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The taxonomy and nutrition of the genus Pseudomonas Burton, Margaret Olive 1947

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THE TAXONOMY AND NUTRITION OF THE GENUS PSEUDOMONAS -by-Margaret Olive Burton A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE in the DEPARTMENT OF DAIRYING The University of B r i t i s h Columbia Apri l 1947. \ ACiOTOWIEDGMENT I wish, to extend my sincere thanks to Dr. B. A« Eagles for his guidance and encouragement during the preparation of this thesis, and to Dr. J. J, R. Campbell for his valuable criticisms and suggestions i n the experimental work, I am grateful also to the National Research Council of Canada for the grant made available for this work, and to those who so kindly contributed cultures for this study. TABLE OF CONTENTS Page No* INTRODUCTION 1 PART I THE TAXONOMY OF THE GENUS PSEUDOMONAS 2 REVIEW OF LITERATURE 2 The Genus Pseudomonas 2 Classification Within the G-enus Pseudomonas 7 Distribution of Pseudomonas Bacteria in Nature 10 EXPERIMENTAL 13 Methods 13 Cultures employed 13 History of species being tested 14 Tests applied 20 Results 25 DISCUSSION 26 SUMMARY 29 PART II THE AMINO ACID REQUIREMENTS FOR PYOCYANIN PRODUCTION BY PS^ AERUGINOSA 30 METHODS 31 EXPERIMENTAL 32 SUMMARY 41 PART III THE MINERAL REQUIREMENTS FOR PYOCYANIN PRODUCTION 43 METHODS 44 EXPERIMENTAL 45 Essential Salts 45 Optimum Concentrations of Salts 46 Essential Ions 49 SUMMARY 51 BIBLIOGRAPHY 5 3 THE TAXONOMY AMD NUTRITION OF THE GENUS PSEUDOMONAS  ABSTRACT Part I A detailed study of 35 representative microorganisms of the genus Pseudomonas has been made. The results obtained have been compared with those recorded by other workers em-ploying the same species. As a result of this investigation, a new cl a s s i f i c a -tion of the members of the genus has been proposed. This classification i s based on the a b i l i t y of the organisms to re-duce nitrates in synthetic medium, lipolyse fat, liquefy gelatin, and ferment carbohydrates. Part II A study of the amino acid requirements of Pseudomonas  aeruginosa for the production of the blue pigment pyocyahin has been made. A synthetic medium consisting of dl-alanine or glycine at 0.4% concentration combined with 0.8% 1-leucine, 1.0% glycerol, and a salt mixture has been shown to be the most suitable medium for pyocyanin production by Ps. aeruginosa Part III The salts essential to pyocyanin formation have been ascertained and their optimum concentrations i n a synthetic medium established. The following mineral ions were found to be necessary for the production of the pigment: K, P0 4, Mg, S0 4, and Fe. M. 0. Burton. 1 THE TAXONOMY AND NUTRITION OF  THE GENUS PSEUDOMONAS* INTRODUCTION The present investigation on the taxonomy of the genus Pseudomonas was undertaken with the object of attempting to find a more satisfactory basis for the classification of the species within this genus, A study of the nutritive re-quirements of Pseudomonas aeruginosa and their relationship to pigment formation by thi s species was also carried out. Part I embodies a review of the literature on the genus Pseudomonas, the species within the genus, and the dis-tribution of these organisms in nature, A brief history of some of the more important tests used in differentiating the species i s included and the results obtained employing these tests with known cultures of Pseudomonas are compared with those recorded by other workers. Parts II and III are studies on the nutritional re-quirements of Ps, aeruginosa for the production of the blue pigment, pyocyanin. & This study was carried out under a grant from the National Research Council of Canada, PART I THE TAXONOMY OF THE GENUS PSEUDOMONAS  REVIEW OF LITERATURE The Genus Pseudomonas In 1894, Mlgula (60) proposed the genus name Pseudomonas for those organisms having polar f l a g e l l a , and which rarely formed endospores. The genus was not defined u n t i l 1895 when the following description was published by Migula (61): "Short or long rod-shaped cells which occasionally form small filaments and are actively motile with polar f l a g e l l a . The numbers of f l a g e l l a present at one pole varies with the species between one and ten!7 and i s most frequently one or between three and six. The formation of endospores occurs however, only with a few types. n The f i r s t species assigned to the genus was the "blue pus" fluorescing organism^1 Bacillus pyooyaneus; Gessard (38), which Migula renamed Ps. pyocyaneus. This organism i s now called Ps, aeruginosa, A second species of Pseudomonas was described by Flugge i n 1886 (26) under the name Bacillus fluores oens-llquefaoiens. The name of this organism was later changed to Ps. fluoresceins. The a b i l i t y of organisms of thi s genus to produce 3. a yellow-green fluorescent pigment was emphasized by Migula. It became, therefore 1^ the tendency to group together i n this genus those bacteria excreting a green diffusible pigment which was capable of causing the medium to fluoresce. Kruse i n 1896 (47) however; stated that the members of this group had only the fluorescing a b i l i t y i n common, and otherwise f e l l into widely varied groups* He believed that the a b i l i t y of organisms to fluoresce made them appear similar, but did not necessarily mean that they had more than that property i n common. On the other hand, Ruzicka (7E) i n 1898 believed that there were i n the fluorescent group, bacteria closely related to either the semi-pathogenic organism pyocyaneus or fluorescens-liquefaciens . Only a detailed examination of large numbers of fluorescent bacteria from various sources could disclose whether or not these bacteria form a homogeneous group. Conn i n 1909 (16) stated that the fluorescent bacteria are char-acterized by having a single polar flagellum. Eisenberg i n 1914 (23) found that with seven strains of fluorescent bacteria, there were intergrading,characteristics which made i t d i f f i c u l t to separate them into species and varieties. Tanner in 1918 (87) studied 100 strains of fluorescent bacteria from water. He found that a l l cultures agreed with regard to the production of fluorescent pigment, formation of acid in glucose broth, absence of diastatic action upon potato starch, negative indole formation, and non-fermentation of sucrose and lactose. The fluorescent bacteria in water as indicated by this study, constitute a homogeneous group of bacteria. In 1920, Winslow et a l (97) described the genus Pseudomonas as follows: "Rod-shaped, short, usually motile by means of polar fl a g e l l a or rarely non-motile. Aerobic and facultative. Frequently gelatin-liquefiers and active ammonifiers. No endospores. Gram stain variable, though usually negative. Fermentation of carbohydrates as a rule not active. Frequently produce a water-soluble pigment which diffuses through the medium as green, purple-; brown, etc." Within the genus Phytomonas (Bergey) there are a number of plant pathogens differing from other members of their genus by the production of a water-soluble, green pig-ment known as fluorescin. Ac cording to Burkholder i n 1930 (5), these organisms represent a natural closely-related group with a distinct similarity to members of the genus Pseudomonas of Bergey. Clara in 1934 (12) studied 27 isolates of green fluorescent bacteria which were pathogenic to plants. These organisms were found to be non-spore forming rods having one to several polar f l a g e l l a , and showing weak fermentation of carbohydrates. Thus a striking resemblance to members of the genus Pseudomonas was found. With the species studied i n this investigation, no separation could be made of the two genera other than on the basis of pathogenicity to plants. Ps. fluorescens furthermore showed tendencies to break down this separation by causing infection of the f r u i t of the pear, Tu r f i t t in 1936 (92) studied the chromogenic function of species of Pseudomonas i n relation to classification. He believed that so distinctive a property as the production of a diffusible green fluorescent pigment i n the culture medium would be a very convenient basis for classification of the respective organisms provided that the organisms are not widely differentiated by their morphological and cultural characteristics, that the chromogenic function i s sufficiently invariable, and that the green pigment i s of the same chemical composition i n each case. In addition to the three stock cultures Ps,  pyooyanea, Ps, fluorescens-liquefaciens, and Ps. fluorescens  non-llquefaciens. he studied 100 fluorescent bacteria from s o i l , water, faeces, etc. He showed that these organisms were alike culturally and morphologically, and that their chromo-genic characteristics were remarkably constant on certain specified media. In 1937 (92) the same worker isolated the fluorescent pigment from cultures of the organisms mentioned in his 1936 paper. Considerations of properties and of analytical and spectrophotometry data point to the identical nature of the green pigment obtained from the various organ-isms selected. Therefore, on the basis of morphological similarity, identity of pigment and constancy of pigment pro-duction, T u r f i t t recommended the classification of a l l green fluorescent bacteria within the genus Pseudomonas. It i s necessary to remember, however, that he did not study any 6. known species of the genus Phytomonas. However, he did be-lieve that further work on the fluorescent plant pathogens would probably result i n their being placed among the Pseudomonas. The description of the genus Pseudomonas by Bergey in 1939 (3) i s a summary of the classifications reviewed up to this time. "Principally water and s o i l bacteria producing a water-soluble pigment which d i f -fuses through the medium as a green, blue or yellow-green pigment. Motile or non-motile. Gram negative." Loohhead i n 1943 (54) stated that the red chromo-genic halophilic bacteria, Serratia salinaria and Serratia  cutirubra, should be included i n the genus Pseudomonas. These organisms, because they have polar flagella, and lack the a b i l i t y to produce acid from carbohydrates, should be separated from the Serratia in spite of their having a red pigment. However, the inclusion of red-pigmenting halophilic bacteria i n the genus Pseudomonas naturally requires a broadening of the generic description. In the same year, Starr and Weiss (83) proposed changing the generic name of the plant pathogen species from Phytomonas to others now considered more appropriate. To the genus Pseudomonas, they proposed the addition of the green-fluorescent pigment-producing phytopathogenic bacteria. Tobie i n 1945 (89) suggested a biochemical basis for classification of the genus Pseudomonas* Those rod-shaped bacteria which produce either pigments which are phenazine derivatives regardless of their colour, or water-soluble fluorescent pigments, or both, would comprise the genus Pseudomonas, Since these pigments apparently have a significant role in the l i f e of the bacteria which secrete them, the proposed classification would be a rational one from a biochemical and physiological point of view. Classification Within the Genus Pseudomonas Even before 1900, the literature contained descrip-tions of more than 50 species of bacteria which produced a green fluorescent pigment; Such bacteria were originally assigned to various widely separated groups and after 1895 hafl a l l to be classified within the genus Pseudomonas of Migula, It was discovered that many of the described species were identical, while the characteristics of others graded together so imperceptibly that differentiation was d i f f i c u l t . In 1900, Niederkorn (66) studied 15 strains of fluorescent bacteria i n order to discover satisfactory methods for differentiation. He believed there to be only two con-stant forms, 1. Those producing the blue pigment pyocyanin and fluorescin, 2, Those producing fluorescin only. He considered the others to be variants of these two, Jordan i n 1903 (45), studying 58 fluorescent bacteria from river water, found that 33 of the isolated 8. organisms were able to liquefy gelatin while the remaining 25 did not possess this power. In other characteristics the liquefying and non-liquefying forms were found to be similar. None of the cultures formed the blue pigment pyocyanin. Jor-dan therefore would classify the species of the genus Pseudo-monas as follows: 1* B, pyocyaneus 2. fluoresoens-liquefaciens 3, fluorescens non-liquefaoiens In 1912, Edson and Carpenter (22) isolated and studied 42 green-fluorescent baoteria occurring in maple sap. Along with these unidentified species, they tested the following: Ps. ulba, Ps. fluorescens, Ps. longa, Ps. mesen- terica , Ps, tennuis, and Ps. putrida. They found that no sharp line of differentiation could be drawn between these fluorescent forms. It was believed that the fluorescent bacteria of maple sap, as well as the so-oalled known species l i s t e d above should properly be recognized as strains of the liquefying and non-liquefying varieties of Ps. fluorescens. Tanner in 1918 (8ft) studied 100 fluorescent bacteria from water. On the basis of gelatin liquefaction, nitrate reduction, hydrogen sulphide production, indole formation, peptonization of casein, starch hydrolysis and fermentations, he was able to divide them into 27 groups. Haines i n 1938 (36) attempted to classify 29 Pseudo-monas isolated from egg rot. He stated that i t was d i f f i c u l t to devise a classification within the genus partly owing to the scanty literature and partly due to the lack of any pronounced characteristics of the organisms, Bergey in 1939 (3) based his primary division of the species on the number of polar f l a g e l l a . This characteristic i s a d i f f i c u l t one to determine accurately and has been shown with some organisms to vary with the method of culture (15). He l i s t s 31 species of Pseudomonas, In 1942, Reid et a l (70), examining the microflora of soils and plants, obtained 4000 strains of Gram negative fluorescent bacteria. In addition, they studied 100 stock oultures of Pseudomonas and Phytomonas. From these, 640 re-presentative cultures were selected, and their morphological and biochemical characteristics studied. Of the 640 cultures, 613 were found to be lophotriohous and 27 to be monotrichous. The latter were identified as Ps. aeruginosa. The 613 lopho-triohous organisms were separated further into two groups. I Gelatin was liquefied by 603 cultures. These organisms conformed to the description of Bact.  fluorescens-liquefaciens (Fliigge) although the group includes isolations of known species ob-tained from other labs. These species include: Phyt. tobaci Phyt. vignae Phyt. angulata Phyt. syringae Phyt. primulae Pseud, fluorescens Phyt. cerasi II Gelatin was not liquefied by the ten cultures which conform to the description of Bact. putidum (Fliigge). These organisms were a l l isolated from the s o i l , Seleen^and Stark i n 1943 (80), studying 199 green-fluorescent bacteria were able to divide their cultures into 14 groups on the basis of their a b i l i t y to grow at 5°C and 42°C, action on milk, gelatin liquefaction, and reduction of nitrates. The names Ps. aeruginosa, Ps. fragi, Ps. putida, and Ps. ovalls were attached to the groups which resembled Bergey»s descriptions of these organisms. In 1945, Munoz (64) studied a representative number of strains of Pseudomonas to determine whether or not there i s any serological relationship among strains of this genus. He found that the known strains of Pseudomonas could be easily differentiated by serological means. He concluded that sero-logical methods are essential for the identification and classification of members of genus Pseudomonas. Distribution of Pseudomonas Bacteria i n Nature An outline of the literature reporting the occur-rence of the members of the Genus Pseudomonas is given below. Although i t is not intended to be complete, i t does serve to indicate the widespread a c t i v i t y of this group of micro-organisms. Since Schroeter in 1872 (77) f i r s t isolated and described an organism producing a fluorescent pigment, this type of bacteria has been reported from many sources. Schmelck in 1888 (76) found that Pseudomonas were the predominating organisms i n gl a c i a l waters. Harrison (38) found fluoresoent 11. bacteria present in h a i l on two different occasions. Fuller and Johnson (29), Jordan (45), and Tanner (87) found Pseudo-monas to be one of the predominating types i n water. Conn, in 1910 (17), attributed certain cases of rancidity i n butter to the presence of fluorescent bacteria. Jensen (42), and Garrison and Hammer (30) also reported fluorescent bacteria i n butter. Thoni (88) reported the presence of fluorescent bacteria i n 15 samples of lemonade, and Conn (18) isolated large numbers of fluorescent bacteria from s o i l . Patrick and Werkman (68) reported four species of Pseudomonas isolated from diseased and normal snakes. They l i s t Ps. visoosaV Ps. jaegeri. Ps. smaragdina. and a new species, Ps. purls. Caldwell and Ryerson (7), while investi-gating the cause for death of reptiles used as controls in experimental work> repeatedly isolated a pathological organism of the genus Pseudomonas. The organism differed from any of the 31 species described by Bergey i n 1939 and from organisms thus far described in the literature. They named the new speoies Ps. reptilivorous. Goresline in 1932 (34) isolated a number of organ-isms from an experimental t r i c k l i n g f i l t e r receiving creamery waBtes. These organisms were distinctive in that they digested the agar medium upon which they were grown. Two of the organ-isms so isolated were Gram negative rods producing yellow-coloured growth and were motile by polar f l a g e l l a . As a survey of the literature showed that they had not been previously described, they were designated as two new species, Ps. laounogenes and Ps. segne. In 1939, Claydon and Hammer (13) obtained three samples of high quality butter which developed a skunk-like odor on holding at 21°C. The causative organism was isolated and described, and the name suggested for i t was Ps. mephltlca In 1941, Lee and Chandler (48) investigated the heavy bacterial growth i n oil-water emulsions used as lub r i -cants and cooling agents i n the cutting and grinding of metals This contamination was thought to be causing skin infections among the workers. One species of bacteria described as a gram negative, fluorescing rod with polar flagella', was found to be present almost to the exclusion of a l l others. This organism, a new species, was named Ps. oleovorans. Prior to extraction of rubber from the guayule plant, i t i s necessary for biological decomposition of the plant to take place (retting). Allen et a l (1) obtained from the retting shrub, four isolates of Pseudomonas which were capable of decomposing guayule resin. These organisms were classified as Ps. boreopolis. In the same year, Naghski et a l (65) studied the aerobic decomposition of crushed guayule shrub. The profuse microflora was tested for i t s biochemical reactions and the predominating organisms were identified. Three members of the genus Pseudomonas were isolated and identified as Ps. incognita. Ps. putida. and Ps. aeruginosa. 13. EXPERIMENTAL A thorough, study of the literature dealing with the characteristics of the genus Pseudomonas revealed a lack of uniformity i n the methods employed for purposes of c l a s s i f i -cation by different workers i n this f i e l d . For this reason, i t was considered desirable to subject as many representative known cultures of Pseudomonas as could be procured to a series of established routine tests with the object of correlating the results obtained with the published work of other investi-gators and i f possible to establish a system of classification based on the findings obtained. To this endj, 35 known cul-tures of Pseudomonas were selected for study. METHODS The following cultures were employed i n this investigation: Ps. aeruginosa - American Type Culture Collection 9027 Ps. aeruginosa - American Type Culture Collection 8689 Ps. pyooyaneus - V 21 Cornell University Ps* aeruginosa - R.M. University of B r i t i s h Columbia PsT aeruginosa - 256 * Ps. aeruginosa - 257 * Ps. aeruginosa - 260 .* Ps. aeruginosa - Pa Ps. fluoresoens - Ps. 21 Cornell University Ps. fluorescens - 949 * Ps. mildenbergii - American Type Culture Collection 598 Ps. mildenbergii - 795 J PsT mildenbergii - 598 a Ps. fragl - American Type Culture Collection 4973 Ps. fragi - 4975 * ^ £ These cultures were obtained through the courtesy of Dr. M. Scherago, Department of Bacteriology, University of Kentucky. 14. Ps. niferifaoiens - 1 £ Ps. nigrifaciens - 6 ^ Ps. nlgrlfaoiensi - 30 *| Ps. nlgrlfaolens - 9M ~~.  Ps. putrefaolens - 375 ** Ps. putrefaolens - EM_^> ^ Ps* putrefaolens - 8SS Ps. putrefaolens - 65A ¥s7 putrefaolens - 8SS(R) Ps. muoidolens - 4686 * 6 ,_, %g Ps. muoidolens* - 4687 J Ps. graveolens - 4683 A PsT grayeoiens - 4684 Ps. oavlae - B * Ps.. caviae - C * . P*s7 pavonaoea - 957 * . 557 riboflavina - 6 R 6151 "** PsT putida - 4359.* P"sT ovalis - 950 * A brief history of the species studied in this investigation i s presented below. Ps. aeruginosa A blue pigmenting organism was isolated by Schroeter i n 1872 (77) and named Bacterium aeruginosum. Gessard i n 1882 (32) isolated and described a similar organism which he named Bacillus pyooyaneus after the blue pigment pyocyanin. In 1895 Migula (61) assigned the organism to the genus Pseudo-monas and named i t Ps. pyocyanea. However j" Schroeter»s prior-i t y i s recognized today, and the organism is called Ps.  aeruginosa. A. These cultures were obtained through the courtesy of Dr. M. Scherago, Department of Bacteriology, University of Kentucky. Alt These cultures were obtained through the courtesy of A. H. White, Division of Bacteriology and Dairy Research, Department of Agriculture; Ottawa, Ont. A&fr The culture :of Ps. riboflavina was very kindly sent by H. B. Woodruff, Head Research Section of the Microbiologi-^aj Dept., Research Laboratory, Merk and Co. Inc., Rahway, 15. Jordan in 1899 (44) separated the species Ps.  aeruginosa into four varieties: (a) pyocyanogenio and fluores-cigenic, (b) pyocyanigenio only, (o) fluorescigenio only, and (d) non-chromogenic. However, Header et a l i n 1925 (57) con-cluded from their investigation that a l l t y p i c a l strains of Ps. aeruginosa produce three water-soluble pigments, the f l u -orescent pigment, pyocyanin and pyorubia (red pigment). In 1918, Brown (4) isolated a pseudomonas which was the causative agent in a disease of lettuce. It was charac-t e r i s t i c of the genus, and produced a bluish pigment i n addi-tion to fluorescin. She thought i t to be a new species and therefore named i t B. marginale, A similar isolant was ob-tained by Paine and Branfoot i n 1924 (67), Mehta and Berridge (58) studied the morphological and cultural characteristics of B. pyocyaneus and B^ marginale and found them to be identical i n every respect. Clara in 1930 (11) isolated the causal organism of a new bacterial leaf disease of tobacco and named i t Phyt*  polyoolor. Elrod and Braun in 1942 (25) proved the identity of Phyt. polycolor with Ps. aeruginosa and in addition, showed them both to be pathogenic for small animals and the tobacco leaf, Ps, fluorescens This species of Pseudomonas was originally named Ps,  fluorescens-liquefaciens by Fliigge in 1886 (26) and was sub-sequently called Bacterium fluorescens by Lehmann and Neumann i n 1896 (49), However, when the organism was placed in the 16, genus Pseudomonas, i t was named Ps, fluorescens, Ps, fluorescens has been reported as being pathogenic to both plants and animals. In 192a (39) and 1933 (73) i t was recorded that a specific discolouration of halibut was due to the a c t i v i t y of the chromogenic bacterium, Ps, fluorescens. In 1934, Clara (12) found striking similarities between Ps, fluorescens and the fluorescent plant pathogens. Aside from pathogen!cityV no separation could be made between Ps, fluo- rescens and fluorescent Phytomonas, Ps. fluorescens showed tendencies to dissolve even this difference by causing infec-tion in the f r u i t of the pear, Ps, mildenbergii Mildenberg in 1922 (62) isolated an organism causing blue milk. Bergey, in 1923 (2) gave the name Ps, cyanogena to the species. However, because of the possibility of confusing the organism with Ps, oyanogenes (37) Bergey later changed the name to Ps, mildenbergii, Ps, fragi In 1902 Eichholz (24) isolated and described an organism from milk which had been held several days at 3° to 7°C. He named this organism, which produced a strawberry-like odour, Bacterium f r a g i , Griiber (35) in 1902 isolated a similar organism which he named Ps. fragariae• Hussong, in 1932 (40), concluded that the organisms were identical and belonged to the genus Pseudomonas. He thus named the species Ps. f r a g i , Hus-song et a l , i n 1937 (41), were able to isolate the organism from dairy products having a May apple odour, or i n some cases from rancid products, Ps, nigrifaciens In 1940, White (95) isolated an organism causing dis-colouration of print butter. He described the organism and assigned i t to the genus Pseudomonas with the name Ps, n i g r i - faciens. In a later investigation (96), he stated that a l l attempts to isolate Ps, nigrifaciens from water and s o i l had been unsuccessful. The organism was recovered in some creameries from swab cultures of the f l o o r , drains, etc. In isolating and culturing Ps, nigrifaciens, i t i s necessary that the media contain 1,5$ s a l t , and that the i n -cubation temperature be between. 15° and 20°C, These conditions are particularly important i f the black pigment is desired, Ps, putrefaciens. In 1931, Derby and Hammer (21) isolated from surface taint butter an organism which was tentatively designated as Aohromobacter putrefaolens. Further studies on the organism by Long and Hammer (55) i n 1941 revealed that i t belonged to the genus Pseudo-monas, and the name therefore was changed to Ps, putrefaolens, These investigators were able to isolate the organism from water, s o i l , and raw milk supplies. In 1942, Wolochow et a l (98) published some further data on a large number of strains of Ps. putrefaolens isolated from butter and water. Both Woloerhow et a l , and Long and 18 Hammer emphasized the variations i n the fermentative a b i l i t i e s among the different strains of this species* Ps* graveolens and Ps* mucldolens In 1932, Levine and Anderson (51) investigated the cause of mustiness in eggs* The organisms responsible were isolated and among them were two new species of Pseudomonas* These organisms were described, and named Ps. graveolens, and Ps. mucldolens* Ps. caviae In 1936, Scherago (74)(75) obtained isolates of this species from a young guinea pig which had died of a sep-ticemic disease. He was then able to reproduce the disease in young guinea pigs by injecting strains of the isolated organ-ism (Ps. caviae) into the animal. The organism i s reported to resemble Pa. aeruginosa i n morphology and staining reactions except that Ps. caviae i s encapsulated. However, i t differs from Ps. aeruginosa in i t s cultural and biochemical character-i s t i c s . Ps* pavonacea In 1926, Levine and Soppeland (52) isolated an organism from creamery wastes which was dis t i n c t l y green on solid media', but showed no fluorescence* Because of the variegated colours of growth obtained on solid media,' the species name Ps. pavonacea was suggested* Ps* riboflavina Foster i n 1944 (27) discovered a localized area of s o i l i n Rahway with an uncommonly high content of riboflavin. Small portions of this s o i l were added to flasks containing a solution of 0.1% riboflavin and a small amount of phosphate and magnesium sulfate. A variety of bacteria, yeasts, and fungi were isolated and one organism was singled out for detailed study. This culture produced a bright yellow appear-ance when grown on riboflavin-containing agar. Further i n -vestigation revealed that the yellow appearance of the growth on riboflavin was due to a l o c a l concentration of a yellow crystalline substance identified as 6, 7-dimethyl alloxazine (lumichrome) • The a b i l i t y of this particular organism to oxidize riboflavin to lumi chrome was believed to be a distinctive enough physiological property to warrant designating the organism as a new species. The name Ps. riboflavinus was originally used, but in a later publication (28) was changed to Ps. riboflavina. Ps. putida This organism was originally named Bacillus  fluorescens-putidus by Flugge in 1886 (26). However, i t was renamed Ps. putida by Migula i n 1895 (61). Ps. ovalls An organism was isolated in 1896 by Ravenel (69) from cultivated s o i l at a depth of two feet. He gave i t the name Bacillus fluorescens ovalis. Chester i n 1901 (10) renamed the organism Ps. ovalis. In 1926, the organism was isolated by Levine and Soppeland (52) from "buttermilk and skim milk, Ps,  ovalis was among those bacteria isolated by Stuart and Goresline (85) from fermenting egg white. The organism was also iso-lated by Seleen and Stark i n 1943 (80), The morphological and biochemical tests employed i n this investigation are outlined below, Flagella Stain The method of Knaysi (46) was employed. The smear i s covered with a tannic acid mordant for ten minutes, washed, and flooded with carbol fuchsin. As soon as the dye i s added, the preparation can be observed under the microscope as a wet mount. Production of Fluorescent Pigment The a b i l i t y of Pseudomonas to form the yellow-fluorescent pigment varies with the medium employed. There-fore to determine the a b i l i t y of an organism to fluoresce, a medium capable of stimulating fluorescin production must be obtained, Sullivan i n 1905 (86) postulated the simplest synthetic medium for the production of fluorescin: Asparagine 1.0$, MgS04 0.02$, K 2HP0 4 0,1$. Georgia and Poe (31) agreed with Sullivan that some nitrogen-containing organic compound such as asparagine or ammonium succinate is necessary for fluorescin production. Their medium differs from S u l l i v a n ^ only i n percentage composition. Lourie i n 1945 (56) showed that a modification of these two media was superior for the 21. production of fluorescin. 0.3$ 0.05$ 0.05$ 0.2$ 2.5% Lourie^ medium was used for the detection of fluo-rescin i n this investigation. As a check, another medium capable of supporting the production of fluorescence was employed. This medium was composed of casamino acids (0.5$) and glycerol (1.0$)• medium and the casamino acid medium, and incubated at 30°C for two weeks. Periodic observations for fluorescence were made under ordinary as well as ultra-violet l i g h t . Litmus Milk Cultures growth was established at 30°C, were incubated at room tem-perature. The cultures were observed periodically up to six weeks. Nutrient Gelatin Stab Cultures temperature. The cultures were observed up to a period of six weeks. Methyl Red and V.P. Tests The standard tests were employed. Hitrate Reduction duction by microorganisms has been to detect the presence of The organisms were cultured on Lourie*s synthetic Tubes of litmus milk were inoculated, and after The gelatin deeps were inoculated and held at room The standard procedure for detecting nitrate re-n i t r i t e i n cultures growing on nitrate agar. When n i t r i t e i s not found present, i t is assumed that nitrate has not "been reduced. However, Zo Bell i n 1932 (99) reported that care-f u l scrutinization of a large number of cultures under d i f -ferent conditions showed that many bacteria including certain strains of Pseudomonas destroy the n i t r i t e s as rapidly as they are formed. Only at irregular intervals does the con-centration of n i t r i t e become sufficiently high to give a positive test even with the most sensitive methods. This worker recommended that a test for nitrates invariably be made i n conjunction with that for n i t r i t e when the latter is negative. Meiklejohn i n 1940 (59) studied nitrate reduction by two species of Pseudomonas. She employed a synthetic liquid medium and tested daily for the presence of nitrate, ammonia, n i t r i t e , and nitrogen gas. The medium had the following composition: KN03 0.1% Glucose 0.35% Neutral basal solution: - K 2HP0 4 0.05% K H O P O A 0.05% CaClg 0.01% NaCl 0.01% MgS04 0.03% FeCls traoe The basal solution and nitrate were autoclaved together, the glucose added, and the pH adjusted to 7.0 or 8.0. The com-plete medium was measured out into the flasks to be used i n the experiment and steamed for one hour. In this investigation, two procedures for determining the reduction of nitrate were employed. 1. The organisms were grown on nitrate agar and the production of n i t r i t e was detected in 48 hours with sulfanilic acid arid^napthylamine. 2. The organisms were cultured i n 50cc quantities of Meiklejohn*s synthetic medium. Species i n -capable of growth on this medium were supplemented with added peptone. Daily tests were made for the presence of n i t r i t e and, i f negative, for nitrate. The production of nitrogen gas waw observed by employing test tube cultures of MeikleJohn's medium containing gas vi a l s . Lipolytic Power Comparatively l i t t l e i s known or recorded about microbial act i v i t y on fats. In Bergey 1s manual, rarely i f ever i s oxidative or hydrolytic activity on fats mentioned, and apart from a few general and widely scattered references, there appears to be no place i n the literature where informa-tion regarding the action of specific baoteria on fats i s recorded. The nile-blue sulfate agar of Turner (93)(94) which seems to be one of the most satisfactory methods for the detection of lipase-*producing bacteria, was employed in this investigation. Carbohydrate Fermentation The basal medium used in fermentation tests by Pseudomonas has received considerable attention in the l i t e r a -ture. Two per cent peptone has been employed extensively i n fermentation work with these organisms. However, Sears i n 1916 (78) reported that cultures of B^ pyooyaneus growing on 2$ peptone showed a gradual increase in free ammonia after the 24, f i r s t 24 hours. De Bord i n 1923 (20) showed that Ps. aeru- ginosa i n 2% peptone broth containing 1% glucose oxidized a l l the sugar without acid production. Therefore, the hydrogen ion concentration may not be an index of the destruction of glucose in bacterial cultures. Sears and Gourley in 1928 (79) concluded that the acid end products of the reaction were masked by the products of nitrogen metabolism. Consequently, in order to avoid this masking action these investigators concluded that i t should only be necessary to reduce nitrogen metabolism to a minimum. Therefore, when they employed 1% or less peptone and 1% glucose, the pH dropped and remained on the acid side. However, when other sugars were used in place of glucose, acid production was not evident even though many of the sugars were u t i l i z e d . Burkholder i n 1932 (6) studying the carbohydrate fermenting a b i l i t y of the Phytomonas, u t i l i z e d synthetic basal medium to which the various carbohydrates were added. This medium contained no amino acid which could be converted into ammonia. The medium employed was that recommended i n the Manual of Methods for Pure Culture Study of Bacteria; - (81): N H A H O P O A 0.1% MgS04 0.02% KCI * 0.02% Sugar 1.0% Agar 1,5% The pH was adjusted to 7.2 and the indicator brom cresol purple added. Stein and Weaver in 1942 (84) recommended the use of a synthetic medium containing carbohydrate as the only source of energy and carbon as the best routine method for the deter-mination of carbohydrate u t i l i z a t i o n by members of the genus 25. Pseudomonas* In this investigation, the medium, and procedure re-commended by Burkholder was employed. The sugars were added aseptically at lf0 concentration to the ster i l e synthetic basal medium. Incubation was at 30°C. RESULTS Tables I to YI (Part I) record the results obtained with the 35 organisms i n the above tests along with the findings reported by other investigators employing the same species and similar tests* Table I Biochemical Reactions of Pseudomonas aeruginosa GELATIN AUTHORS PIGMENTS LITMUS MILK LIQUE- M.R. . 7.P. NITRATE I . I P 0 L - CARBOHYDRATE FERMENTATIONS* FACTION REDUCTION YSIS n CD CD CD a i H CD CD *C <D CD CD CD CQ CO •H O 43 CD CO a 6 H CO CD H H CD CO CO O O CO PI CO cd a •H CD 13 CD © g O O CO O O CD CQ CD O CD CD CD ; O i PI Pi TH 4> O a CD 0 H a +> CD + > T j U CD fl O +> 43 CO O CO 4> CO CO CO p| d i r f ^ r l r l •H s» U O p H 0 cd O -H CD Oft CD co •H Pi •H •H O 4> O O OO O i H •H I U O O O N .Q 0 O p ccf •p U P U g f-l O 0 & PS .PI 0 0 , cd . u +> +> <M rH +3 U -H H •i-t 0 P 1-4 cd M Pi 43 <d +> h 4 3 co >» H cd cd p| P P rH O H O SH p \ M cd H H |H H >> H CD 10 0 rH CD •H CD -rH O •H aj rH l» 1^ O cd cd U H cd : P cd cd cd Pi I CD 4> Cd CD ! CD CD « <4 O c5 CQ m n CK O CQ 3 a rt H l O CQ CQ O ^ 1 i I , | 1.Present I n v e s t i -T~"" "'• • i : i 1 g a t i o n 1 J 9027 + + + + + - - + + + + + + + + — - + + + , + - — — mm .0 — mm ' mm 4 8689 + + 4 + + - - + + + + + + + + - _ + + + ; + - ~ — ~ - - ' ~ 4 V21 + + 4 + + - - + + 4 + + + + + - - + + + i + - 4 R . M . + + + 4 4 - - + + + + + 4 + + - - + + + ! + - - - 4 256 + 4 + + - - + + + + + + + + - - + + + + + - - - — — = — — ~ + 257 — + 4 + + - - + + + + + + + - - ' + + + + + - — - - — — — - — + 260 + + + + - - + + + + + + + - - + + + + ! + - + Pa + + 4 4 4 - - + + + + 4 + 4 + - - + + + 4 ; + - 4 2.Mehta & Berridge (58) 1924 Ps. pyocyaneus 1 . B. Marginale + + 4 + + + — + + J . C l a r a (11) 1950 Ps. P o l y c o l o r + + + 4 + + + + + + 1 4.Lehmann & Neu-mann (50 )1931 + + 4 + + + 5 . C l a r a (12)1934 + + 4 + + + + + + — + + 4 + 4- - _ - 4 6.Topley & Wilson (90) 1936 + + 4 + 4 + - - - 4 - - - - 0 — 7.Bergey (3)1939 + + + + + + + + - + -8 . C a s t e l l & Ger-r a r d (9)1941 C a s t e l l (8)1941 + 9 . S t a r r & Burk-hol d e r (82)1942 l O . S l r o d & Braun (25) 1942 + -s- + 4 + - + _ _ 11.Stuart A Gore-s i ine (85)1942 + + + + + + + + 12.Seleen & Stark ( 8 0)1943 + ! + 4 4 + + | 1 + 4 1 [ 1 4 Carbohydrate f e r m e n t a t i o n s : + » a c i d formation, no gas; - = no a c i d or gas formation. O P 4 o «l Pi 4 P cf CD H> (D 4 CD c+ P c+ H* o CO I + ii a ts P o o H-P P" O H->cl P^4 O Hi P" o s 4 o 0 c+ a- o - o ts o p o OQ P 0<3 CO p • co H1  ca VO (-» H* IV) 13 CD H O a CO c»-p 4 — p o 4 NO P 4 oo ta ro e K i oo U a CD O 4 H — H H H- fcf ^- co H NO 8» a P i+ H a vO H P •e* 4 p H ro & t"« P a> t) I* P — 0 o — »• H so S CO vo NO hj ^ co vo • ro H 4 CD CO CD P c+ H ti «4 CD CO + I + + + + + + i i + + + + i + + + + + + + i i + i + + + + + + + + + i i i i i t I 8 I I i t i i I 9 I D I I + + Fluorescin Fluorescent under Ultra Violet Light Non-chromo-genic  Reduced Coagulated Alkaline Peptonized Nitrate Reduc ed Nitrite Formed Nitrogen Gas Formed Glycerol Xylose Arabinose Rhamnose Sorbitol Mannitol Mannose Fructo se Glucose Galactose Sucrose Maltose Lactose Raffinose Inulin Dextrin Starch Salicin Cellobiose Melizitose Melibiose a o w 03 1-3 CO H 1 m H Oil) H •3 <3 £ O I _ > o w <3 o •3 Cfi O f M f M co I O > w o a & E t?J 125 M O 125 03 M W H- O O tc CD H» O P H CD P O ct- H* O CO 1-3 O P >-* O* »X3 CD CO CD p- o o t» p CO H) H (3 O 4 CD co a CD co H Table I I I Biochemical Reactions of Pseudomonas m i l d e n b e r g i i , Pseudomonas f r a g i t and Pseudomonas pavanacea AUTHORS PIGMENTS +y CO + > H 03 0 CO +3 U UO O O H 3 > d o r-l etH LITMUS MILK G2LATIH !L I N -ACTION Li. R. V. P. NITRATE REDUCTION LIP-[>LY-IS CARBOHYDRATE FERMENTATIONS35 cd a CO CO u c5 CO CD +> CO N ct) rx •i-CD rH •H e O rH o 3 < 0 W> 00 -p •d -H CO •M e CD O O H CD o "I CO r d +3 CO Cd o n 2 p r d ' H CD 3 (4 CO • H © rH 6 • P rH • H O !25 <H el CD O CO - P u • H O a 'rH CD w ; CO r H H O o co. O O rH CD C o 43 +3 CD w • H a • H • H O o £> ' rO el S» cd cd rH el H >» U O cd 3 o j CO © CQ O el el cd CD co o +3 o fH H © CQ O O © o 43 O cd r - l cd © © CQ CO O O U 43 O r H p i Cd O CO ^ © CQ O 43 O cd Hi © CQ O el •H <4H <rH cd el •H H el u 43 M , © © 4) ! CQ CO © 1 O 0 t o Fi H p 0 • H J=> rH «H o O O N . O fH <H r H • H >H cd H H +> .cd © © © CO to O Ps. mildenbergii 1 .Present In-v e s t i g a t i o n 598 + - + 7 9 5 + -- + + - -2 . Topley & Wil-son (90 )19?6 3 . Bergey (3)1939 - + a s . r ragT Present In-v e s t i g a t i o n 4 9 7 3  + + + s+ - =. + + + + + + - - + 4 9 7 5 + • + + - - + + + I + S1+ 2.Hussong et al. ( 4 1 ) 1 9 3 7 + + - + • + 3.Seleen & Stark ( 8 0 ) 1 9 4 3 . Ps. pavonacea 1 . Present In-v e s t i g a t i o n 957- •••• 2 . Levine & Soppeland ( 3 2 ) 1 9 2 6 . + + Bergey ( 3 ) 19 39 . + + ^Carbohydrate fermentations + = p r o d u c t i o n of a c i d . - s no p r o d u c t i o n of a c i d . S+ = slow p r o d u c t i o n of a c i d . S1+ = only s l i g h t p roduction of a c i d , t ~ a c i d p r o d u c t i o n v a r i a b l e . Table IV Biochemical Reactions of Pseudomonas n i g r i f a c i e n s & P_s. p u t r e f a c i e n s . PIGMENTS LITMUS MILK JELATIN LIQUE-FACTION M. R. V. P. NITRATE REDUCTION LIP-3LY-SIS CARBOHYDRATE FERMENTATIONS* Black Fluores-cent under U.V. light Reduced Acid P w cd o o CP Pi H —i cd H •H PI o •p p <D P-i Nitrate seduced Nitrite formed •Nitrogen gas formed Glycerol jXylose Arabinose j Rhamnose Sorbitol Mannitol Mannose J Fructose Glucose Galactose Sucrose Maltose [Lactose w o Pi H H in cd H H P Pi H Dextrin Starch Salicin i o c o c—ICQ H C CCn-O.C HCO c o c j Meiibiose P s . n i g r i f a c i e n s 1. Present i n -v e s t i g a t i o n 1 + + + + + S1+ S1+ SL+ Si- S1+ + + + + + - - - - - - 31+;- - - I S1+ S1+ S1+ S1+ S1+ S1+ 31+ ;- - - !S1+ S1+ - - - -22 + + + + + - - - - - 31+1 - - S1+ S1 + S1+ S1+ S1+ - S1+ |_ - -JS1+ S1+ - - - -30 + + + I + + - — - — — 31+, - — — — S1+ - — S1+ S1+ — Si- '- - - 6l+ si-» - - -9M + + + + + - — — - - - 31+; - - - - S1+ S1 + S1+ S1+ S1+ s i * si+ - - |S1+ S1+ - - — -2.White ( 9 3 ) 1940 + + + + + i i Alk. Alk. Alk Alk Al Ps.putrefaciens l o Present i n -vest i g a t ion 375 + + + + + 1 i S1+ S1+ - S1+ s n S H 2M + + + + + - -8SS(S) + + + - - + I + I - — - | - — ' — | — — - — s m - — S1+ - - - - — - SL+ — — 65A + + + - - S1+ - - I - si+! - S1+ S1+! 8SS(R) + + + - - - | -IS1+- • S1+; - S1H si+;- 1 2. Derby & Hammer (21) 1931. Achromobacter p u t r e f a c i e n s + I + + I I + + I 1 | ! + I + i I _ 3» Long & Hammer (55) 1941 1 ! +! * j i I • + + + + + ' * + + + + > j J 4. G a s t e l l (8 ) 1941, Achrbmobae,t¥r p u t r e f a c i e n s . 1 - i r ~ I C3 .... 1 . I + I * !* -- — -—-Carbohydrate fermentations: + = v a r i a b l e a c i d p r o d u c t i o n . + = a c i d p r o d u c t i o n . S1+ = s l i g h t production of a c i d . - = no a c i d p r o d u c t i o n . A l k . = a l k a l i n e r e a c t i o n . Table V Biochemical Reactions of Pseudomonas muc i dolens. Pseudomonas graveolens t Pseudomonas caviae, PIGMENTS LI THUS II ILK GJfiLiiTIN LIQUE-FACTION I'd . R. NITRATJJJ R E D U C T I O N LIP-3 LY-S I S CARBOHYDRATE FERMENT AT I O N S S fl •i-i a co CO u o P I—I P>4 Ps.mucidolens 1. Present i n -vest i g a t ion 4 6 8 6 +3 P d . f l fl U W © P - H O H H ra fD CD P U U CD O CD i—l P T j o N p> 1 ' d ; CD CD p ESI - d cd • H CD r H PI O 1 P o P 6 0 p >d I ^ Cd P4 CD . O CD « ; O P4 - d CD CQ (H cd O c5 F--) CQ Pi CD <d CD CD P CD P M ' d cd o • H O CD h p J-4 U S p <d P • P h •H CD •H •H O SI CD CQ o 1 o u i CD , P i CD i CQ •H O O £> >. i H cd —i >» H 3 h< - i — -a} CD IQ O Pi cd & (3 r H :rH O O P P • H - H .£> P i Pi o i cd — — I — © IQ O P i P i cd © © © CQ CQ CQ © O © i CD © O Pi O CQ - P CQ . 03 CQ Pi P o , O o ; O O • H • H o o cd fn : p P <H r H 0 p H O H o <M P u r H cd P , cd 1 * cd Pi CO : 1 - i M ' P i I P i ' •P © o rH cd - p CO Pi • H O • H r H cd to © CO o •H o r H I—I © o © CQ O - P •H N • H r H H © © CQ O • H X> •H r H © >-1 + + f + , + + + + + + + - I -4TBT + + 2. Levine & Anderson (51)1932 - + + + + .+ + + + + + 3 . C a s t e l l ( 8 ) 1941 J L Ps.graveolens 1. Present i n -v e s t i g a t i o n 4 6 8 3  4 6 8 4 + •, -+ • - |+ ; + + - + + + - 1+ + + + + I i 1 — — 1 - ,+ - + Levine & Soppeland (52)1932. Ps, caviae 1. Present i n -vest i g a t i o n - + + + =. - , _ + + + - + + + - _ Scherago ( 7 4 ) ( 7 5 ) 19 3 6 and 19 37 - + + + ! •*Carbohyflrate f e r m e n t a t i o n s : + s a c i d p r o d u c t i o n - = no a c i d p r o d u c t i o n . I + ii a 3 P o o C9 p, O p. 4 o •d P" 4 d o a p. c+ pj H-o o o pi CD 4 B — CD a c+ p c+ {r-- CO r-» r-» K + e II II O p is H H WVJ P H CQ H. H pi H" CD OQ 4 cr CD P W O 4 cr O H- P> o e 0 C3 • c+ H« O tl O r+, P o cr« —~ CO CQ CO cr CD O P H — 4 © P? © H B VO CP CO OO O c+ VJ1 4 © w M H v© H-pi ro © 1  bd vo © vM 4 vo otj © \J4 — CQ vn O ro *d —"d © H vo P ro p ON p. cx p NO < ON © pi © ON NO NO <j rrj Vr, © 4 O W CD cr CO H* © CK2 d P CT cr O d d I CQ CQ cr © P H 4 © Pf © d oo 8° o ro NO © VM 4 vo (ft © v>J © \-n co vo cr w p C+ H- O PS |co ro ON <j •x) 4 NO O td © 4 H-*» CO ON co © cr CT © O © H* © H> 4 H* OQ P* M P cr P cr <4 ro H-H-H-O d PJ PJ 1 P co CQ CQ (-• +— CQ ! e to H + CQ !+ CQ + JMO Pigments Fluoresc in Fluorescent under Ultra Yiolet light Reduced Acid Coagulated Alkali ne Peptonized Nitrate Reduced "Nitrite" Formed Nitrogen Gas Formea Glycerol Xylose Arabinose Rhamno se Sorbitol Kannitol Hannose Tructo se' 'Gluco se~~ 'Galactose Sucrose Salt ose Lacto se Raf1inose Inulin Dextrin" Starch Salicin Cellobiose Llelizitose Melibiose H CP K) CD r1 H t-3 en R M *1 t"* CP r* M td O ,p H fel H O I "WW •d <J te! M Ci W o > •3 r^ M W o co o t-) H C** H M KM td H* O O d* © o p © P o cr o W o © © Pi P* o 3 o B V o l-t» H r3 p p pi © p • < 1—1= CO © p! P" o o Pi p CO •d p: c+ o H-to Pu P trJ o »> © © r3 P: A- o ^1 3 H o t* p CO o It-<1 re p M M O S © iK • DISCUSSION The species of Pseudomonas studied i n this investi-gation were a l l shown to possess polar f l a g e l l a . In this respect they conformed to the findings of other workers. The study of fluorescent pigments indicated that with some species a yellow-green fluorescent pigment was pro-duced which could be observed under ordinary light. However, cjther members of the genus did not form this pigment visible i n daylight, but did exhibit a blue fluorescence when observed by ultra-violet light. Those cultures which produced the yellow pigment exhibited the blue fluorescence under ultra-violet light before the yellow pigment appeared, and a green fluorescence after the yellow pigment was v i s i b l e . A l l species studied, with the exception of Ps. ribo- flavina produced one or both types of fluorescence. Ps.  riboflavina has been reported capable of oxidizing riboflavin to lumichrome, a non-fluorescent substance (28). Therefore, i f fluorescin i s identical with riboflavin as suggested by Orla-Jensen (43) this would explain the lack of fluorescence in cultures of Ps^ riboflavina. When a synthetic medium was employed to determine nitrate reduction, the reactions of Pseudomonas were found to vary considerably according to the species tested. The fol-lowing tive types of reactions were observed: 1. Nitrate reduced to n i t r i t e and nitrogen gas 2. Nitrate reduced to n i t r i t e only 27. 3. Nitrate util i z e d with .no detectable formation of n i t r i t e or nitrogen gas 4. Nitrate reduced to n i t r i t e , only i n the presence of an additional nitrogen source such as peptone 5. Nitrate not reduced When the results of different workers are compared i t is to be seen that the greatest variance in findings occurs i n the case of the carbohydrate fermentation studies. This inconsistency can probably be traced to the different basal media employed by the respective investigators. However, the results obtained by recent workers using synthetic media cor-respond more closely to the fermentations recorded in this investigation. The results obtained i n this study for various members of the same species obtained from different sources seem to exhibit a remarkable consistency. The species of Pseudomonas employed i n the present study were found to exhibit the following generic character-i s t i c s : polar fla g e l l a , a b i l i t y to fluoresce (with the exoeption of Ps. riboflavina) and weak fermentation of carbo-hydrates. A tentative classification of the species studied based on the findings recorded i s suggested. Suggested Classification Nitrates Reduced A. Nitrate used as sole source of Nitrogen 1. Ni t r i t e and Nitrogen formed Ps. aeruginosa  Ps. fluorescens  Ps. muoidolens 2. N i t r i t e only formed a) l i p o l y t i c - Ps, caviae b) non-lipolytic - Ps. putida 3. Nitrite not formed a) Litmus milk acid and coagulated -Ps. graveolens b) LTxTmus mi He alkaline and not peptonized - Ps. mildenbergii Ps. ovalis B. Nitrate not used as sole source of Nitrogen Ps. putrefaoiens Nitrates Not Reduced A. Lipolytic 1. Ferment glycerol, sucrose, and maltose -Ps. fragi 2. Do not ferment glycerol, sucrose, and maltose - Ps. pavonacea B. Non l i p o l y t i c 1. Liquefy gelatin - Ps. nigrifaoiens 2. Do not liquefy gelatin - Ps. rlboflavina SUMMARY OF PART I A detailed study of 35 representative microdrganisms of the genus Pseudomonas has "been made. The results obtained have been compared with those recorded by other workers employing the same species* As a result of this investigation, a new basis for the classification of the members of the genus has been proposed 30 PART II THE AMINO ACID REQUIREMENTS FOR PYOCYANIN PRODUCTION BY PS. AERUGINOSA Various synthetic and semi-synthetic media have been used i n studies of pyocyanin production by Pseudomonas aeru- ginosa. Gessard (33) showed that glycerol peptone agar pro-vided an admirable medium for pyocyanin formation. Jordan (44) reported that the ammonium salts of succinic, l a c t i c , acetic or c i t r i c acids could serve as adequate carbon and nitrogen sources for pyocyanin production. Lourie (56) found that acetate, although suitable for the production of the fluores-cent pigment would not support pyocyanin development. Robin-son (71) stated that the yield of pyocyanin by Ps. aeruginosa from various synthetic media had never been found to equal that from Bacto-peptone. Seleen and Stark (80), in studies on the nutritive requirements of Ps. aeruginosa. were unable to devise a medium more suitable for pyocyanin production than the glycerol peptone agar of Gessard. The object of the present investigation was to as-certain whether or not amino acids could replace peptone for the production of pyocyanin and to determine which are essential to pigment formation. METHODS' The organism employed in this work was Ps. aeru- ginosa A.T.C. 9027. A preliminary study of various media showed that formation of pyocyanin equal to that produced on glycerol peptone agar could he obtained when the following medium was employed* Casamino acids 1.0$ (Bacto acid hydrolyzed casein) Glycerol 1.0$ Salt Solutions A and B* 0.2 ml. of each per 100 ml. medium pH 7.2 Agar was found not to be essential for maximum pyocyanin formation i f the medium was aerated during growth i n tubes or i f a thin layer of the medium in flasks was employedC10 ml. medium i n 125 ml. erlenmeyer flasks). The latter procedure gave more reproducible results. A l l cultures were incubated at 30°C for four days. For the determination of the amino acids essential to pigment formation, the general technique of Mueller (63) was employed. The casamino acids were separated by the Dakin method (19) into three fractions: 1. Monoamino monocarboxylic acids 2. Proline 3. Diamino and dicarboxylio acids ft Salt Solution A: K 0 H P O 4 0.05 gms. per ml. KH 2P0 4 0.05 gms. per ml. Salt Solution B: ngSO^.THpO 0*05 gms. per ml. KC1 rf 0.02 gms. per ml. FeS04.7H20 0.0002 gms. per ml. 32 These fractions were employed as the source of nitrogen re-placing the casamino acids used i n the medium described above. The extent of pyocyanin production was measured by means of the Fischer Electrophotometer. Ten m i l l i l i t r e s of culture were shaken with four successive 2-ml. portions of chloroform. The red acid pyocyanin was separated from chloro-form by the addition of 5.0 ml. of 0.1 N HC1. Five m i l l i l i t r e s of 0.1 N NaOH were then added and the resulting blue pjjrocyanin solution was diluted with an equal volume of d i s t i l l e d water. The density of pigment was measured electrophotometrically. The effect of dilution of pigment on photoelectric readings was found to be a linear relationship. EXPERIMENTAL Fractions 1, 2, and 3 recombined end added to the basal medium of glycerol and salts in the following amounts: Fraction 1, 0.5 gms., Fraction 2, 0.18 gms., and Fraction 3, 0.63 gms. per 100 ml., resulted i n pyocyanin formation equal to that obtained when the unfractionated casamino acids were employed as the source of nitrogen. In order to determine which of the fractions were essential to pyocyanin production, media whose nitrogen sources consisted entirely of individual fractions, or of various combinations of the respective fractions at different percentage concentrations, were prepared. Glycerol and Salt Solutions A and B at the percentages employed i n the 33 preliminary study were used throughout. Table I l i s t s the media used and the amount of pyocyanin produced from each medium. TAB335 I The Effect of Fractionated Casamino Acids on Pyocyanin Formation t % Medium Fractions Added Pyocyanin Medium Fractions Added Pyocyanii l a 1 0.5 29.3 lb 1 1.0 75.5 2a 2 0.18 0 2b Z 1.0 0 3a 3 0.63 0 3b 3 1.0 0 4a 1 0.5 4b 1 0.74 2 0.18 31.5 2 0.26 70.5 5a 1 0.5 5b 1 0.44 3 0.63 68.7 3 0.56 51.7 6a 2 0.18 6b 2 0.22 3 0.63 0 3 0.78 0 7a 1 0.5 -2 0.18 67.2 Casamino acids 1.0 61.0 3 0.63 It i s seen from Table I that when Fraction 1 i s omitted from a medium no pyocyanin formation occurs. When this fraction i s present to the extent of 1.0$ even though i t i s the sole source of nitrogen pyocyanin production equal to that ob-tained when unfractionated casamino acids are employed at 1.0$ concentration i s obtained. At low.er cojuoeat rat ions (0.5$) Fraction 1 employed as the sole source of nitrogen resulted i n & Basal medium: glycerol 1.0$ Salt Solutions A and B 0.2 ml. of each per 100 ml. medium. && Pyocyanin expressed as reading on logarithmic scale of Fischer Electrrophotometer. 34 pyocyanin formation but i n smaller amounts. The addition of Fractions 2 or 3 to the lower concentrations of Fraction 1 re-sulted in formation of increased amounts of pyocyanin. A medium made up of the nine monoamino monocarboxylic acids present in Fraction 1 and combined in the proportions i n which they are present in casein to total 1.0% was next pre-pared. The composition of this medium, designated as Medium A, i s given below: A MEDIUM A Amino Acids in Percentage Equivalent proportion to fraction 1 in Casein total 1.0% (1 gm./lOO ml.) Glycine 0.45 0.011 dl-alanine 1.85 0.046 dl-valine 6.2 0.155 dl-isoleucine 6.4 0.16 1-leucine 9.9 0.248 dl-phenylalanine 5.2 0.13 1-tyrosine 5.7 0.14 dl-serine 0.5 0.016 dl-threonine 4.2 0.105 40.5% 1.01 gms. Nine different media, identical with Medium A except for the omission of one of the respective amino acids consti-tuting Fraction 1, were also prepared. A medium consisting of Fraction 1 at 1.0% concentration served as a control. The amounts of pyocyanin produced from these media are presented & Basal medium: glycerol 1.0% Salt Solutions A and B 0.2 ml. of each per 100 ml. medium. 35 in Table I I. TABLE II The Substitution of Fraction 1 by the Mono-amino Monocarboxylic Acids of Casein Medium Constituents of Medium Pyocyanin A. 9 amino acids of Fraction 1 57.0 1. Medium A minus glycine 38.0 2. n n alanine 46.7 3. « n tt valine 34.7 4. tt n tt isoleucine 34.5 5. it tt tt leucine 34.0 6. tt tt « phenylalanine 67.0 7. tt tt tt tyrosine 54.0 8. t! tt « serine 51.0 9. tt tt tt threonine 34.0 Control Fraction 1 (1. 0%) 77.0 , The results show that a synthetic mixture of the nine amino acids as a source of nitrogen was satisfactory for pyo-oyanin production and that more than one amino acid i s concerned with pigment formation. The, results obtained when phenyla-lanine was omitted from the medium suggest that the presence of this amino acid may exert an inhibitory effect on pyocyanin production. In order to study the effect of individual amino acids on the microbial synthesis of pyocyanin, mine media were pre-pared, employing each of the respective amino acids of Frac-tion 1 as the sole source of nitrogen. Except i n the case of tyrosine, which was used at 0.6% concentration, the amino acids were made up at 1.0%. The results of this experiment are re-36 corded i n Table I I I . TABLE III Pyocyanin Production from Individual Amino Acids of Fraction 1 : Medium Pyocyanin 1. Glycine 1.0$ 25.5 2. dl-alanine 1.0$ 38.0 3. dl-valine 1.0$ 29.0 4. 1-leucine 1.0$ 0 5. dl^-isoleucine 1.0$ 0 6. d l - threonine 1.0$ 0 7. dl-phenylalanine 1.0 $ 0 8. dl-serine 1.0$ 0 9. 1-tyrosine 0.6$ 18.5 While growth of Ps. aeruginosa was obtained in the case of a l l media employed, pyocyanin production occurred only in the presence of glycine, alanine, valine or tyrosine as the source of nitrogen. Alanine gave the highest yfeld of pyocyanin which, however, did not equal the amount obtained when either casamino acids or combinations of the amino acids were employed. Even when alanine was employed up to a con-centration of 2.0$ no greater yiel d of pyocyanin was obtained. With the object of determining the amino acids es-sential to maximum production of pyocyanin, media whose nitro-gen sources consisted of various combinations of the pyocyanin producing and non pigment forming amino acids of Fraction 1 were prepared. The media used i n this experiment and the re-sults obtained are given i n Table IV. TABLE IV The Effect of Various Combinations of Monoamine* Monocarboxylic Acids on Pyocyanin Production d i - 31- 1- 1- a i - d l - dl-phenyl- a i -Glycine a l anine valin< tyrosine leucine isoleucine thr eonine alanine eerine Pyocyanin 1. 0.2% 0.2% 0.2% 21.0 2* 0.4% 0.4% 0.4% — * 40.0 3. 0.2% 0.2% 0.2% 0.6% — 87.5 4. 0.2% 0.2% 0.2% 0.6% — _ ™ — 33.5 5. 0.2% 0.2% 0.2% . 0.6% 53.0 6. 0.2% 0.2% 0.2% . — * 0.6% 0 7. 0.2% 0.2% 0.2% 0.6% 24.0 8. — 0.6% mm mum 12.0 9, •>— 0.6% 0.2% 0.2% 0^2% mm an mm- 10.5 10. = 0.6% — * 0.2% 0.2% 0 SI 38 Whereas no pigment i s formed when leucine i s em-ployed as the sole source of nitrogen, the inclusion of this amino acid i n a medium containing glycine, alanine and valine results i n a marked increase i n pigment production. Leucine i s , however, without influence on the pigment forming a b i l i t y of tyrosine. The addition of phenylalanine to a mixture of the pigment forming amino acids results i n the complete sup-pression of pyocyanin, confirming the observation recorded i n Table I I . As the results obtained from these experiments showed clearly that the concentrations of the amino acids used in media were of marked significance with respect to the pro-duction of pyocyanin, media made up of various concentrations of alanine and leucine were prepared. Fraction 1 at 1.0% concentration again served as the control medium. The results are given i n Table V. TABLE Y essaaaaa The Effect of Various Combinations of Alanine and Leucine on Pyocyanin Production Medium Alanine i Leucine Pyocyanin 1. 1.0 — 41.0 2. 1.0 0 3. 1.0 0.2 66.0 4. 0.8 0.4 76.0 5. 0.6 0.6 68.0 6. 0.4 0.8 93.0 7. 0.2 1.0 78.0 Control Fraction 1 at 1.0% concentration 75.5 39 The maximum y i e l d of pyocyanin was obtained when 0.4$ alanine and 0,8$ leucine were incorporated i n the medium, sur-passing pyocyanin production i n the control, Fraction 1, A comparative study of the effect of glycine", alanine and valine, employed as sole sources of nitrogen at two d i f -ferent concentrations and combined with leucine, on pyocyanin formation i s presented i n Table 7T. TABLE VI The Stimulatory Effect of 1-leuoine.on Pyocyanin Production from Alanine', Glycine and Valine  Medium Constituents of Medium Pyocyanin 1. 2. 3. Alanine 0,4$ Alanine 1,2$ Alanine 0.4$ 4- 1-leucine 0.8$ 35.5 43.0 96.0 4. 5. 6. Glycine 0.4$ Glycine 1.2$ Glycine 0.4$ 4- 1-leucine 0.8$ 18.0 8.5 95.0 7. 8. 9. Valine 0.4$ Valine 1.2$ Valine 0.4$ + 1-leucine 0.8$ 11.5 27.5 33.0 The stimulating effect of 1-leucine on pyocyanin pro-duction i s shown to be most marked i n the case of glycine and alanine and, as a result of these studies, synthetic media for maximum production of pyocyanin by Ps. aeruginosa A.T.C. 9027 have been developed. Either of the media detailed below yielded pyocyanin in quantities greater than those obtained on any other medium. TABLE VII Comparison of Pyooyanin Production by Different Strains of Ps» aeruginosa Medium Constituents of Medium ~P5T aeruginosa -pgV • 11 aeruginosa '" Ps. pyocyaneus — p a r aeruginosa Ps. 1 — ~ aeruginosa A.T.C. 9027 8689 V21 R.M. Pa.* 1* Fractions 1 and 2 0 0 0 0 0 2* Glycine 1.0$ 17*0 12.0 21*5 28.0 16*5 3* dl-alanine 1.0$ 38.0 26.0 30.0 23.0 18.5 4* dl-valine 1.0$ 29.0 25*0 18.5 20.5 9.5 5. 1-tyrosine 0.6$ 9*5 10.5 7*5 10.0 8.0 6. . dl-alanine 0.4$ 1-leucine 0.8$. 97.0 50.0 38.5 50.0 18*0 7* Casamino acida 1.0$ 67.0 31.0 25*5 51.0 20.0 & We are indebted to Dr. M* Sherago, Department of Bacteriology, University of Kentucky, for culture 0?f Ps. aeruginosa Pa. 41 1. Glycine 0.4$ 2. dl-alanine 0.4$ 1-leucine 0,8$ 1-leucine 0,8$ Glycerol 1.0$ Glycerol 1.0$ Salt Solutions A and B Salt Solutions A and B 0.2 ml. of each 0.2 ml. of each per 100 ml. medium per 100 ml. medium In order to ascertain the general s u i t a b i l i t y of a synthetic medium for pyocyanin production by Pseudomonas  aeruginosa i t was considered essential to study other repre-sentative strains of the species. In Table VTI pyocyanin production by five strains on seven different media are com-pared. (Table vTI on previous page) The pyocyanin forming a b i l i t y of the respective strains of Ps. aeruginosa is shown to vary markedly. The synthetic medium in which alanine and leucine are employed as the source of nitrogen i s , however, shown to be equal or superior to casamino acids for pigment formation by a l l strains studied. SUMMARY OF PART II A medium consisting of acid hydrolyzed casein, glycerol and a salt mixture has been shown to yi e l d pyocyanin by Pseudomonas aeruginosa equal i n amount to that obtained from glycerol peptone agar. The monoamino monocarboxylic acids fraction obtained from casamino acids (Bacto) has been shown to be the source of nitrogen essential to pigment formation. Glycine, dl-alanine, dl-valine, or 1-tyrosine have been shown to produce pyocyanin when employed as sole sources of nitrogen. The addition of 1-leucine to media containing gly-cine or dl-alanine markedly increased pyocyanin formation. The addition of dl-phenylalanine to a synthetic medium inhibited pigment production. A synthetic medium consisting of dl-alanine or glycine at 0.4$ concentration combined with 0*8$ 1-leucine, 1.0$ glycerol, and a salt mixture has been shown to be the most suitable medium for pyocyanin production by five repre-sentative strains of Pseudomonas aeruginosa. 43 gAlf III THE MIHERAL REQ.UIRBMENTS FOR PYOCYANIN PRODUCTION A study of the mineral requirements for pyocyanin production has been hindered by the lack of a satisfactory synthetic medium capable of supporting the production of this pigment. Consequently, there i s very l i t t l e to be found in the literature dealing with the essential salts required for pyocyanin formation* Robinson i n 1932 (71), when studying the nutrition o f Ps. aeruginosa "established" the salt requirements for pyo-cyanin production, and i n addition, l i s t e d some of the common minerals and ions which he found not to be essential for the formation of the pigment. He stated that both P0 4 and Mg were necessary, while Na, K, Ca, CI, S0 4, and C0 3 were unessential. For optimum pigment production5, he recommended 0.09% KHgPO^ and 0.05% MgS04. He stated further that the formation of both fluorescin and pyocyanin required only those substances neces-sary for growth. Lourie i n 1945 (56), stated that P0 4 and S0 4 were necessary for growth of Ps. aeruginosa, and employed both K gHP0 4 and MgS04 in media used for the production of pyocyanin. In the study of the amino acid requirements for pyo-cyanin production (Part II of this investigation) a rather f u l l complement of salts was employed to ensure that the mineral supply would not be a limiting factor i n the production of the 44 pigment* Only after the carton and nitrogen components of the synthetic medium had been established was i t possible to dem-onstrate which of the mineral constituents of the medium were essential to pigment formation and to establish the concentra-tions of salts required for optimum yield of the pigment. To this end, the following work was undertaken. METHODS A synthetic medium composed of glycerol and specific amino acids, and minerals was found to be satisfactory for the production of pyocyanin. The composition of the medium desig-nated as Medium B was as follows: Glycerol 1.0$ Glycine 0.6$ 1-leucine 0.6$ MgS0 4.7H 20 ©.€0$ KrflPG, 0.01$ KH9PO4 0.1 ' KCI * 0.004$ FeS04.7H20 0.0004$ By omitting each salt respectively from this medium those which are required for pyocyanin formation were deter-mined; by varying the percentage of each salt i n the medium, the optimum levels of concentration of the respective salts were ascertained; and f i n a l l y , by eliminating each respective ion from the medium those which are essential to pigment for-mation were established. The methods of culturing the organism (Ps. aeruginosa A.T.C. 9027) and extracting the pigment were the same as those employed i n Part II of this investigation. Ten ml; quantities 45 of media were cultured i n 125 ml. erlenmeyers and th.e pyo-cyanin after extraction with chloroform was measured i n a Fischer Electrophotometer. EXPERIMENTAL Essential Salts The relative importance of the salts i n Medium B to pyocyanin production was determined by omitting each salt successively from the medium. The pyocyanin obtained from these media was measured and compared with that extracted from the complete medium. Table I shows the salt composition of the media employed and the amount of pyocyanin resulting i n each case. TABLE I The Effect on Pyocyanin Production of Omitting Various Salts from the Medium Medium Pyocyanin* Medium B 40.5 Medium B minus KoHP04 24.0 Medium B minus KELP04 26.0 Medium B minus KgHP04 and KH PO, 0.0 Medium B minus MgS^ >4 2 4 0.0 Medium B minus KC1 40.0 Medium B minus FeSO^ 0.0 From this experiment i t was concluded that growth £ Pyocyanin expressed as reading on logarithmic scale of Fischer Electrophotometer. 46 does not occur when either MgS04 or both phosphate salts are omitted from the medium. When iron i s absent the growth is equal to that obtained in Medium B but pyocyanin i s entirely replaced by large quantities of fluorescin. KCI does not ap-pear to be essential to either growth or pyocyanin production. Optimum Concentrations of Salts 1. KCI The previous experiment indicated that the presence of KCI i s not required for pyocyanin production i n Medium B. However, i t was thought that different concentrations of the salt might influence the production of pigment. Although concentrations of KCI ranging from 0.0001$ to 0.1$ were em-ployed, no effect on the production of pyocyanin could be detected. S. K2HP0A and KHgP04 In order to simplify the medium K2HP04 alone was substituted for the two phosphate salts. From Medium B, both phosphates were omitted and a range of concentrations of the salt K gHP0 4 was employed. The percentage of KgHPO^ added to the medium, and the pyocyanin resulting are recorded in Table I I . (Next page) The medium containing 0.0001$ KgHP04 was capable of supporting only a trace of growth. The maximum amount of pyocyanin occurred at a level of 0.03$ K 2KP0 4, at which per-centage no fluorescin was observed. Above this concentration of phosphate, the production of fluorescin increased propor-47 tionally to the decrease in pyocyanin. At 0.1% K 2HP0 4 there was considerable fluorescin produced but no pyocyanin. TABLE XT The Effect of Varying Concentrations of K 2HP0 4  on Pyocyanin Production ^ % KoHP04 in Medium B Pyocyanin 0.0001% 0.005% 0.01% 0.03% 0.05% 0.07% 0.10% 0.0 IE.5 20.5 50.0 43.5 17.5 0.0 3. MgS04.7H80 In Medium B, 0.01% MgSO. .7H 0 was replaced with a * 2 range of concentrations of the sa l t . Table III l i s t s the per-centages of MgS04.7Hg0 employed and the pyocyanin obtained i n each case. TABLE III The Effect of Varying Concentrations of MgSO^THgO on Pyocyanin Production % MgSO^THgO in Medium B Pyocyanin 0.0001% 0.0 0.005% 29.5 0.01% 45.5 0.05% 59.5 0.07% 60.0 0.10% 61.0 0.50% 60.0 48 Only a slight amount of growth occurred when 0.0001$ MgSO^ was employed. Maximum pyocyanin production was obtained with 0.05$ and higher concentrations of MgS0A. No drop i n pigment production occurred even up to 0.5$ of the salt and no fluorescin formation was observed within the range employed. 4. FeS04.7H20 A series of concentrations of FeSO^THgO were sub-stituted for the 0.0004$ of the salt in Medium B. The con-centrations employed, and the pyocyanin obtained are recorded i n Table IV. TABLE IV The Effect of Varying Concentrations of FeSO^.THgO on Pyocyanin Production  $ FeS0/.7H20 in Medium B 0.00001$ 0.00005$ 0.0001$ 0.000?$ 0.0005$ 0.005$ 0.01$ 0.03$ Pyocyanin 0.0 9.5 19.5 33.0 45.0 47.0 22.0 0.0 At a concentration of 0.00001$ FeS0A.7H20 there was abundant production of fluorescin but no evidence of pyocyanin. However, fluorescin production decreased proportionally with the increase in pyocyanin as the percentage of FeSOA approached 0.0005$. At this concentration and higher, no fluorescin was 49 observed. Maximum pyocyanin occurred at 0,0005% and 0,005%, Growth was inhibited i n the presence of 0.03% FeS0 4. The foregoing experiments have indicated the optimum concentrations of the essential salts for pyocyanin production A medium was next prepared employing glycerol, glycine, and 1-leucine as in Medium B, plus 0.03% K 2HP0 4, 0.10% MgS04.7H20, and 0.0005% FeSO^.THgO. A pyocyanin reading of 68.0 was ob-tained from this culture, which shows considerable increase over the reading of 40.5 obtained with Medium B. This in-crease in pyocyanin production was due to the optimum balance of salts employed. Essential Ions With the salts employed i n the foregoing media, one or both of the ions may be essential to pyocyanin formation. Therefore, i t was necessary to use salts, each of which contain only one of the following ions: K, P0 4, Mg, S0 4, and Fe. By employing such sal t s , each ion respectively can be omitted from the medium. Molar equivalents of the respective ions were cal-culated and supplied i n other salt combinations as follows: 0.022%-K01 and 0.029% CaHP0/,2Hj>0 are equivalent to 0.03% K 2HP0 4 * 0.04%,MgCl2.6HcO and 0.036% CaS04.2H20 are equivalent to 0.05% MgS04.7H 20 0.00048% FeCl 3 . 6 H o O i s equivalent to 0.0005%, FeS04.7H20 Media were then prepared containing the same five ions as previously employed but the ions were supplied i n different salts. In addition, f i v e media in which each ion respectively was omitted, were prepared. The media i n each case contained glycerol (1.0$) glycine (0.6$) and 1-leucine (0.6$) and had the following mineral composition: 1. Original salts K„HP0 A 0.03$ MgSO * 7 H o O 0.05$ FeS04.7B|0 0.0005$ 2. K«HP0 A replaced by KCI and CaHP0A.2H 0 * * KCI 0.02$ * 2 CaHP04.2HgO 0.03$ MgS04,.7HP0 0.05$ FeS0A.7H^0 0.0005$ 3. MgSOA.THpO and FeS04.7HP0 replaced by MgCl 2, ^CaS04.2H20 andT'eClg.eHgO K 2HP0 4 0.03$ CaS04?2HP0 0.04$ M g C l o.6H£0 0.04$ FeCl3.6Hg0 0.0005$ 4. P0 A omitted KCI 0.02$ MgS04.7HgO 0.05$ FeS04.7H^0 0.0005$ CaHP0 4 «2HgO 0 • 5. K omitted MgS0AT7HPt) O.l FeS0j.7Hg0 0.0005$ 6. S 0 4 omitted K 2 HP0 4 0.03$ MgCl 2 .6H P 0 0.04$ FeCl 3 . 6 H £ 0 0.0005$ 7. Mg omitted K 2 HP0 4 0.03$ C a S 0 4 . 2 H o O 0.035$ FeS0 4 . 7H 2 0 0.0005$ 8. Fe omitted >HP04 0.03$ J 1 0 4 . 7 H 2 0 0.05$ The pyocyanin readings 'Obtained with extracts from 51 these media are recorded in Table V. TABLE V The Importance of the Ionic Constituents of Salts in Pyocyanin Formation  Medium Growth Obtained Pyocyanin 1. Original Salts 2. KpHPO^ . replaced 3. M g S 0 4 and F e S 0 4 re 4. P 0 4 omitted 5. K omitted 6. SO4 omitted 7. Mg omitted 8 . Fe omitted 4444' 4444 placed 4444 4 4444 +444 44 4444 67.0 43.5 56.5 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 The fiv e ions under investigation, P0 4, K, Mg,' S0 4, and Fe are required i f growth and pyocyanin production are both to be obtained. However, for growth without pyocyanin production, K, S0 4, and Fe do not appear to be essential. As was previously noted, the omission of iron from the medium results i n abundant production of fluorescin, but no pyocyanin. SUMMARY OF PART III An investigation of the salt requirements for pyo-cyanin production has been carried out. In a basal medium of glycerol (1.0%), glycine (0.6%), and 1-leucine (0.6%), the following salts i n the prescribed concentrations give a maximum yield of pyocyanin with Ps. aeruginosa A.T.C. 9027: K 2HP0 4 0.03% 52 MgSO, .7HP0 0,10% FeS0|.7Hg0 0,0005% The five ions represented i n these three salts were shown to be essential for growth and pyocyanin production 1 3 7 P 3« aeruginosa. A survey of the literature reveals that neither K nor Fe have been previously associated with pyo-cyanin production. 53 BIBLIOGRAPHY 1. Allen, P.J., Naghski, J., and Hoover, S.R., J. Bact. 47: 559 (1944). 2. 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