..7H20 (Warring and Werkman 1942). Apparently this amount is supplied as contaminants of glassware, reagents and water used since the organisms could grow and produce pigment when no iron was added and the addition of up to 0.005; had little effect on the actual amount produced. The low amount which occurs in the control is quite possibly less than 0.00001% itself. If any Fe requirement for pigment production is shown, it is rather most likely a requirement for growth instead. Over 0.005%, however, results in a dramatic inhibition of pigment formation but not of growth. The next experiments were devised to answer certain ques-. tions about the necessity of the ions so far studied. Prior to this point it had been only complete compounds which had been studied. Which portions of these are the important ones? Tables XVIII, XIX, and xx are the results of attempts to answer this question. (See next page for tables). from tube 5 (Table XVIII) and its control,7, apparently either'NH. ion is inhibitory or K ion is required. That K may 84 TABLE.XVIII EFFECT CF VARIOUS IONS ON GROWTH AND THE PRODUCTION OF RED PIGMENT %Concen- KgH- Kgs- Rel. tration P0. 0. Pig. Control for: Gro- Conc. wth 1 €8C12.6520 .68 + - 0 Ng-standard + 2 'agSO. .58 + - 75 SCI-Standard + 5 a01 .48 e - 0 Tubes l,2(NaCl)- 4 (NH,)gSO. .52 + - 0 SO.-Standerd - 5 (NH.)2HP0. .0092 - + 0 HPO.-Standard - 6 H.Cl .45 + - 0 Tube 4 (NH.) - 7 H.01 .0075 - + 0 Tube 6 (NH.) - 8 thC. .708 + - 58 504-3tandard + 9 Cl .505 4 - 0 Tube 8 (K) - 10 standard - + + 155 Control + be required is seen in tubes 2,4 and 8 where 2 and 8 produce a small amount of pigment and while 4 does not. The only dif- ference between these tubes is the presence of K in 2 and 8 but not in 4. That h is not the only factor required is seen in tube 9 where all the ingredients except Mg and 30. are present. gince the only difference between 9, 2 and 8 is the absence of o0., this may be required. That Mg is not required is seen from 2 and 8 which form pigment with no added Mg, and tube 1 which has kg added, no s0. and no pigment. No evidence on requirement for 3P0. ion was obtained. The conclusions drawn from this experiment are as follows: K.may be required or NH. may be inhibitory. Instead of K, 50. may be the requirement, and finally Ng is not required. Phosphate requirement is unknown as yet. In order to clarify these questions further, another 6Xperiment was set up to test the effects of these various ions and,in addition, some other ions reported to suppress Pyocyanine formation; an, in, Cu and As. These latter ions 85 were arided.as sulfates (except Na2A30.) and therefore the 53.50,.‘71-130 content was reduced. Both P"g30., and Kai-11304 were present in all tubes in contrast to those recorded in Table XVIII. 'The results of this experiment will be found in “able XIX. TABLE XIX EFFECT ON THE PRODUCTION OF RED PIGMZLINT AND GROWTH BY VARIOUS IONo' IN THE PRESENCE OF MgSC‘ Tub Salt Salt Relative Growth 5 concen- Pigment t§ation Concent. 0 1 QC]. .4 245 + 2 Nchl .0075 190 + 5 (NH‘)gSO‘ .6 245 + 4 M8012 .6 190 + 5 NaaSC. .6 240 + 6 NaCl .6 210 4 7 K250. .6 290 + 8 ZnSO. .6 0 - 9 MnSO. .6 0 - 10 (“1304 .1 0 - ll N82ASO4 .5 0 - __¥ 12 Control - 155 + tfirom this table it can be seen that the ammonium ion does not ithibit pigmentation (tubes 1,2 and 5); but, on the con- trary, stimulates it to some degree. The same may be said of the Cl and Mg ions. sulfate also seems to stimulate pigment Production (tubes 3,5 and 7) while heavy metals inhibited b°th growth and pigmentation at the concentrations used. The contlwsl (tube 12) produced very little pigment which may have been-<1ue to the low amount of MgSO. present in the medium unsupported by other minerals. 1"I'om the last two experiments we may conclude that the 30* 1Januwas required for pigmentation while the Mg ion enhanced 86 the amount of pigment produced but was not required. The NH. ion is not required but enhances pigmentation, as does the 01 ion, provided the medium is otherwise sufficient for pig- ment formation. If the medium is not complete these last two ions may be slightly inhibitory. This experiment still gave no information on the relatve importance of the K and RFC. ions, specifically. The results will be found in Table XX. TABLE XX RELATIVE EFFECT OF K AND HPO‘ IONS ON THE PRODUCTION OF RED PIGNENT so an experiment was designed to determine these ions Tubd Salts % Conc- KaHPO. Relative Gro- h’ entrat- added Pigment wth ion Concent. l (NH.)aHPO. .0074 - o - 2 KG]- 001 " O '- 3 (NHa)aHPo. .0074 _ K01 . 01 } 184 * 4 (m ) QI'IPO‘ . 2 _ 26 + K01 .01 5 K01 .01 + 174 + 6 NazflPO. .0082 - 0 - 7 NEQIiPO‘ 00082 - ' KCl .01 } 150 + 8 Control + 185 + y From this experiment it can be seen that both K and HPO. 1°38 are required for growth, and pigmentation as a consequence. Neither would produce either pigment or growth in the absence 01‘ the other. That it is phosphate which limits the amount of Pigment produced may be seen in tube 4 where a large amount 8'7 of phosphate reduces the amount formed to a very low level. 1116 results of the Optimum initial pH may be found in table XII. .TABLE XXI EFFECT OF INITIAL pH ON PIGMENT PRODUCTION AND ON THE FINAL 23 OBSERVED Initial Final Relative pH pH Pigment Concent. 5 5 0 6 7.9 155 7 8.0 155 8 8.0 108 9 8.1 12 ]?t can be seen from Table XXI that an initial pH of betWeen 6 and 7 produces the cptimum amount of pigment. Below PH ‘5 Ito pigment is formed, in fact, no growth occurs, while at 51 IXH,of 9 growth occurs but pigmentation is very poor. The frn‘ilL ng of a growing culture on this medium is at 8.0. The Optimum pH appears to lie in the vicinity of 7.0 Discussion lProm this section several conclusions can be drawn: IL. 30. ion is required for pigmentation at a concentration of 0.5% MgSO;.7HgO; 23. Mg is not required but improves the amount of pigment formed; 3. K‘fHPO. are required for growth alfi thus for pigmentation. Neither alone will produce either growth or pigment. Optimum pigmentation occurs at a concentration of 0.05% and completely inhibits pigment formation at a concentration of 0.07%. This inhibition is due to the 88 3P0. ion rather than the K ion. 4. Fe is required for growth and in this sense probably for pigmentation. Sufficient Fe occurs in distilled water and ordinary reagents to produce both pigment and growth. F650..7H30 in excess of 0.005% inhibits pigmentation but not growth. 5. NH. and 01 ions appear to stimulate pigmentation some- what. 6. Ions of Cu, Zn, Mn, and A30. inhibit both growth and and pigmentation at concentrations at which Hg stim- ulates both. 7. The Optimum initial pH of the medium lies between 6 and 7, while the final pH after pigment is formed is 8. From the above conclusions and those from the previous section, the following medium was adOpted for further studies on isolation and characterization: DL-alanine 1.0% MgSO..7H20 0.5 glycerol 0.5 KgHPO. 0.01 pH 7.0 The results using 00pper are interesting by way of the fact that this organism will grow on 0.01% CuCl in a glycerol- tyrosine base producing a brown pigment. However, on 0.1% CuSO., the organism.was completely inhibited. The percentage of KgHPO¢ chosen was deliberately lower than the Optimum concentration. A 0.01% concentration produced abundant pigment and because of the organisms very sensitive response to increases in phosphate content, it was thought 89 more desirable to choose the lower concentration as a safety factor against the possible presence of this ion in reagents, water, glassware, etc. The Fe level was ignored because of the wide range of concentration over which the pigment was produced and the fact that it did not matter whether Fe were added or not. It is felt that the phosphate level or the Fe level would be the most likely explanation for the variation exper- ienced when using various peptones as a substrate, and indeed, is probably the primary cause of the inhibition of pigmentation seen with beef extract. On the other hand, peptones may be poor pigment substrates because of a lack of free or utilizable amino acids which are required for pigment production. The medium chosen has amply demonstrated its suitability in.producing large amounts of pigment and also in its reproducability from eXperiment to eXperiment. 90 Please note: Page 91 seems to be lacking in nwhbering only. UNIVERSITY I-ZICPDFIUJZS, INC. IV ISOLATION AND CHARACTERILATION The isolation and characterization of this pigment are included in the same section because,for this particular sub~ stanca,the method of isolation is, in I sense, a characteristic. There will be some attempt to keep the two separate but the isolation is dependent on the characteristics of the pigment and the characteristics depend on the efficiency of isolation. The section will be divided into three parts. The first will deal with some characteristics of the pigment in whole culture which will lead to the second part; the actual isolation and control of purity; and, finally the characteristics of the isolated pigment. a. The Pigment In Culture as briefly mentioned already, the pigment was very rarely found in liquid medium and when it was it occurred as a leuco form which became red upon shaking. The red color disappeared again upon standing, provided the concentration was not too high. Prior to the appearance of the red pigment the broth was yellowish, but apparently this was not the fluorescent pigment. 0n solid media the following changes occurred: Very early in the life of the culture (prior to 24 hours) the medium under went a transformation to yellow, then to grass green and finally, at 24 to 48 hours, turned red. The green pigment is never seen again and has not been isolated. It is not choloroform soluble and has been separated from the culture but not purified. These pigments will be discussed again with the pure red pigment. Under ultraviolet light the medium fluoresces a brilliant red when the red pigment is present. When grown on solid media or by means of the filter tech- nique, the red pigment is found abundantly. In the early stages it appeared like an extremely dilute solution of safranine 0, but in older cultures it assumed.the color of port wine with, a slight bluish tint. The absorption spectrum of this pigment in cell free culture had an absorption maximum at 525 mu when grown on alanine (Figure 1). In culture medium this pigment can be diluted and the optical density reading is proportional to the relative con- centration (Figure 2;. The red pigment cannot be separated from fluid culture by any water immiscible solvents. It is soluble in those solvents miscible with water only if water is present and in amounts which depends on the solvent. When a culture is dried only methyl alcohol, acetic acid, and phenol (othhe solvents tested) would dissolve the pigment. Nethyl alcohol would remove only a portion of the pigment present, leaving the rest as an insoluble residue. I Paper chromatography of the complete culture medium con- tainng the pigment resulted inacontinuous streak of Spots of various colors, overlaid by a continuous streak of ninhydrin reacting substances. It was found that if the culture was 93 dehydrated and then dissolved in methyl alcohol that a char- acteristic chromatogram could be develOped using 1N NH‘OH as the solvent. This technique will be discussed more fully when isolation procedures are discussed. Acid or base had little effect on the pigment when in culture, but,due to the blue tint, a change toward the blue could not be recognized and was overlooked. This will be eXplained more fully when the isolated pigment is discussed. B. Isolation It was first thought that paper chromatography would af- ford a good means of isolating the pigment, but it was soon observed that the technique was too limited in the amounts one could isolate. Several techniques were examined; paper electrOphoresis, ion exchange resins, absorption on activated charcoal and almmina, among others. Each was discarded as un- satisfactory for one reason or another. The substances did not separate well by electrOphoresis or in ion exchange resins which required a relatively purified substance to begin with. Alumina would not absorb the pigment while charcoal would but from which it could not be removed. It was not until column chromatography was attempted that any success was attained. The most effective adsorbent was found to be powdered filter paper and several solvents. This technique suffered also from the fact that a relatively pure substance was required. The following procedure was the one develOped as moat effective in isolating and purifying the pigment. 94 1. Experimental The columns were glass, 3 cm in diameter by 40 cm in length, with a removable sintered glass joint on the bottom held on by Springs. The columns were packed by adding a slurry of powdered filter paper to the column while suction from a water aspirator was being applied from the bottom of the col- umn. as the paper was pulled to the bottom of the column, it was tamped gently into place with a long wooden stick. The column was filled by this means to the desired level, usually within 5-8 cm of the tOp. The filter was then washed with distilled water for 12 hours to insure that it was clean. This was followed by the particular solvent to be used. The column was closed off at the bottom by a clamp and a rubber hose as soon as the solvent had cleared the bottom of the column. Pigment was checked for purity during the Operations by visible means, under ultraviolet light and by the use of paper chromatography. Visible control was for non-red contaminants appearing upon dehydrating. The ultra violet technique involved examination of the column and column washings for the presence of fluorescing as well as visible substances. washing proceeded until these were removed. The ultraviolet lamp used was a Westinghouse floor model with a screw type bulb (GEBH4-100W) which produced near-visible wave lengths. Paper chromatography was used as follows: Large sheets of whatman #1 filter paper were spotted along a base line and placed in the trough of a Recco Chromatocab. It was then allowed to come to equilibrium in an atmosphere of the solvent 95 which was 1N NH40H for 6-12 hours. The solvent was then poured into the trough and the cab closed up for 3 -3 l/2 hours at which time the paper was removed and dried, either in a 100 C oven or before an air fan. The dried paper was then examined under visible and ultraviolet light for the presence of any spots. These were marked and the paper sprayed with 0.1% ninhydrin in n—butyl alcohol and placed in an oven for 5 min- utes at 105 C at which time any ninhydrin reacting Spots would appear. Other reagents could be used similarly. The absence of all spots other than the red pigment was one indication of purity. The actual procedure of isolation was as follows: The organism was grown on filter pads floating on the basal medium presented at the end of the last section. Cultures were allowed to grow until virtually dry at which time distilled water was added and the pigment collected at no more than 500 ml per 1 liter flask. To the culture fluid was added solid B3(OH)2 which was shaken and allowed to stand until no reaction could be obtained with BaNOa and base applied to a small filtered sample. This step was necessary to remove all of the Mgso. as BaSQ‘ and wg(oH)g. The solution was then filtered through double Whatman #1 filter paper and collected in a vacuwm flask;. The clear red solution was then evaporated on a 57 0 Water loath under suction produced by a water aSpirator. This temperature was chosen because higher ones altered the pigment SOmBWhEit, especially when dry. When the volume had been reduced to 25-50 ml 11; was ready for the next step. 96 .1145? , Hi ..... i.— ‘UHSJDI.’H>I T The first column was prepared as described earlier ex- cept that the column height was 10 cm. The solvent in this case was pure acetone which was run through the column leaving an excess of one half the remaining column height on top. The small volume of pigment-in-culture was then poured gently and slowly into the excess acetone on the t0p of the column. The pigment precipitated in the acetone as an oily or granular mass. acetone was then added to the t0p of the column and it was allowed to pass through the pigment and other material precipitated on the tOp of the column. acetone continued to be added to the tOp of the column until all visible or ultra— violet fluorescing substances ceased coming through the column. The acetone was followed by three column volumes of choloroform and this was follcwed by 200 proof ethyl alcohol until all visible and fluorescent materials had ceased coming off. Then two column volumes of 15% water in acetone were used. Following this was 30% methyl alcohol in acetone. Sach of these was added until no more visible or ultraviolet fluorescing sub- stances came ofl‘ of the column. The red pigment was finally removed by washing it offthe column with pure anhydrous methyl alcohol. This was the crude preparation. The methyl alcohol-red pigment band was collected from the column in a vacuum flask and evaporated to dryness in a 57 C water bath under suction. The residue was picked up in methyl alcohol again, transfered (filtered if necessary) to another evacuating flask and evaporated to dryness as before. This procedure was repeated until little or no residue remained 97 when the pigment was picked up in methyl alcohol. The final pick up with methyl alcohol was in as small a volume as pos- sible (certainly no more than 10 ml). This amount was transfer- ed to the tap of the second column which contained methyl alcohol as the solvent. The above evaporations were found to be advantageous in removing certain unidentified compounds which were soluble to some extent in methyl alcohol. It was quite effective in removing most of these substances. The second column had methyl alcohol as the solvent. The level of solvent was allowed to dI‘Op until the meniscus was Just at the tOp of the column. The very small volume of methyl alcOhol containing the pigment was then added carefully to the t0p 0f the column. '! he height of this band should under no conditions exceed 2.5 cm and ideally should bemuch less than this- The pigment band was allowed to pass slowly into the 001% until the meniscus was Just at the top of the paper. I'ethyl alcohol was then added slowly while the red band moved down the column. a reservoir of the solvent was attached to the top 01' the column and the flow out the bottom adjusted to L-8--:l..(: ml per mine e. The movement thru the column separated several visible bands and some visible only under ultraviolet light . Eventually the red pigment passed out the bottom and was <3Ollected in a vacuum flask and evaporated at 37 C as before. The pigment was picked up in methyl alcohol, transfered to a‘no‘bher flask and evaporated as before. This was repeated until little or no residue change occurred (2-3 times). The final pick up was with m NH40H, again in a minimal amount. 98 The small amount pigment was added to the tOp of the last column which had 1N NFi‘OH as its solvent. The pigment was added in the manner indicated for the second column. The amount of pigment solution used should not exceed 2.5 cm of the column height and should be much less. The reservoir of 1N NIi‘OH was attached and the flow out the bottom again adjusted to 0.8-1.0 ml per minute. Separation occurred and the pigment was collect- / ed in a. vacuum flask and evaporated to dryness as before. The pigment was then taken up in water or methyl alcohol as was desired. A small amount (50 microliters or less) was spotted on a chromatogram paper and checked for fluorescing and nin- H- hYdI'in reacting spots. If any were observed, the entire process was repeated as if the pigment were in a crude extract. When the red band on the final column was collected there were no con- taminants present by the detection methods used. Culture Bfl(0H)2 Pgecipitatee filter >f11trate isc-‘al‘ded wash with- levaporate to 20ml remove in lfiaficetone’ precipitate on anhydrous‘e CHCla acetone column 011.30}: 200 proof ethyl alc. 15, water in acetone 30% CH30H in acetone Cl uh (f: (‘5 “’31) orate (2x) 01—13 011 solvent 1r CH30H\ ghrgmatograph 3 CH30H evaporate (Z-Sx) 1N NH.0H with onson _ ficnaon solvent so vent chroma ographed in lb. NH..0H Pure pigmente evapgrate and 1N NH.OH pick up in H20 or CH30H solvents 99 2. Results When the culture medium was precipitated on the first columun and washed with the various solvents, the following substances were removed: 1. acetone removed a yellow-green compound showing blue fluorescence which acid did not affect; 2. CHCla did not remove anything fluorescent or visible; 3. 200 proof ethyl alcohol removed a yellow compound which fluoresced blue. Acid removed the fluorescence and base restored it. There was no alteration in color when Zn and acid were added; 4:. 1533 water in acetone removed a colorless non-fluorescent substance which appeared as aprecipitate when more acetone was added; 5. 30% methyl alcohol in acetone removed an orange pig- ment Showing yellow fluorescence. Acid changed the color to red and destroyed the fluorescence which could be restored by adding base; 6 . CH30H removed the red pigment; '7 . P120 removed a red-brown pigment which was insoluble in CHaOIi. This portion seemed to be associated with a ninhydrin reacting substance on paper chromatograms. Only the red pigment, soluble in methyl alcohol was stud- ied ful‘ther. z"Hush chromatographed on the second column with methyl alco- h°1 es the solvent, the following observations were made; 1- A dark red band which showed a violet tint preceded the main red band. This dark red band fluoresced a reddish- 100 purple color under ultraviolet light. It showed a strong positive reaction with ninhydrin and when chromatographed in 1N NH4OH it showed several distinct yellow pigments as well as the usual red. When dry it appeared red-brown. 2. A yellow, a blue and a green fluorescent band may trail the red band but these have not been isolated nor studied to any degree. They may be residuals left from incom- plete extraction on the first column. 0n the last column five bands appeared which may be impor- tant in isolation. l. The first was a yellow band whose dilute solution appeared greenish. It has not been isolated or studied. 2. The second was again a brown-red band giving a reaction with ninhydrin. Apparently it was the red pigment associated with an amino acid or peptide. 3. The main red band. 4. The very end of the red band tailed or stretched out. then it was collected separately, it gave a very slight reac- fion with ninhydrin indicating an association with an amino acid or peptide. 5. ‘inally, half way or more back up the column (at the time the red bandwas leaving the bottom) a faint yellow band, which in.fresh batches of pigment was usually not seen. This may be a breakdown product of the pigment. 'The main red band was collected and found negative for ninhydrin reacting substances, gave no test for Mg,Ba or so. ions, was carbohydrate free as tested for by ammoniacal silver 101 nitrate, resorcinol and triphenyl tetrazolium chloride reagents, and gave a single spot when chromatographed in lN NH40H or 03305. The pigment was thus considered pure. C. Characterization The purpose of this investigation was not definitely to determine the structure of the pigment under study but at least to determine sufficient characteristics so that future investi- gators may recognize the pigment and be able to compare their results with the ones set forth here. With this in mind certain prOperties of the pigment have been deter- mined. These characteristics may be conveniently divided into three groups although they are not completely separate since overlapping does occur. The groups may be identified as: 1. Determination of possible biological action. 2. Chemical reactions of the pigment. 5. studies of the pigment by use of the photometer and absorption Spectra. 1. Experimental The electromotive potential Was determined at pH 8.0 with the Beckman pH meter model G using the platinum and calomel electrodes. The following protocol was used to determine the ability of the red pigment to act as an electron acceptor in an oxi- ¢ktion~reduction reaction carried out by living organims. The enzyme system used was the succinic dehydrogenase system of yeast. Fleischman's Activated Dry Yeast was prepared so that 102 a milliliter ofits solution contained a weight of 0.0475 gm. sodium succinate M/SO was the substrate and M/lo KHgPO. and NagHP04 at pH 7.4 was the buffer. Fethylene blue was used as a control with each ml containing 0.02 mg of dye. Each ml of the red pigment contained 0.052 mg of pigment. The following schedule was observed: Tube y yeast buffer dye vol- substrate volume volume ume l 1 ml 2 ml 2 ml M.B. 2 ml succinate 2 l 2 2 " 2 ml water 3 l(boiled) 2 2 " 2 ml succinate 4 l 2 2 pigment 2 ml succinate 5 l 2 2 " 2 ml water 6 l(boiled) 2 2 " 2 ml succinate The yeast solution added to tubes 3 and 6 was boiled to destroy the enzyme system and thus constituted a negative control. Tubes 2 and 5 were autogenous controls without added substrate. After all solutions were added together, mineral oil at least one inch in depth was layered over the mixture in order to insure anaerobic conditions. The chemical studies were a series of more or less unrelated reactions and will be described as the results are discussed. Determination of ionic activity was made on hhatman ”1 filter paper sheets with an applied electrical current. The sheets were 29 cm wide x 26 cm long with small strips extending downward 6 cm on each side to dip into the electrode dish. The paper was wetted in the buffer to be used, placed in the trough of the Chromatocab and the 6 cm edge pieces placed in 30 ml beakers of the buffer which acted as electrode dishes. The power supply was that suggested in Scientific American (1955) 103 which delivered 250 volts 3.0. The electrodes were platinum wires placed one in each beaker. Current was observed constantly on an ammeter, giving a range from the time the spot was placed until the eXperiment ended. The current was applied and ad- Justed to a range between 10-50 milliamperes. when this was accomplished the power was turned off and the pigment applied to the top of a verticle line,which had been plumbed to insure that it was vertical, until it was visible to the eye. There- upon the Cab was closed and the current applied. The time of the run was determined by the Speed of downward migration of the spot. Below is an outline of the materials used: Buffer pH Current in Minutes milliamps of run 0.1N HCl+ 1M NaCl 1.0 55-50 75 1M KHzPO‘J- NaOH 5.5 58 180 ll: m2P0‘+ NaOH 7.0 15-20 660 1M K2HPO. 9.0 20-25 560 0.1N NaOH+ 1M NaCl 15.0 15-25 180 Determination of the absorption spectrum of the pure pig- ment was made in a Beckman DU recording spectrOphotometer from a wavelength of 250 mu to one of 600 mu (Figure 9). other spectra were determined on the Bausch and Lomb spectronic (‘0 0 already mentioned 2. Results The electromotive potential was determined at a pH of 8.0 and found to be +0.215 volts. This pH was chosen because 104 it was that obtained when the dry pigment was added to freshly boiled and cooled water. The E; of methylene blue at this pH is about +0.0005 volts while that of cytochrome c at pH 7.0 is +0.262 (Lardy 1949). The study of the pigment as an electron acceptor showed that while it can be reduced by the yeast-succinate system, it required a great deal longer than methylene blue in the same system. hethylene blue was completely reduced in 10 minutes while the red pigment was only half reduced in 56 minutes. The red pigment was completely reduced on standing overnight. Meither tube containing boiled yeast showed any reduction on standing overnight. The pigment therefore may act as an electron acceptor in a biological system although it is less efficient than methylene blue in the same system. It is interesting to note that the color or the reduced pigment was a weak yellow- green whereas the methylene blue tubes were colorless. The first chemical study with the red pigment concerned its action in acid and base. Acid when added to yield a pH below 5 gave a violet color identical in shade to that produced when safranine 0 or neutral red is acidified. The color change was noted to occur between pH 4.0 and 5.0. Base produced no change, as was observed in the case or:wmranine 0, while neutral red became yellow. The pure pigment itself assumed the color of a neutral solution or neutral red and was also similar in shade to safranine C. The pure pigment fluoresces a brilliant orange-red under ultraviolet light as does safranine when base is added. 105 The molecule would appear to be rather small since it diffused through a dialysing membrane within a few minutes. Beating the dry pigment to 100 C caused a breakdown leaving a red-brown residue only part of which was soluble in water. when left at this temperature long enough, it was completely destroyed. an empirical analysis of a sample performed by licro-Tech Laboratories, Skokie, Illinois showed the following: C 26.44%, H 5.7%,N 4.6% and an ash content of 46.65%. The sample sent for analysis gave no test for Ba, Mg or 50‘ ions. However, when tested with strong base a slight floc was noted which was probably rg ion and at the same time the prOperties of the pigment were altered. Two possibilities exist for explaining the high ash content; one that hgso. remained as a contaminant in spite of the tests performed or, second that Mg ions formed a complex with the pigment and the ash content represented MgCOs. The percentages given above indicate an empirical formula of C5H12N104- The oxygen was determined by difference. The value is a little higher than might be expected for hydrogen. When dissolved in water, unbuffered at pH 7.0, the pig- ment causes a shift to pH 8.0 indicating a basic nature. The results of the experiment to determine how the pigment ionizes will be found in Table XXII on the following page. From.the information in this Table it can be seen that the pigment is a cation at all pH values from 1 to 14, since the molecule moves toward the negative pole in all cases. 106 TABLE XXII RESULTS OF PIGMENT MIGRATION IN AN ELECTRIC FIELD AT VARIOUS pH VALUES pH minutes Centimeters Rate of Centimeters of run of vertical Vertical of lateral movement Novement movement Cm/min (- to anode) 1.0 75 9.5 0.124 -l.l 5.5 180 15.5 0.075 -2. 7.0 660 9.0 0.014 -0.7 9.0 560 14.0 0.059 -l.5 14.0 180 16.5 0.090 -0.8 Another interesting fact is that at the extremes of pH the pigment migrates downward much more rapidly than at neutrality indicating that least solubility is at the neutral point or very near it. since the molecule favors the anode it probably does not contain a zwitterion. The pigment was chromatographed and sprayed with various reagents to test for different groups. The reagents used to test for the various types of compounds and the results ob- tained with them are as follows: Carbohydrates; 0.1% naphtholresorcinol in 0.4; HCl and 80% ethyl alcohol, 0.1% triphenyl tetrazolium chloride in 2N NaOH, ammoniacal silver nitrate- all negative Protein, amino acids, amines; 0.1% ninhydrin in n-butanol- no reaction. Acids; ammoniacal silver nitrate- no reaction. Phenols; ammoniacal silver nitrate, ihrlich's‘reagent, FeCla - no reaction. To summarize the above, the pigment gave no reaction for carbohydrates, amine groups, acid groups or phenolic groups. These may occur, however, and be masked by such things as ester or ether groups, or tertiary or quaternary amine groups 107 thus being non-reactive. The above tests indicate that these compounds and groups do not exist free as such in the pigment. These same tests were used to demonstrate the purity of the pigment sample by chromatography and Spraying. The effects of the various oxidizing and reducing agents are the last chemical studies to be mentioned. Bromine water and H202 oxidize the pigment completely to a colorless sub- stance which cannot be reduced back to the red color, while Fe(CN)E" had no effect. Reducing agents were mixed in their reactions. Acid plus zinc only partially reduced the pigment. Ascorbic acid had no effect. sodium hydrosulfite (Na2320.) had the most prompt reaction, completely removing the red color. Reduction by this method did not result in a colorless solution, however. The solution turned a lemon yellow when it was concentrated and a yellow-green when it was more dilute. Before leaving the subject of chemical reactions, several peculiarities must be noted. The red pigment when formed in culture is wine red which turns a very slight violet on the addition of acid. A strong light must be used to observe this or the color change, which is so very slight, is likely to be missed. Weak solutions, as observed on complex media, do not demonstrate this readily. Base has no effect on the color regardless of the normality. When allowed to stand overnight in the presence of one normal strong base, the red pigment lost its characteristic red color and became yellow-orange. This occurred only to a very slight extent in NH40H at one normal. The addition of 108 acid caused the violet color, normally associated with the acid form,to appear. Addition of base caused the yellow-orange color to reappear again. These new acid-base color changes were also characteristic of neutral red except that the basic color was more yellow with this compound. Reduction of the new form of the pigment with N823204 still resulted in the lemon yellow color. The indicator properties were restored on shaking with air. Below is a schematic outline of the conditions so far observed: I N8232O4 reduction H+ pH 3 lemon yellow< s.Pigment as isolatedzs —4*violet oxidation Red with red fluor- OH‘ no F air escence (F) IN N H 12-2 hours NaQSQOQ Alreduction . HI pH 5 Lemon yellow ‘ ,Yellow-orange pig-{ 5 violet oxidation ment, orange F OH' no F air Apparently the exposure to 1N NaOH caused some group to split off of the pigment molecule which allowed it to become an indicator. Apparently the group which splits off, in some manner prevents expression of the basic property or color. whether this is a complex with Mg or some other group is as yet unknown. The color of the pigment as it is isolated is probably that which occurs near the pH of the color change- 1.e. pH 3-4. None of the compounds so far described are soluble in chloroform which would tend to eliminate such pigments as 109 pyocyanine and its oxidation product, l-hydroxyphenazine (pyoxanthose). Furthermore, the color changes, although reminiscent of l-hydroxyphenazine and chlororaphine, are not identical to these. It demonstrates certain prOperties of chlororaphine in that the fully reduced chlororaphine is orange-yellow. When a sample is chromatographed on paper it is important that the pigment be pure since various components of the medium tend to retard or hold back its progress, especially ninhydrin reacting substances. When pure, with 1N NH4OI as the solvent, the red pigment has an R1. value of 0.68 while the basic form of the pigment after treatment with NaOH has an Rf of 0.27. The use of the spectrOphotometer to determine an absorption spectrum is not new, and it can result in a very characteristic 'curve frequently identifiable only with a particular compound. It might be noted here that the pure pigment can be diluted and the relative concentration is still prOportional to the Optical density reading (Figure 8) for most of the scale. From Figure 9 one can see that the red pigment under study, in its pure isolated form, has absorption peaks (at pH 8.0) at 51C, 592, 281, and 254 while in acid these are 558, 580, 287, and 255. The peak at 281-287 may be due to a hydroxy- phenyl group such as found in tyrosine. The peak at 580-592 may be due to the phenazine ring as seen in Figure 10, which shows the Spectrum of phenazine N§§ in concentrated acid which is N4¢ fully ionized and in hexane in which it is unionized. Base 110 then might shift the molecule to an unionized form and cammquently to shorter wave lengths. Further close relationships to the phenazines may be seen in Figure 11 which shows the acid forms of neutral red, pyo- cyanine and the red pigment (after treatment with NaOH) and also in Figure 12 which compares the red pigment at pH 8.0 (before NaOH treatment) with safranine 0 and acid pyocyanine. Figure 15 compares the basic forms of the red pigment (after NaOH treatment), neutral red and pyocyanine. From these figures one can see the similarity of absorp- tion,at nearly the same wave lengths, of neutral red, acid pyocyanine, and the red pigment; and the similarities of pyo- cyanine and the red pigment in the 580-590 mu area. The die- similarities of these compounds, the basic color of pyocyanine and the lack of a band at 590 mu for neutral red must also be noted. The Similarities of the absorption bands of basic neutral red and basic NaOH treated pigment are inescapable, indicating the possibility of a phenazine nucleus for the red pigment. Figure 14 shows the spectrum of the red pigment (not treated with NaOH) and the reduced form of this same pigment (reduced with Hagsgog). One other piece of evidence, especially for the identi- fication of the organism, must be produced. The culture obtained from from the National Collection of Type Cultures, London V5085 gs, aeruginosa variety erythrogenes and called #8 in this study, produced a red pigment almost identical 111 to that of the organisms studied here. Using the same medium, method of culture and procedure of pigment isolation, a red pigment was obtained in pure form. It gave an absorption curve identical to that of culture 17-1 (Figure 15). Other prOperties of the pigment were identical. Below is summarized some of the principle characteristics of the two pigments: Characteristic 17-1 8 Absorption peaks 590,515 590,515 Color as isolated-Acid violet violet -Base red red UV fluorescence -Acid none none as isolated-Base red red NaOH treated -Acid violet violet -Base orange-yellow orange-yellow UV fluorescence -Acid none none NaOH treated -Base orange orange Rf in iN NH.0H ' Red (as isolated) .68 .68 Yellow ( NaOH treated) .27 .25 Reduced color lemon-yellow lemon-yellow It was concluded that the red pigments of cultures 17-1 and 8 were identical and that the organisms were the same, i.e. Pseudomonas aeruginosa var. erythrogenes (Gessard). V Gh ZRAL SUMMARY The amino acid, carbohydrate and mineral requirements for the production of a red water soluble pigment (pyorubrin) by an organism which has been shown to be identical with.§§, aeruginosa var. erythrogenes have been determined. It was found that alanine and glycerol were the most efficient in producing the red pigment, while NgSO. and KgHPO‘ were also required, the former in high concentration and the latter in a very low concentration. The final medium decided upon was: DL-alanine 1% MgSO..7H20 0.5 glycerol 0.5 K2HP0. 0.01 pH 7.0 This medium produced large quantities of pigment and was the basis for pigment production in isolating and character- izing the pigment. The pigment was isolated by a series of complex extrac- tbns and column chromatography which resulted in an isolated Pigment as pure as available tests could indicate. The empir- ical determinations indicated 46.65% ash which could either be contamination of the pigment with residual NgSO. or a complex.between the pigment and Mg. Evidence of the presence of Mg could be seen in the formation of a floc in the presence 0f strong base (but not in weak base), however, this treatment a180 altered the pigment somewhat, so that the evidence is inconclusive. 113 In the presence of NaOH for 12-24 hours, the pigment was altered in such a way that the basic form was now yellow instead of its usual red, while the color of the acid form remained the same (absorption data indicated a slight shift toward the shorter wave lengths giving a slightly more red and less violet color although it remained the same to the eye). The pH at which the pigment changed color remained the same after NaOH treatment as it was before, 5-4. The only difference was the cplor. When freshly isolated the pigment was wine red above pH 4 and violet below pH 5. After NaOH treatment it was yellow above pH 4 and violet below pH 5. These changes indicate the possibility of some group (Mg, amide, ester, etc.) which masked the basic reaction by holding the portion of the molecule involved in the color change in a form which can only express a neutral reaction i.e. cannot take part in the shifting of double bonds required to produce the basic color. This reaction can be observed in the unextract- ed culture if the normality of the base is increased and the time extended. The similarities between the absorption spectrum of the pigment under various conditions and those of various phen- azine derivatives has been pointed out and it is the authors contention that this red pigment (pyorubrin) is another Variety of the phenazine pigments which occur quite widely in this genus of organisms. The structure and positive identi- fication of its phenazine nature remains to be worked out. It would not be too suprising to find that the green fluores- cent pigment, produced by most species of pseudomonads, is 114 also a phenazine derivative. This view was also expressed by Hugo and Turner (1957) who pointed out that its acid absorption peak was at 570 mu.(also Turfitt 1957) which is somewhat in agreement with the phenazine curve in hexane and HC1(Figure 10). The author's view on the sequence of events observed in a culture of this organism is as follows: The organism forms a yellow,water soluble, chloroform insoluble pigment which may become grass green. This latter pigment is only very weakly soluble in chloroform (this is not like pyocyanine) when in high concentration. This green pigment may be on the the sequence leading to the red pigment or it may be a side reaction. It never occurred ina concentra- tion Sufficient for isolation and study. The red pigment apparently is normally formed directly from the yellow, and if a green pigment is on the sequence it is So transitory as to not be seen in most cases. The yellow itself may be tran- sitory Since it is rarely seen in the l6-l,2 and 17-l,2 cultures While it is much more prominent in the English fi8 culture. Addition of a small amount of base (NH.0H or NaOH) seems to SPeed up the transition from the yellow to the red; however, the true effects remain unknown. The red pigment may be the result of an association with an kg ion to form a complex or Perhaps some group masks the portion of the molecule involved in the change. The pigment has been characterized to some extent, most n0tably in determining its absorption Spectrum. Cne other 115 Characteristic which may be of importance is the fact that this pigment can and does act as an electron acceptor with an electromotive potential at pH 8.0 of +0.215 volts which puts it in a class with cytochrome c. 116 117 118 0 0. 0. hufiudoa Hcowaflnnvu Wave Length mu 119 120 . 7H30 Berlin. 1951) 124 125 126 127 and 1957) 128 LITERATURE CITED Acklin, 0. 1925. Zur Biochemie des Bacterium pyogyaneum. Biochmm. Z. 164:512-570. Aoki, K. 1926. Agglutinatorisch:Einteilung von Pyocyaneus- Bazillen welche bei verschiedenen Menschenerkrankungen nach- geweisen worden. Zent. Bakteriol. Parasitenk. Abt.I Orig. 98:186-195. Arnaud, A. and Charrin, A. 1891. Recherches chimiques sur les secretions microbienne. Transformation et elimination de la matiere organique azoteé par le bacille oggocyanigue un milieu le culture determines. Compt. ran -75 Aubel, E. and Colin, H. 1915. Nature de l'aliment azoté et pro- duction de pyocyanine par le Bacille pyocyanique. 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