{"@context":{"@language":"en","Affiliation":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","AggregatedSourceRepository":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","Campus":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","Creator":"http:\/\/purl.org\/dc\/terms\/creator","DateAvailable":"http:\/\/purl.org\/dc\/terms\/issued","DateIssued":"http:\/\/purl.org\/dc\/terms\/issued","Degree":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","DegreeGrantor":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","Description":"http:\/\/purl.org\/dc\/terms\/description","DigitalResourceOriginalRecord":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","FullText":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","Genre":"http:\/\/www.europeana.eu\/schemas\/edm\/hasType","IsShownAt":"http:\/\/www.europeana.eu\/schemas\/edm\/isShownAt","Language":"http:\/\/purl.org\/dc\/terms\/language","Program":"https:\/\/open.library.ubc.ca\/terms#degreeDiscipline","Provider":"http:\/\/www.europeana.eu\/schemas\/edm\/provider","Publisher":"http:\/\/purl.org\/dc\/terms\/publisher","Rights":"http:\/\/purl.org\/dc\/terms\/rights","ScholarlyLevel":"https:\/\/open.library.ubc.ca\/terms#scholarLevel","Title":"http:\/\/purl.org\/dc\/terms\/title","Type":"http:\/\/purl.org\/dc\/terms\/type","URI":"https:\/\/open.library.ubc.ca\/terms#identifierURI","SortDate":"http:\/\/purl.org\/dc\/terms\/date"},"Affiliation":[{"@value":"Land and Food Systems, Faculty of","@language":"en"}],"AggregatedSourceRepository":[{"@value":"DSpace","@language":"en"}],"Campus":[{"@value":"UBCV","@language":"en"}],"Creator":[{"@value":"Triggs, Rosalie Elizabeth","@language":"en"}],"DateAvailable":[{"@value":"2012-03-07T20:24:48Z","@language":"en"}],"DateIssued":[{"@value":"1950","@language":"en"}],"Degree":[{"@value":"Master of Science - MSc","@language":"en"}],"DegreeGrantor":[{"@value":"University of British Columbia","@language":"en"}],"Description":[{"@value":"Methods for the isolation of Leuconostoc mesenteroides and the establishment of its mineral requirements have been studied.\r\nNine natural materials were employed as probable sources for the isolation of species of Leuconostoc. Sauerkraut and ensilage yielded typical cultures. No cultures were obtained from cheese, milk, soured potato, cow manure and sliming cabbage, lettuce and cucumber. The microorganisms were isolated by diluting and then plating onto solid media. Yeast Tryptic Digest Agar, Tomato Juice Agar and Yeast Tryptic Digest Gelatin, each at pH 6.9, 6.2 and 5.6 were employed as the plating media. The Yeast Tryptic Digest Agar at a pH between 6.2 and 6.8 proved to be the most effective medium for the isolation of species of Leuconostoc.\r\nAn enrichment technique was also carried out employing a variety of media. The tubes were held under both aerobic and anaerobic conditions. However, this method resulted in failure. Proteolytic bacteria predominated to such an extent that the relatively weaker Leuconostoc, if present at all, were unable to survive even under the favourable anaerobic conditions employed.\r\nThe cultures isolated from sauerkraut and ensilage were found to be similar in all respects to known species of Leuconostoc mesenteroides.\r\nDue to the difficulties involved in the isolation of the genus Leuconostoc a selective method for the isolation of lactic acid bacteria employing hydrogen peroxide was evolved. Preliminary conditions for isolation were worked out using S. lactis and E. coli. It was found that the minimum amount of E. coli cells (.0058%) and the maximum amount of S. lactis cells (19.2%) remained viable when treated with hydrogen peroxide at a concentration of 0.05% at 15\u00b0C for sixty minutes. A concentration of 0.2% hydrogen peroxide, while destroying all E. coli after sixty minutes treatment was also very deleterious to S. lactis. It was found that with an increase of temperature over 15\u00b0C, the rate of destruction of hydrogen peroxide and the rate of growth of the bacteria served to complicate the findings obtained. \r\nFresh raw milk, employed as the medium of suspension, was found to be necessary for obtaining the optimum effect with hydrogen peroxide. Reconstituted milk and nutrient broth appear to lack the unknown protective factor present in fresh raw milk. \r\nUsing the conditions worked out for S. lactis and E.coli, eight additional organisms were tested for the effect of hydrogen peroxide inhibition. It was demonstrated that these conditions permit the isolation of other catalase negative organisms and at the same time drastically reduce the number of catalase positive bacteria present. The two Micrococci cultures studied are an exception to the general observation that catalase positive organisms are sensitive to peroxide. \r\nThe efficiency of this method when mixed cultures were employed was tested. It was found that all E. coli cells were destroyed while 5,700,000 S. lactis cells remained viable. \r\nTo determine the practical application of this technique, a sample of fresh cow manure was treated with hydrogen peroxide. By using this agent the percentage of catalase negative bacteria arising on a plate increased from 3% to 40-45%. \r\nThe mineral requirements of a defined strain of Leuconostoc mesenteroides have been determined. By depleting the basal medium (McLeod and Snell, 1947) with the strain of L. mesenteroides understudy the essential nature of manganese but not magnesium or iron could be shown. However, by depleting the basal medium with A. niger for twelve days the need for magnesium was demonstrated. Manganese must be present before the magnesium has a significant influence. \r\nA combination of A. niger and L. mesenteroides depletion had no significant advantage over the depletion by the mould alone. \r\nNo stimulatory effect could be observed by the addition of calcium, zinc or cobalt at a concentration of 1.0 ppm., copper at a concentration of 0.05 ppm., and molybdenum at a concentration of 0.02 ppm. Molybdenum and zinc showed a slight inhibitory effect. \r\nBasal medium and depleted basal medium were extracted with 8-hydroxyquinoline and chloroform. Part of both media underwent further treatment; hydrolysis for thirty minutes with HCI (final concentration of 0.1N). In all cases the effect of added iron at a concentration of 0.1 mg. FeSO\u2084\/10 ml. medium was very pronounced if manganese was also present. The addition of 0.01 FeSO\u2084 was as influential as 0.001 mg. or 0.01 mg. FeSO\u2084)10 ml. medium. \r\nThe additional procedure of hydrolysis apparently removes some factor other than iron, manganese or magnesium which is necessary for the optimal growth of L. mesenteroides. It was shown that this factor was not tryptophane, zinc or copper. \r\nThe effect of iron on two other heterofermentative and two homofermentative bacteria was determined. One heterofermentative organism (species of Leuconostoc) showed stimulation with the addition of iron and manganese; however, the other one belonging to this type (E. coli)and the two homofermentative bacteria (S. lactis and S. faecalis) gave very little response even after the addition of manganese, magnesium and iron. \r\nRespiration studies of L. mesenteroides were carried out. No difference in acid production or carbon dioxide evolution per cell could be observed with varying amounts of manganese added to the growth medium.","@language":"en"}],"DigitalResourceOriginalRecord":[{"@value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/41199?expand=metadata","@language":"en"}],"FullText":[{"@value":"L 1= \/5 ? METHODS FOR THE ISOLATION OF LEUCONOSTOC MESENTEROIDES AND A STUDY OF ITS MINERAL REQUIREMENTS - by -Rosalie Elizabeth Triggs A Thesis Submitted In Pa r t i a l Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the DEPARTMENT OF DAIRYING THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER 1950 ABSTRACT Methods f o r the i s o l a t i o n of Leuconostoc mesenteroldes and the establishment of i t s mineral requirements have been studied. Nine natural materials were employed as probable sources f o r the i s o l a t i o n of species of Leuconostoc. Sauerkraut and ensilage yielded t y p i c a l cultures. No cultures were obtained from cheese, milk, soured potato, cow manure and sliming cabbage, lettuce and cucumber. The micro-organisms were i s o l a t e d by d i l u t i n g and then plating onto s o l i d media. Yeast Trypti c Digest Agar, Tomato Juice Agar and Yeast Trypti c Digest Gelatin, each at pH 6 .9 , 6.2 and \u00a3.6 were employed as the pla t i n g media. The Yeast Tryptic Digest Agar at a pH between 6.2 and 6.8 proved to be the most e f f e c t i v e medium f o r the i s o l a t i o n of species of Leuconostoc An enrichment technique was also c a r r i e d out employing a v a r i e t y of media. The tubes were held under both aerobic and anaerobic conditions. However, t h i s method resulted i n f a i l u r e . P r o t e o l y t i c b acteria predominated to such an extent that the r e l a t i v e l y weaker Leuconostoc. i f present at a l l , were unable to survive even under the favourable anaerobic conditions employed. The cultures i s o l a t e d from sauerkraut and ensilage were found to be si m i l a r i n a l l respects to known species of Leuconostoc mesenteroldes. Due to the d i f f i c u l t i e s involved i n the i s o l a t i o n of the genus Leuconostoc a selective method for the isolation of l a c t i c acid bacteria employing hydrogen peroxide was evolved. Preliminary conditions for isolation were worked out using S. l a c t i s and E. c o l l . It was found that the minimum amount of B. c o l i c e l l s (#005\"8$O and the maximum amount of S. l a c t i s cells (19.2$) remained viable when treated with hydrogen peroxide at a concentration of 0.05$ at 15\u00b0C for sixty minutes. A concentration of 0*2$ hydrogen peroxide, while destroying a l l E. c o l i after sixty minutes treatment was also very deleterious to S. l a c t i s . It was found that with an increase of temperature over 15\u00b0C, the rate of destruction of hydrogen peroxide and the rate of growth of the bacteria served to complicate the findings obtained. Fresh raw milk, employed as the medium of suspension, was found to be necessary for obtaining the optimum effect with hydrogen peroxide. Reconstituted milk and nutrient broth appear to lack the unknown protective factor present i n fresh raw milk. Using the conditions worked out for S. l a c t i s and E.coll. eight additional organisms were tested for the effect of hydrogen peroxide inhibition. It was demonstrated that these conditions permit the isolation of other catalase negative organisms and at the same time drastically reduce the number of catalase positive bacteria present. The two Micrococci cultures studied are an exception to the general observation that catalase positive organisms are sensitive - 3 -to peroxide. The e f f i c i e n c y of t h i s method when mixed cultures were employed was tested. I t was found that a l l E. e o l i c e l l s were destroyed while 5 .700 .000 S. l a c t i s c e l l s remained v i a b l e . To determine the p r a c t i c a l a p p l i c a t i o n of t h i s tech-nique, a sample of fr e s h cow manure was treated with hydrogen peroxide. By using t h i s agent the percentage of catalase negative bacteria a r i s i n g on a plate increased from 3$ to 40 - 4 5 $ . The mineral requirements of a defined s t r a i n of Leuconostoc mesenteroldes have been determined. By depleting the basal medium (McLeod and S n e l l , 1947) with the s t r a i n of L. mesenteroldes understudy the e s s e n t i a l nature of manganese but not magnesium or i r o n could be shown. However, by depleting the basal medium with A. nlger f o r twelve days the need f o r magnesium was demonstrated. Manganese must be present before the magnesium has a s i g n i f i c a n t influence. A combination of A. nlger and L. mesenteroldes depletion had no s i g n i f i c a n t advantage over the depletion by the mould alone. No stimulatory e f f e c t could be observed by the addition of calcium, zinc or cobalt at a concentration of 1 .0 ppm., copper at a concentration of 0 . 0 5 ppnu> and molybdenum at a concentration of 0 . 0 2 ppm. Molybdenum and zinc showed a s l i g h t i n h i b i t o r y e f f e c t . - 4 -Basal medium and depleted basal medium were extracted with 8-hydroxyquinoline and chloroform. Part of both media underwent further treatment; hydrolysis f o r t h i r t y minutes with HCI ( f i n a l concentration of 0.1N). In a l l cases the e f f e c t of added i r o n at a concentration of 0.1 mg. FeSO^\/lO ml. medium was very pronounced i f manganese was also present. The addition of 0.01 FeS04 was as i n f l u e n t i a l as 0.001 mg. or 0.01 mg. FeSO^lO ml. medium. The a d d i t i o n a l procedure of hydrolysis apparently removes some factor other than i r o n , manganese or magnesium which i s necessary f o r the optimal growth of L. mesenteroldes. I t was shown that t h i s factor was not tryptophane, zinc or copper. The e f f e c t of i r o n on two other heterofermentative and two homofermentative b a c t e r i a was determined. One hetero-fermentative organism (species of Leuconostoc) showed stimu-l a t i o n with the addition of i r o n and manganese; however, the other one belonging to t h i s type (E. coll)and the two homo-fermentative bacteria (S. l a c t i s and S. f a e c a l i s ) gave very l i t t l e response even a f t e r the addition of manganese, magnes-ium and i r o n . Respiration studies of L. mesenteroldes were carried out. No difference i n acid production or carbon dioxide evolution per c e l l could be observed with varying amounts of manganese added to the growth medium. I wish to extend my sincere thanks to Dr. J. J. R. Campbell for his encouragement and assistance during the course of this thesis, and to Dean B. A. Eagles for his valuable suggestions and criticisms i n the experimental work. R.E.T. TABLE OF CONTENTS INTRODUCTION . 1 PART I SECTION I THE ISOLATION OF LEUCONOSTOC MESENTEROIDES Introduction \u2022 \u2022 . .2 Methods . 4 Results and Discussions . . . . . . . . . . . . 7 PART I SECTION I I THE USE OF HYDROGEN PEROXIDE FOR THE SELECTIVE ISOLATION OF LACTIC ACID BACTERIA Introduction . . . . . . . . . . 1 0 Methods . . . . . 13 Experimental and Discussion \u2022 \u2022 . \u2022 \u2022 14 Summary . . . . . . . . . . . . . . . . . . . . 24 PART II THE MINERAL REQUIREMENTS OF LEUCONOSTOC MESENTEROIDES Introduction 25 Methods . 37 Experimental \u2022 .42 Discussion. \u2022 \u2022 56 Summary . . . \u2022 \u2022 \u2022 \u2022 . . \u2022 \u2022 . \u2022 \u2022 . \u2022 \u2022 \u2022 \u2022 \u2022 6 0 PART I I I RESPIRATION STUDIES Introduction . . . . . . . . . . . . . . . . . . 61 Methods \u2022 . 63 Experimental and Discussion 65 ADDENDUM 70 BIBLIOGRAPHY 71 - 1 -INTRODUCTION When one considers the r e l a t i v e importance of the Genus Leuconostoc. not only i n i t s use i n the dairy industry but also as a t o o l i n the f i e l d of n u t r i t i o n a l research, i t i s d i f f i c u l t to understand why more attention has not been paid to t h i s group of microorganisms. The c l a r i f i c a t i o n of the place of these organisms I n the c l a s s i f i c a t i o n of bacteria has been c a r e f u l l y reviewed and ascertained by Sucker and Pederson (1930). The requirements of defined species of t h i s genus f o r c e r t a i n of the amino acids for optimal growth has been investigated (1947, 1948). However, i n comparison with the amount of e f f o r t that has been spent i n the study of the l a c t i c acid bacteria, as a whole toery l i t t l e attention has been devoted to the heterofermentative members of the group. The present work was undertaken with the object of studying i n d e t a i l the mineral requirements f o r growth of a defined s t r a i n of Leuconostoc and to compare t h i s information with data available on the homofermentative types. - 2 -PART I. SECTION I. THE ISOLATION OF LEPCOHOSTOC MESENTEROIDES Two of the major distinguishing characteristics of the Genus Lenconostoc are their a b i l i t y to produce slime on sucrose medium and their a b i l i t y to form carbon dioxide from carbohydrate. However, over a long period of time stock cultures tend to lose these distinctive properties. For this reason i t was considered desirable to make fresh isolations of cultures of Leuconostoc from natural sources. Regardless of the fact that species of Leuconostoc have been Isolated frequently, the literature supplies only scanty details as to the methods employed. Pederson (1930) isolated Leuconostoc species from sauerkraut samples by plating on Yeast Extract Agar. He found that pH 6.0 was optimum for their isolation; pH 7.0 permitting Gram negative baeteria. to predominate and pH 5*0 allowing only more acid-tolerant toacteria to grow. Leuconostoc colonies failed to appear on an agar medium prepared with sauerkraut juice, that had been fermented two days. It was also found that members of this genus developed only i n the earlier period of fermentation of sauerkraut. Hohl (1942), working on the fermentation of lettuce, isolated species of Leuconostoc on Tomato Juice Tryptone Agar. In other work (1940) he observed that the addition of yeast extract to the isolation medium was advantageous. Sadler et a l (1930) described species of Leuconostoc from Kingston cheese, which were isolated on Peptic Casein Digest Agar and on Yeast Extract Agar at pH 6.8. Orla Jensen (1919) isolated Leuconostoc from such sources as sugar beets, cheese, milk, cow manure, and sauer-kraut. Two types of media were employed, namely, deep agar tubes and gelatin plates. I n addition, an enrichment technique, i n which the organic matter was added to s t e r i l e milk, was carried out. By incubating these tubes at definite temperatures for defined periods of time, transfer-ring to fresh tubes of milk and reincubating, certain organ-isms were permitted to grow which otherwise were crowded out* The i n i t i a l stages of the work reported upon herein were concerned with the isolation of the desired microorganisms and the various procedures employed by Pederson, Hohl and Orla Jensen were followed. METHODS Samples of the following materials were obtained as probable sources of species of Leuconostoct cheese, milk, soured potato, cow manure, and sliming cabbage, lettuce and cucumber. Sauerkraut and ensilage were prepared i n the laboratory. Preparation of sauerkraut. Alternate layers of shredded cabbage and salt ( f i n a l concentration of two percent) were firmly packed into a t a l l enamel container and a heavy, snugly f i t t i n g weight placed on top to ensure anaerobic conditions. The fermentation was carried out at room temperature. Preparation of ensilage; The same procedure as far sauerkraut employing pea shells, corn kernels and corn stalks was carried out. Approximately one percent molasses were added i n the case of the ensilage fermentation. Daily samples were taken from the brine solution of both preparations and plated on a variety of media. Two general methods were carried out i n an attempt to isolate species of Leuconostoc from these natural materials. The f i r s t method involved the diluting and plating of the natural materials onto a variety of media. Three basic media were employed for this purpose; namely, yeast Sryptie Digest Agar, Tomato Juice Agar, and least Tryptic Digest Gelatin \u2022 Flasks or tubes of each of these three media were adjusted to pH 6 .9) 6 . 2 and *>.6 respectively, before autoclavlng; making i n a l l , a total of nine plating media. Both pour plates and - 5 -streak plates were prepared in the case of all samples. The incubation temperature was 25\u00b0C. The second method employed was a modification of Orla Jensen's enrichment technique. One gram of material was weighed out and aseptically transferred to each of six tubes of a specified medium. The media employed were Cabbage Broth, Yeast Tryptic Digest Broth, Tomato Juice Broth, Milk and Yeast Peptone Milk. To obtain anaerobic conditions in half of the tubes, sterile paraffin wax was melted and added to three tubes of eaeh medium. Members of the Genus Leuconostoc have a relatively low optimum temperature and therefore, stand a better chance of outgrowing other bacteria at the lower temperature ranges, especially under anaerobic conditions* To this end one aerobic and one anaerobic tube of each medium were incubated at each of three temperatures - 20\u00b0C, 25\u00b0C and 30\u00b0C. At 48 hour' intervals, 1 ml. from each aerobic tube was transferred to a fresh tube of the same medium. Transfers were made from the anaerobic tubes after gas formation had appeared. When transfers were made plates of Yeast Tryptic Digest Agar and Yeast Tryptic Digest Agar Gelatin were inoculated. Typical colonies of lactic acid bacteria Isolated by both methods were transferred into Yeast Tryptic Digest Broth, pH 6.8. Catalase tests were carried out and al l tubes giving positive results were discarded. From the catalase negative tubes transfers were made onto Sucrose Gelatin Agar Slopes to test for slime production, and into Yeast Peptone Milk and 6 Yeast Sucrose Broth (both containing st e r i l e paraffin,plugs) to test for gas production* Only those cultures producing gas and slime were retained. These cultures were purified by repeated plating and picking of typical colonies. To ascertain the genus and species to which the purified cultures belonged, the characteristics were established by the routine tests .employed for the classification of the Lactic Acid Bacteria. - 7 RESULTS AND DISCUSSION. Isolates of Leuconostoc were obtained from only two of the nine materials employed; namely, sauerkraut and ensilage. Such a predominance of catalase postive, proteolytic bacteria were present i n five of the remaining 7 materials that the relatively weaker Leuconostoc. i f present at a l l , were unable to survive even under the favourable anaerobic conditions employed. Although isolates from milk and cheese were undoubtedly l a c t i c acid bacteria none proved to be of the gas production variety. Leuconostoc species were found to be relatively easy to Isolate from sauerkraut and ensilage. In agreement with Pederson (1930) this genus predominated i n sauerkraut between the second and fourth days of fermentation. At less than two days proteolytic bacteria were the main type,while at greater than four days acid tolerant l a c t i c acid rods were present. With ensilage, however, i t required seven days before Leuconostoc became established. The cultures were isolated by the f i r s t method described. This method i s based on the fact that i n certain materials; such as sauerkraut, organisms arise i n a definite sequence and no elaborate enrichment technique i s necessary for their isolation. The second method i s designed to aid i n the Isolation of a specific type of organism from a mixture of several types. By varying the pH of the medium, the length of incubation, the temperature, etc., conditions w i l l be, made favourable for the development of specific groups of organisms* Although Orla Jensen and other workers have obtained results with this method, a l l attempts to use this techniqure for the isolation of Leuconostoc met with f a i l u r e . Yeast Tryptic Digest Agar would appear to be the most favourable medium for the isolation of members of the genus Leuconostoc for i t was on this medium that a l l cultures obtained for purification were isolated. Gelatin i s usually added to stock medium because of i t s protective action for bacteria. However, this characteristic of gelatin was not effective for the isolation of Leuconostoc i n this study. She results obtained indicate that a pH between 6.2 and 6.8 i s optimum for members of this genus. Cultures from ensilage arose at either of these two pH's while those from sauerkraut came up only at the higher pH. A pH lower than 6.2 i s definitely too acidic for the growth of Leuconostoc. The characteristics of the three cultures retained for investigation are given i n Table I. From the results of the f i r s t six tests employed for purposes of classification and from the fact that the three organisms show a marked yeast incidence, i t can be concluded that the organisms isolated belong to the genus Leuconostoc. Because of their ability to ferment sucrose they can be classified as either Leuconostoc mesenteroldes or Leuconostoc dextranlcum. As they are also capable of fermenting the pentoses, arabinose and xylose, i t can therefore be concluded that a l l three - 9 -cultures are stra i n s of Leuconostoc mesenteroldes* TABLE I Cha r a c t e r i s t i c s of the Isolated Species of Leuconostoc T4 T7 T14 Source of Culture Ensilage Sauerkraut Sauerkraut YTDA pH6.2 YTDA pH6.8 YTDA pH6.8 7 days 2 days 4 days Morphology small d i p - small d i p - large d i p -l o c o c c i ; l o c o c c i ; l o c o c c i ; chains chains chains Satalase _ - -Gas Production - YPM Gas Production - YSB + Slime Formation - SGA Optimum Temperature 25\u00b0-30\u00b0C 25\u00b0-30\u00b0c 25\u00b0-30\u00b0c F i n a l pH i n Flucose 4.4 4.4 4.4 NH. from Arginine - \u2022 \u2014 Sodium Hippurate S p l i t - - \u2014 Aesculin S p l i t \u2022 G e l a t i n Liquefied - - -Type of L a c t i c Acid L a e v o - Laevo- Laevo-rotary rotary rotary Yeast Milk 6.8 6.8 7.0* Milk 1 .8 2.0 1.5 Xylose 3.4 3.2 3.2 Arabinose 2.5 2.3 2.7 Sucrose 4.5 4.5 4.5 Maltose 3.6 3.6 3.2 Lactose 2.5 2.7 2.7 Raffinose 3.3 3.2 3.6 S a l i c i n 2.3 2.3 2.3 Trehalose 4.1 4.1 4.1 A l l figures given are grams of l a c t i c acid produced per l i t e r ef medium. - negative r e s u l t . \u2022 p o s i t i v e r e s u l t . - 10 -PART I. SECTION XI. THE USE OF HYDROGEN PEROXIDE FOR THE SELECTIVE ISOLATION OF LACTIC ACID BACTERIA. The d i f f i c u l t i e s encountered i n the Isolation of species of the Genus Leuconostoc when employing either of the tech-niques as described i n Section I led to experimental work whieh resulted i n the adoption of a selective method for the isolation of l a c t i c acid bacteria. The necessity for work-ing out some such method became apparent when i t was observed that except for certain materials i n which bacteria arise i n a definite sequence, i t had proved impossible by the methods previously adopted, to r i d the natural materials of proteo-l y t i c types of microorganisms with which the heterofermenta-tlve l a c t i c acid species, being present i n much smaller quantities, were i n competition. The use of hydrogen perox-ide as a selective agent to inhibit or k i l l off certain groups of microorganisms was attempted and a method employing this reagent evolved. The bactericidal effect of hydrogen peroxide has been known for some time. As early as 1893 Travgott showed that various pathogenic organisms differed i n their degree of sensitivity to hydrogen peroxide. Freer and Novy (1902) found that active organic peroxide; such as, benzoyl acetyl or acetyl peroxide, were at least one hundred times as active as hydrogen peroxide. Using peroxide i n a concentration of \u20220% active oxygen a l l S.pyogenes and B.pyocyaneous c e l l s were k i l l e d i n fifteen minutes. E. c o l l was found to be more resistant than the Streptococci, some cells remaining v i a b l e a f t e r s i x t y minutes of treatment. M'Leod and Gordon (1923) proposed a c l a s s i f i c a t i o n of bacteria on the basis of catalase production and peroxide s e n s i t i v i t y . According to t h i s c l a s s i f i c a t i o n evolved by these workers the l a c t i c acid bacteria f a l l i n t o Group I I ; i . e . , those bacteria which are devoid of catalase and are only moderately sensitive to hydrogen peroxide. A number of workers i n I t a l y (1943,1945,1948) have published data on the use of hydrogen peroxide f o r the s t e r -i l i z a t i o n of milk. They found that o.2# hydrogen peroxide, added to milk, gave a permissable b a c t e r i a l count i n 19-15 hours. However, t h i s s t e r i l i t y was only an apparent one because af t e r 30 hours the b a c t e r i a l count was greatly increased over the o r i g i n a l . The i n t e n s i t y of the action * o depended on the temperature; 15-17 C being the most e f f e c t i v e range. Hydrogen peroxide has been employed to destroy E . c o l i i n milk used i n cheese manufacturing. Niven (1949) held milk with added hydrogen peroxide at 40\u00b0C f o r 40 minutes. A f t e r t h i s treatment catalase was added promptly to destroy any remaining peroxide. He found that i n t h i s way coliform organisms could be eliminated, leaving the majority of l a c t i c acid bacteria unharmed. Recently, Nambudripad and Laxminarayana (1949) in v e s t -igated the b a c t e r i c i d a l e f f i c i e n c y of perocide on seven species or organisms Isolated from milk. They found that 100 ppm hydrogen peroxide, f o r 100 percent destruction of - 12 c e l l s , required 2*5 hours for S.lactis and S.bulgaricus. hut only 45 minutes for E.coli and 30 minutes for A*aerogenes. B.subtilis and B.megatherium, however, required 18 hours treatment before being k i l l e d * . , The relative insensitivity of the l a c t i c acid bacteria to the action of hydrogen peroxide was used to advantage i n the development of an isolation technique for this group of organisms* The conditions requisite to the isolation of laetie acid bacteria i n the presence of hydrogen peroxide are described below* 13 METHODS In order to determine the conditions best stilted for the selective isolation of this broad group of organisms studies were at f i r s t carried out on a typical strain of S.laetls. For comparative purposes, parallel studies were made employing E.coli as the test organism. Unless other-wise stated fresh raw milk was the medium used for the suspension of the organisms during the period of inhibition. The milk was dispensed into test tubes i n 10 ml quantities to which were added the appropriate amounts of a 1$ solution of hydrogen peroxide and o. l ml of a 6 hour culture of the particular organism under study. This procedure gave an i n i t i a l bacterial count of from one to ten million per ml. The tubes containing the peroxide and microorganisms were placed i n a constant temperature water bath for incubation, after which, samples were removed to determine the number surviving. Counts were made i n accordance with the Standard Methods Plate Count. EXPERIMENTAL AND RESULTS In that a f i n a l concentration of 0 .2$ H9O2 was used by the Italian workers for s t e r i l i z i n g milk and o.05% H 2 0 2 was employed by Niven i n the manufacture of cheese, these two concentrations were compared with respect to their effective-ness i n destroying calalase positive microorganisms. The temperature employed was 28\u00b0C. Samples were removed at 30 and 60 minutes intervals and after 18 hours incubation. From the results recorded i n TAble II, i t i s seen that E.coli was almost completely destroyed whereas nearly 80% of the ce l l s of S.lactis remained viable after one hour of treatment with 0 . 0 H . 2 0 2 . A concentration of \u00a9.2$ H 2 0 2 was even more effective i n the destruction of E. c o l i . This concentration was also more active against S. l a c t i s when held for 60 minutes or longer. Since 0.05$ ^ 2^2 S i v e s a clearer indication of the trend of the percent of surviving organisms at the various stages of treatment, this concentra-tion was employed i n subsequent experiments. Increased temperatures usually intensify the action of an inhibition; however i n the present case the picture i s complicated by the rate of destruction, of the peroxide and by the influence of temperature on growth rate of the organ-isms. For this reason an experiement on the influence of temperature on the germicidal action of peroxide was carried out, Table III. These results confirm the Italian report that 18\u00b0C i s optimum for the Inhibitory effect of peroxide. The findings TAPES U The Influence of Hydrogen Peroxide Concentration on the Survival of S. l a c t i s and E. c o l l i n Raw Milk Escherichia c o l l Streptococcus l a c t i s H 2 \u00a9 2 3 cone. % 'ime of In-hibition Survivors % H 2 \u00b0 2 cone. % Time of In-hibition Survivors % \u00a9.\u00a95 o , o 5 o.\u00a95 30 min. 60 min. 18 hrs. 4.26 0.0031 0 . 0 0 0 0.05 0.05 0.05 30 min. 60 min. 18 hrs. 6 5 . 3 7 9 . 0 7.9 0*20 0 . 2 0 0.2\u00a9 30 min. 60 min. 18 hrs. 0.0149 0 . 0 0 0 0 . 0 0 0 0 . 2 0 0 . 2 0 0 . 2 0 30 min. 60 min. 18 hrs. 6 9 . 0 4 3 . 6 \u00a9\u2022\u00a900 - 16 T&BT.R TTT The Influence of Temperature on the Germicidal Action of .05$ Hydrogen Peroxide. Escher Lchia c o l i Streptococcus l a c t i s Period of Inhibition Temperature of Reaction Survivors Period of Inhibition Temperature of Reaction oc Survivors * 30 min 15 .0244 30 TBJTl 15 27.10 30 min 20 .088? 30 min 20 22.30 30 min 25 \u20220620 30 25 25.00 30 min 30 .0431 30 30 40.60 60 min 15 .0058 60 min v> 19*20 60 min 20 .0188 60 min 20 15.00 60 min 25 .0191 60 min 25 27.50 60 min 30 .0093 60 min 30 13.33 4 hrs 1? .0015 4 hrs 15 18.4 4 hrs 20 .0065 4 hrs 20 12.7 4 hrs 25 \u2022006 4 hrs 25 10.0 4 hrs 30 \u2022006 4 hrs 30 2.2 - 17 reported upon herein indicate that B. c o l i i s most sensitive to peroxide at 15\u00b0G. S. l a c t i s . on the other hand, after one hour, appears to be favoured at both 15\u00b0C and \u00a75\u00b0C. However, the results obtained after four hours treatment show that 15\u00b0G i s the optimum. Therefore, on account of the sensitivity of E. c o l i and the relatively strong resistance of S. l a c t i s at 15\u00b0C, this temperature was ehosen for further experimental work. Italian workers have claimed that milk must be absolutely fresh i f peroxide i s to be expeeted to k i l l bacteria. For this reason an experiment was set up employing raw milk held at 15\u00b0C over a period of 24 hours. At definite time intervals a portion of the milk was removed and used as the medium of suspension. Observations were made of a l l samples after sixty minutes treatment with hydrogen peroxide. The results given i n Table IV indicate that as the age of the milk increases, the germidical action of the peroxide decreases i n the case of E. c o l l and S. l a c t i s . However, as interest l i e s i n destroying catalase positive bacteria while retaining catalase negative ones, i t would appear desirable that fresh raw milk be employed for experimental purposes. Fresh raw milk was also compared with nutrient broth and reconstituted milk. It i s evident from the results recorded i n Table V that raw milk i s a superior f l u i d for the selective isolation of l a c t i c acid bacteria. Fresh raw milk would appear to possess - 18 TABLE i y The Influence of the Age of Raw Milk Employed on the Survival of S\u00bb la c t i s and E. c o l l i n the Presence of .05% Hydrogen Peroxide Age of Raw. Milk Hours E. c o l l S. l a c t i s Survivors $ Survivors % 0 0.086 39.1 2 0.684 61,1 4 0.378 79.0 16 0.186 64.5 24 1.22 85.0 19 -a protective action for S. l a c t i s . which i s not to he found i n either reconstituted milk or nutrient broth. The nature of this protective factor i s unknown. TABLEJ\/. Influence of Suspending Fluid on the Qermldical Action of .05$ Peroxide Suspending Fluid E. c o l i S. l a c t i s Survivors t Survivors Raw Milk 0.179 49.1 Reconstituted Milk 20.30 5.8 Nutrient Broth 0.03 15.5 Using the optimum conditions described above; namely, .05$ hydrogen peroxide at 15\u00b0C for 60 minutes i n fresh raw milk, eight additional organisms were tested for the effect of hydrogen peroxide inhibition. Negative organisms employed were L.mesenteroides. S. bovis. Tbm. casei and S. faecalis. while the catalase positive were A. aerogenes. Ps. aeruginosa and two strains of Micrococci. From the data recorded i n Table VI i t would appear that conditions established for S. l a c t i s and E. c o l i w i l l permit the isolation of other catalase negative organisms and at the same time drastically reduce the number of catalase positive bacteria. The two Micrococci cultures tested are an exception to the general rule that catalase positive organisms are sensitive to peroxide. The normal habitat for Micrococci i s TABLE VI Germicidal Action of Hydrogen Peroxide on Microorganisms Organism Survivors % Streptococcus l a c t i s 52.4 Leuconostoc mesenteroides 22.5 Streptococcus bovis 29.1 Thermobacterium easel 49.9 Streptococcus faecalis 71.7 Tetraooccus sp. 69.5 Micrococcus sp* 30.0 Escherichia c o l i 0.18 Aerobacter aerogenes 8.6\u00a9 Pseudomonas aeruginosa 0.21 very similar to that for the catalase negative organisms employed; namely, milk and products therefrom. Thus i t i s quite possible that this i s the reason why the Micrococci resemble the lacties rather than the catalase positive bacteria i n this respect. Although a method i s found to be effective for single cultures of bacteria i t does not necessarily follow that i t w i l l be just as efficient when mixed cultures are employed. To determine whether this technique can be used to isolate l a c t i c acid bacteria from a mixed culture, 8. l a c t i s and E\u00bb c o l i were added to raw milk and treated with o.05$ hydrogen peroxide and the usual determinations carried out. The results are given i n Table VII. TABLE YIX Germicidal Action of .05$ Hydrogen Peroxide on a Mixed Culture of E. c o l i and S.lactis I n i t i a l . Count After Peroxide Treatment S. l a c t i s E. c o l i S. l a c t i s and E. coll 8,200,000 8,000,000 13,700,000 5,700,000 0 5,700,000 From the counts obtained before and after peroxide treat-ment i t would appear that i n the mixed culture a l l E. c o l i c ells have been destroyed while 5,700,000 S. l a c t i s c e l l s reamined viable i n each ml. of suspension. Tubes of B r i l l i a n t Green B i l e Broth were inoculated i n parallel to the plates and - 22 -the data from this test confirmed the conclusion that a l l cel l s of E. c o l i had been k i l l e d . To test the usefulness of this procedure for isolation purposes, a sample of fresh cow manure was treated with hydrogen peroxide. The general method was carried out with the exception that either one or two ml. of a age to ten dilution of the manure was added to the ten ml. of milk. Previous attempts to isolate Leuconostoc from manure resulted i n failure because of the overwhelming presence of catalase positive organisms. However, from the data compiled i n Table VIII, i t would appear that, by the use of hydrogen peroxide, the percentage of catalase negative bacteria appear-ing on a plate increases from approximately 3% to 40-45$ when peroxide i s added. The apparent discrepancy i n the results with B r i l l i a n t Green B i l e Broth i s probably due to the fact that a good portion of the catalase postive bacteria remaining are Micrococci. The organic nature of manure w i l l tend to supply a protective coating for a l l bacteria present. Thus the plate counts are not reduced after treatment with peroxide as drastically as one might suspect. T A B L E ; viia; The Influence ef Hydrogen Peroxide on the Survival of Microorganisms i n Cow Manure Number c e l l s \/ Survivors B.6.B.B. Catalase Test ml* % 5 tubes % Positive % Negative 1 ml. Manure 1,220,000 5+ 96.9 3.1 1 ml* Manure & fQ5% H 20 2 270,000 22.1 2+ 55.5 45.5 1 ml. Manure & \u2022 2$ H 20 2 47,500 3.89 63.4 36.6 2 ml* Manure 2,360,000 5* 96.4 3.6 2 ml. Manure & .0% H 20 2 550,000 23.3 3 4 84.3 15.7 2 ml. Manure Sc .2% H 20 2 300,000 12.7 l + 56.6 43.4 24 SUMMARY 1* Strains of Leuconostoc have been isolated from Sauerkraut after fermentation had proceeded for two to four days, and from Ensilage after seven days incubation. 2* The strains Isolated have been shown to be similar i n a l l respects to Leuconostoc mesenteroldes. 3. The most efficient medium for their isolation has been found to be Yeast Tryptic Digest Agar at a pH between 6.2 and 6.8. 4. Hydrogen Peroxide (.05$) at 15\u00b0C for 60 minutes has been employed effectively for the selective isolation of l a c t i c acid bacteria i n the presence of large numbers of other types or organisms. 25 PART I I . TEE MINERAL REQUIREMENTS OF LEUCONOSTOC MESENTEROIDES In an effort to further our knowledge of the part played by specific organisms i n the ripening of Cheddar Cheese a study of the nutritive requirements particularly with respect to minerals, a freshly isolated strain of Leuconostoc mesen-ter old es was carried out. Very l i t t l e information i s available on the trace mineral requirements of these heterofermentative bacteria and for this reason an intensive study was undertaken. The f i e l d concerned with the mineral requirements of microorganisms i s a d i f f i c u l t one i n which to work because of the problem arising from the infinitesimal quantities required by organisms for growth. There are various theories as to why certain minerals accelerate or inhibit bacterial growth. Between 1907 and 1925 the idea of physiological antagonism of ions was brought out. Flexner (1907) showed that sodium chloride was toxic to the Neisseria genus and that toxicity could be neutralized by calcium or potassium salts. Llpman (1909) demonstrated that sodium chloride, i n a concentration of O.LM, stimulated the ammonia production of B.subtilis but that potassium chloride, sodium and magnesium chloride were toxic at this same concentra-tion. However, sodium chloride at 1.3M concentration was also found to be toxic. Antagonism between sodium and calcium, sodium and magnesium, and sodium and potassium, but not between magnesium and calcium, could be demonstrated. Brooks (1919) found that antagonism occurred between - 26 magnesium chloride and sodium chloride with B. suhti l l s hut only slightly between magnesium chloride and calcium chloride. According to Osterhout (1919), those salts which have an opposing effect on the permeability of a c e l l w i l l antagonize each other. Therefore, because sodium chloride produces an increase i n permeability i t w i l l be antagonized by calcium chloride or other salts which produce a decrease i n permeability. It was shown by Holm and Sherman (1921) that the hydrogen ion concentration of the medium modifies the effect of salts on permeability. They found that the accelerating effect of certain salts upon the growth of organisms was due, primarily, to an increase i n the velocity of growth during the period of maximal multiplication, while at the same time the duration of the lag phase was decreased. Falk (1923) and Shaughnessy and Winslow (1927), i n confirmation of the previous workers, found that sodium chlor-ide broadens the optimum zone of hydrogen ion tolerance while calcium salts tend to narrow i t . This i s explained on the basis of a permeability effect. Weaker solutions of sodium or calcium increase permeability while stronger concentrations decrease i t . Closely related to permeability i s the idea put forth by Fabian and Winslow (1929), Winslow and Doloff (1928) and by Winslow and Haywood (1931) that the supposed specific effects of cations at low concentrations are due to the fact that different cations have different \"quantitative potencies\" and that antagonistic or stimulatory effects w i l l show up i n - 2 7 -accordance with the quantitative relationships Involved. This i s borne out by Hotchkiss (1923) who found that at c e r t a i n concentrations a l l cations were stimulatory while at greater concentrations these same cations were i n h i b i t o r y . However, the stimulatory and i n h i b i t o r y concentrations of cations d i f f e r widely one from the other; i n other words, they d i f f e r i n t h e i r degree of potency. As Winslow and Doloff point out, even lead and mercury have been found to be stimulatory i n extremely low concentrations while sodium Is t o x i c i f a s u f f i c i e n t l y night concentration i s employed. Bivalent ions have a \"quantitative potency\" 8 - 1 0 times as great as univalent ions. For example; 0.1M sodium chloride or potassium chloride i s stimulatory while s i m i l a r concentra-tions of calcium chloride or magnesium chloride are t o x i c , while an element such as lead i s t o x i c at .0005M concentration. A d i f f e r e n t theory was proposed by McCalla (1940). He put f o r t h a theory on c a t i o n i c adsorption by bacteria. He states that bacteria contain proteins, are negatively charged and are nearly c o l l o i d a l i n size and that since proteins and inorganic c o l l o i d s possess an adsorption capacity f o r cations, i t i s possible that the b a c t e r i a l c e l l functions as an agent i n the adsorption of p o s i t i v e l y charged ions. As he states: \" I f a b a c t e r i a l c e l l i s placed i n a medium contain-ing various cations there i s an exchange of ions between the solution and the c e l l u n t i l an e q u i l i b -rium i s established comparable with the energy of adsorption of the ions and t h e i r d i s t r i b u t i o n i n the Ions and their distribution i n the solution* If an ion i s used by the organism and becomes an intimate part of the c e l l , i t i s possible that this process could remove an ion from the adsorption sphere. Consequently a further shift i n the equilibrium would be expected and another ion would be removed from the solution to replace the f i r s t . \" 8 Such an adsorption of cations by a bacterial c e l l from solution indicates that the c e l l may be able to concentrate these ions as a store for future use. Many workers report the stimulatory action upon micro-organisms at low concentrations of various minerals; however, essentiality at very low concentrations has rarely been determined. Gottbrecht (1880) was the f i r s t worker to show a stimu-latory effect of a metal on a microorganism. He found that thallium tartarate stimulated yeast growth and fermentation. Buromsky (1912) demonstrated that magnesium was essential for the growth of A. nieer and was not replaceable by calcium, while Frouin and Guillaumie (1926) showed essentially the same effect for B. tuberculomas. Simultaneously with Buromsky, Bertrand (1912) showed that manganese was needed i n the growth of A. nieer cultures. Busk, Lineweaver and Horner (1932) found that magnesium and iron over ranges of 0.1 - 3 mM and .001 - .015 mM, respectively, were highly stimulatory to the growth processes 29 of Azetobacter in both free and fixed nitrogen* In further work by Burk and Horner (1934) i t was observed that magnesium could not be replaced by any other trace mineral* About the same time Mueller (1935) was able to demon-strate that potassium and magnesium stimulated the growth of the diphtheria bacillus* A year later Karasch, Conway and Bloom (1936) showed that large concentrations of copper and manganese (Is 50,000) inhibited Spirillum rubrum while smaller concentrations lead only to a loss in pigmentation* The inhibitory effect could be counteracted by liver extract* Lodge and Hinshelwood (1939) working with B. lactis- aerogenes, demonstrated that a definite concentration of magnesium was necessary for growth of this organism in a medium containing only glucose, ammonium sulfate and mono-potassium phosphate* Furthermore, with an increase in the magnesium concentration the lag phase shortened accordingly* Mclntyre, Riker and Peterson (1941) tested the effect of iron, zinc, manganese and copper in the growth of Phytomonas tumefaeiens. They observed that the first three elements were stimulatory in concentrations of 5, *5, *l\/ml* medium, respectively* Copper was stimulatory under no circumstances, and zinc was found to be effective only in the presence of iron and manganese* Robinson (1932) working with Pseudomcnas. found that magnesium and phosphate were essential for growth, but sodium, potassium and calcium were unnecessary* On the other hand, Pandalai and Roa (1942), although they confirmed the essential - 30 nature of phosphate for Pseudomonas. could find no need for magnesium. However, Burton (194-7) brought forth data agree-ing with Robinson1 s observation that magnesium i s necessary for growth of Pseudomonas. In work on E. c o l i by Young, Begg and Pentz (1944) magnesium and iron were demonstrated to be necessary elements for normal growth. Other elements such as Ca, Co, Nl, Mn, Zn, etc., gave no stimulation either alone or i n combination with magnesium. Work has also been carried out on the mineral nutrition of Bacillus s u b t i l i s . Feeney and Garibaldi i n 194-7 showed that K, Mg, Fe, Mn, and Zn, were a l l essential for growth of this organism. The potassium could be replaced by rubidium; the zinc, by cadmium. Previous studies by Feeney, Mueller and Miller (194-3) on C. tetani and C. dlnhtherlae. showed that iron was necessary for growth. Other investigators have demonstrated iron to be either stimulatory or essential to the growth of bacteria. Bortels (1927), using highly purified solutions found that both iron and zinc were needed by A. nlger. According to Henley (1925) \u00bb Frouin and Guillamie (1924), Long and Seibert (1926) and Reed and Rice (1928) iron influences favourably the growth of the tubercle bacillus. Similar stimulation was found by Karasch, Conway and Bloom (1936) for Spirillum rubrum. and by Elvehjem (1931) for yeast cultures. The latter showed also that the addition of both copper and iron gives a further Increase i n growth. - 31 -Waring and Workman (1942) established the iron require-ments of strains of Ps. aeruginosa. A, lndologenesf A.aerogenes. K. pneumoniae, and E. coli. For maximal growth the latter four require iron in a concentration of about .02 - .03 ppm, while Ps. aeruginosa requires about .0? - .08 ppm. Iron has also been shown to be essential for optimal growth of B. suls (1932), (194-7) and for R. t r l f o l l l (1945) at levels of about .1 ppm, and .06 ppm, respectively. In 1936 Pappenheimer and Johnson observed that optimal growth of C. diphtheriae occurred at the same level of iron as that required for toxin production. Further work by Pappenheimer and Shaskan (1944) established that for C.welchil \u20223 ppm iron, which was optimal for toxin production, was the minimum amount necessary for normal growth. Manganese has been added to media for lactie acid bacteria since 1937, but i t was not until 1939 that the stimulatory effect of this element was definitely noted. Moeller (1939), showed that in a highly purified medium of a synthetic nature manganese was necessary for the growth of Streptobacterium plantarum. Wooley (1941) working on the pantothenic acid content of natural products, observed that some substances produced more rapid growth of the lactic add bacteria than did optimal amounts of pantothenic acid. She effective material in these products proved to be manganese. However, this influence of manganese was only on the rate of growth, not on its extent. - 32 -The nutritive requirements of the l a c t i c acid bacteria i n general have been Investigated extensively by Orla Jensen (194-3)* Using milk, with 12.5 mg\/1. magnesium chloride added, as the medium he found that Thermobacterla and Streptococci grow just as well with as without manganese. On the other hand, a l l Streptobacteria and some strains of Betacoccl were stimulated by manganese. A l l Be. cremoris strains studied needed no additional manganese, while two out of fiv e strains of Be. bovls and one out of five strains of Be. arablnoseus showed stimulation by i t s addition. He states thats \"The possible difference between manganophilic and * non-manganophille bacteria i s more quantitative i n nature; i.e., a l l l a c t i c acid bacteria require manganese, but not i n greater quantities than are already present i n the nutrient medium.\u2022 Whereas, the previous workers have shown that manganese i s stimulatory for various l a c t i c acid bacteria, Barton-Wright (1945) found that the S. faecalis strain employed by him was unique among the lactics i n that i t required only potassium for normal growth. Magnesium, manganese and iron had no influence on this organism. Rogosa (1944) also found that Ibm. easel responded quantitatively to increasing con-centrations of potassium, and that sodium or ammonia could not replace the potassium. Several papers have been published by Snell et a l (1945) (1947)(1948) on the mineral requirements of the l a c t i c acid bacteria. They demonstrated by the depletion method used, - 33 that manganese was necessary for the growth of L\u00bbmesenteroldes, a l l Lactobacilli and probably for S, faecalls. and that man-nesium was stimulatory, but not essential. Tests with ferrous ion showed no influence. It was also found that potassium ion was essential i n comparatively large amounts for a l l the l a c t i c acid bacteria investigated. In a later paper. McLeod <\u00a7fa Snell (1948), working specifically on their potassium requirements showed that the magnitude of the requirements of the bacteria studied (L.arabinosus. L. casle. S. faecalls and L. mesenteroldes) were increased by the addition of sodium and ammonium. A competitive relationship appeared to be present between the essential element, potassium, and these ions; i.e., i t depended upon the ratio between potassium and sodium or ammonium whether or not the latter two were inhibitory. In this paper they also showed that rubidium could replace potassium completely for growth or Sc.faecalls. and L. easel. partially for L. arabinosus. for one strain of L.mesenteroldes. but not at a l l for the other strain. Caesium or lithium did not replace potassium as an essential element for any of the organisms studied. Svec (1948) found that i n a medium giving no test for molybdenum, cobalt or copper, none of the Lactobacilli under investigation were influenced by these ions when added i n concentrations of ,01 - .1 mg\/1., a range expected to give stimulation i f the organisms required the trace mineral. Any concentration over 1 mg\/1 copper, 20 mg\/1 cobalt and 170 mg.\/l molybdenum were inhibitory for a l l three Lactobacilli, - 34 -Zinc i n a concentration not exceeding 0*1 mg\/1. exhibited a s l i g h t stimulatory e f f e c t . Svec could show no influence on the growth of L. casei or L. delbrueckii when manganese and magnesium were omitted. However, the absence of these two elements was detrelmental to L. arabinosus. The only workers to demonstrate any effect of calcium were Meinke and Holland (1948). 0*1 mg* Calcium\/10 ml. medium had a pronounced effect on growth of L. d e l b r u e c k i i . but not on L. mesenteroides. Chattaway, Happold and Sandford (1943) while investigating the influence of the Inorganic content of the medium on r i b o f l a v i n assay, showed that the acid produced can be altered by a v a r i a t i o n i n the concentra-t i o n of calcium. Beyond the optimum concentration (.35 ml* of , % solution of CaCl2) acid production decreased markedly. The r e l a t i v e lack of information about the exact mineral n u t r i t i o n of bacteria i s undoubtedly due mainly to the absence of a medium s u f f i c i e n t l y free from trace elements to reveal marked d e f i c i e n c i e s . Results have been obtained f o r yeast and fungi without the aid of depletion methods, but because bacteria usually require \"less than 1\/10 the amount of mineral needed by yeast and mould\" (Waring and Werkman (1942), work on bacteria has not met with much success. Attempts have been made to overcome t h i s d i f f i c u l t y by the development of various depletion methods. Steinberg (1935) employed the adsorption method f o r the removal of ions. The addition of calcium carbonate to the medium supplies a source of calcium f o r the formation of a - 35 -precipitate to adsorb traces of heavy metals* He found that by using this method, the addition of zinc and iron would produce a f i f t y - f o l d increase i n growth. Elvehjem ( 1 9 3 D also used the adsorption method i n yeast studies* In these cases yeasts and moulds were employed and the adsorption method proved to be successful* However, later workers such as Mueller (1941) , Burk and Horner (1934) and Perlman (1945) have shown that this method i s unsatisfactory for bacteria* Burk and Horner (1934) employed the recrystalization of chemicals for studies i n mineral nutrition. However, practic-a l l y a l l other investigators i n the f i e l d have found i t to be unsuccessful* Another method, which can be used effectively for a simple mineral medium, but which becomes too involved for the more complex types composed of amino acids and vitamins, i s the ion exchange procedure* Perlman (1945) employed this method effect-ively for a study of the mineral requirements of A* aerogenes. The mould purification method brought out by Molisch (1892) and used later by Molliard ( 1929) , although time consuming, has wielded excellent results with A. indologenes (Waring and Work-man, 1942) . A modification of the method has been employed by Bentley et a l (1947) and by McLeod and Snell ( 1947) . Bentley et a l depleted their medium for the microbiological assay of manganese by permitting a manganese requiring organism, L. arabinosus. to grow for 24 hours. McLeod & Snell found that for certain bacteria this depletion was not sufficient. They c a r r i e d out additional depletion using a yeast culture. Waring and Workman (1942) used a method of extraction employing 8-hydroxyqulnoline and chloroform. Rubbo, Goldacre & Balfour (1947) state that \" t h i s type of compound combines with polyvalent metals, such as copper, i r o n , manganese and zinc, and forms complexes lacking the c h a r a c t e r i s t i c properties of metal ions\". Albert et a l (1947) state \"The e f f i c i e n c y of removal of ions such as manganese and magnesium depends on a l k a l i n i t y of the medium, and i t i s improbable that anything more than p a r t i a l removal of magnesium i s possible.\" However, Waring and Werkman obtained excellent r e s u l t s with t h i s method i n t h e i r studies on the i r o n requirements of heterotrophic bacteria. On the basis of the findings reported i n the l i t e r a t u r e the depletion techniques of both Bentley and Molisch were employed i n the study of the mineral n u t r i t i o n of L.mesenteroldes. - 37 -METHODS L. mesenteroldes T7, isolated at two days from sauerkraut was employed for a l l experiments. A repetition of a l l tests one year after isolation showed that to a l l appearances the culture had not varied i n characteristics from those f i r s t determined\u2022 Stock cultures of the organism were refrigerated after incubation for 24 hours at 30\u00b0C. i n medium of the following compositions Tryptone .5$ K p H P 0 4 .2% Yeast Extract . % Glucose .1$ Gelatin 4 % Agar .5-.7% PH 7.0 The organism was transferred at least twice at 24 hour intervals before being used for experimental work. The compo-sit i o n of the transfer medium employed w\u00a7s: Tryptic casein digest 33.3$ by volume. Yeast Extract 1 Glucose \u2022: KoHP04 .1, PH 7.0 A fresh transfer was taken every week to maintain a vigourous, undlssociated culture. The basal medium used throughout the experimental work was that reported by McLeod & Snell (1947), (Table IX) The following solution of salts were also prepared i n the concen-trations indicated: MgS0A.7 Ho0 2 mg. \/ 10 ml. medium. NaCr*\" * .1 mg. \u00bb \u00bb \u00ab FeS0 4.7 HP0 .1 mg. n \u00ab \u00bb M11SO4.7 B2O .1 B \u00bb \u00bb \" - 38 -TABLE IX Composition of Basal Medium (McLeod and S n e l l , 1947) Constituents of Medium Amount per 500 ml, double strength Amount pei 10 ml. f i n a l medium Constituents of Medium Amount per 500 ml. double strength Amount per 10 ml. f i n a l medium Casein-enzymatic Pyridoxine HCI 100 1.0 hydrolysate 200 ml. 100 mg. 1-asparagine 100 mg. 1.0 mg. Thiamine HCI 100 1.0 dl-tryptophane 50 mg. 0.5 rag. Ca pantothenate 200 2.0 Cystine 100 mg. 1.0 mg. Niacin 200 2.0 K 2HP0 4 1 gm. 10 mg. p-aminobenzoic acid 100 1.0 1 gm. 10 rag. F o l i c acid 10 0.05 Adenine sulfate 10 mg. 0.1 mg. B i o t i n 2 1.0 Quanine HCI 10 mg. 0.1 mg. R i b o f l a v i n 200 2.0 U r a c i l 10 mg. 0.1 mg. Glucose 10 gm. 100 mg. Sodium acetate 6 gm. 60 mg. R e d i s t i l l e d water added to make the double strength medium up to 500 ml. 39 -The enzymatic casein digest was prepared i n the follow-ing manner: 120 gm of G.B.I. Vitamin Test Casein was suspended i n 2 l i t e r s of 0.8$ sodium bicarbonate solution and 600 mg* of pancreatln i n 20 ml. of r e d i s t i l l e d water were miced into the solution. Toluene was added, and the mixture incubated at 37\u00b0C for 48 hrs. The mixture was then steamed for 30 minutes; 7 ml. of gl a c i a l acetic acid added, and then f i l t e r e d under suction with the aid of Celite. The f i l t r a t e obtained was stored under toluene i n the refrigerator. Asparagine and tryptophane i n concentrations of 10 mg.\/ml and 5 mg.\/ml. respectively were made up i n 0.5 ml. 20$ HC1 and diluted to 100 ml. with r e d i s t i l l e d water. Cystine was prepared i n 0.2 H HC1 at a concentration of 2.5 mg.\/ml. Due to the fact that i t i s so insoluble, the solution had to be warmed just prior to use. A solution of the purines was prepared by dissolving adenine sulphate, quanine hydrochloride, and u r a c i l , each i n 100 mg. quantities, i n 5 ml. of 20$ HC1 and diluted to 100 ml. The vitamins: pyridoxine H01, thiamine HC1, calcium pantothenate, niacin, para-amino benzoic acid and f o l i c acid were made up i n individual solutions at a concentration such that 10 ml. of each mixed together and added to one solution and diluted to 100 ml. gave the number of micrograms of each vitamin required for 500 ml. of double strength basal medium. Riboflavin was dissolved i n 0.02 N acetic acid at a concentration of 0.2 mg\/ml; while biotin was made up by diluting the contents of one v i a l (25 y\/ml). to 25 ml with - 40 -r e d i s t i l l e d water* A l l solutions of vitamins, purines and the three amino acids were stored under toluene i n the r e f r i g e r a t o r * Glass d i s t i l l e d water was employed throughout f o r a l l solutions. Pyrex glassware was used i n a l l cases and chemic-a l l y cleaned by washing with soap and water, soaking overnight i n 10$ n i t r i c acid, and then r i n s i n g with tap water, d i s t i l l e d water and f i n a l l y r e d i s t i l l e d water. To ensure the removal of the l a s t traces of n i t r i c acid and of the minerals under study the glassware was then f i l l e d with r e d i s t i l l e d water and autoclaved at 15 l b s . pressure f o r 20 minutes* In the experimental work, 5 ml. of the double strength basal medium; the appropriate s a l t s , each i n 1 ml* quantities; and r e d i s t i l l e d water to 9 ml. were added to each tube* These tubes and a 20$ sol u t i o n of glucose were autoclaved at 15 l b s . pressure f o r 15 minutes and the sugar solu t i o n was then added a s e p t i c a l l y to each tube i n 1 ml. quantities so that the f i n a l volume per tube was 10 ml. The glucose was s t e r i l i z e d separately to safeguard against caramelization* For n e u t r a l i z a t i o n of the medium r e d i s t i l l e d NH^OH was employed. The r e d i s t i l l a t i o n was ca r r i e d out by placing concentrated NH4OH i n a d i s t i l l i n g f l a s k connected to a t a l l glass graduate f i l l e d with r e d i s t i l l e d water. Very mild heat was applied to the d i s t i l l i n g f l a s k and d i s t i l l a t i o n was continued u n t i l the apparent saturation point was reached* For inoculation, a 24 hours culture of L.mesenteroides was employed. The inoculum was centrlfuged and washed twice - 4 1 with phy s i o l o g i c a l saline to remove traces of the growth medium. After the f i n a l centrifuging 10 ml. of the physio-l o g i c a l s a line were added to the c e l l s and each tube received one drop of t h i s suspension as inoculum. The inoculated tubes were incubated at 30\u00b0C f o r 48 hours, at which time t u r b i d i t y measurements and t i t r a t i o n s were made. - 4 2 TgYPTgRTMTTOT&T. The major obstacle In studying the mineral requirements of micro-organisms i s the d i f f i c u l t y encountered i n removing every trace of inorganic material. A large part of the work reported upon has thus been concerned with the evolution of s a t i s f a c t o r y methods of rendering a basal medium free of contaminating trace elements. Employing the depletion technique of Bentley et a l the f i r s t experiment was set up to determine whether or not depletion would be advantageous procedure and i f so whether a d d i t i o n a l amounts of vitamins and purines would then be required. The effect of providing a l l the necessary elements except manganese i n the medium pr i o r to depletion was also determined. This was done i n an e f f o r t to ascertain i f greater growth would r e s u l t during depletion and whether t h i s increase i n growth would r e s u l t i n a more complete removal of traces of manganese, thereby providing a medium which could be used to demonstrate r e a d i l y the need f o r t h i s element. Following i n s t r i c t d e t a i l the method employed by Bentley et a l , the culture of L .mesenteroldes was grown f o r a period of 4 8 hrs. i n the basal medium minus the four inorganic s a l t s . The c e l l s were then centrifuged down, the medium dispensed, the necessary additions made and auto-claved. Since t h i s procedure could conceivably deplete the basal medium of constituents other than the inorganic elements, f i v e variations of the technique were carr i e d out and the r e s u l t i n g media, with and without the addition of inorganic s a l t s , tested for t h e i r a b i l i t i e s to support the growth of the - 43 -test organism, Table X. The media used were as follows: 1, undepleted. 2. depleted. 3* readdition of adenine-quanine-uracil, and a l l the vitamins after depletion. 4. Basal medium containing double the normal amount of adenine-quanine-uracil, and a l l vitamins before depletion. 5. FeSC-4, M g S 0 4 , and NaCl added prior to depletion. It can be seen from Table X that manganese has a stimu-latory influence i n a l l five media, even the undepleted one. The media appear to decrease i n suit a b i l i t y for growth and acid production of the culture i n the following orders addition of vitamins and purines after depletion; undepleted; addition of double amounts of vitamins and urines before depletion; depleted; addition of iron, magnesium and sodium chloride before depletion. This trend could be due to the fact that during depletion a l l or certain of the vitamins and purines were used up beyond their optimum concentration, or that with the addition of extra amounts of these factors certain trace minerals, required by the culture, were added to the medium. Medium Ho. 5 i s the least suitable for growth, possibly because due to increased growth during depletion one or a l l of the elements added prior to depletion, or the vitamins and purines i n the medium were reduced to levels below their optimum. However, regardless of the fact that higher yields can be obtained with medium #3, exactly the same TABLE X Influence of Variations i n the Depletion Technique on Demonstrable Inorganic Requirements of Leuconostoc mesenteroldes Uninoculated control Turbidity Acid * Formed Basal medium Turbidity Acid Formed Basal Turbidity Acid Formed Basal \u2022 Mn\"*\" Fe+\u00bb.Na+~.Mg*+ Turbidity Acid Formed I. Undepleted 100 I I . Depleted 100 III. Readdition of a l l the vitamins and adenine,quanine, 100 u r a c i l after deple-tion. IV. Addition of double amounts of a l l the vitamins and adenine]. 100 quanine, u r a c i l prior to depletion. V. FeS04,NaCl, and MgSoTadded prior to 100 depletion 0.1 0.3 0.7 0.0 0.0 90.7 97.5 98.5 98.7 100 4.3 2.1 2.8 2.2 1.4 65.0 69.7 57.7 63.5 76.0 5.7 4.1 6.8 5.0 3.5 59.3 65.0 52.0 58.3 5.3 4.2 6.7 4.9 +Hephelometer reading, percent light transmitted * ml. of N\/10 NaOH required to neutralize 10 ml. medium - 45 -trend can apparently be obtained by the less complicated procedure of simple depletion. Although there was no increase i n acid formation with the addition of iron, sodium and magnesium i n a l l cases a very slight increase i n growth was recorded. Following Bentley 1 s technique, L. arabinosus was employed as the depletion organism. However, with the result-ing medium L. mesenteroldes failed to grow even after the addition of sterile yeast extract or vitamin solutions. In further studies on the depletion of the basal medium a strain of the mould Aspergillus nieer.was tested. The double strength basal medium with 0,5% glucose added, was Inoculated with spores of the mould, and incubated for 12 days at 30\u00b0C. The mat was then removed and the extraneous growth centrifuged off. As i n the previous experiments the depleted medium was also tested for the effect of the purines and the vitamins on growth and acid production. The follow-ing media were employed: 1. Mould depleted. 2. Readdition of purines and a l l vitamins after mould depletion. 3. Basal medium contains double quantities of purines, and vitamins before mould depletion. The media were neutralized, dispensed and autoclaved and their a b i l i t i e s to support the growth of L.mesenteroldes determined. The results are given i n Table XI. TABLE XI Influence of Depletion by A. nieer on Demonstrable Inorganic Requirements of Leuconostoc mesenteroldes. Depleted Medium Turbidity^ Acid* Formed Readdition of Purines and Vitamins aft e r Depletion  Turbidity Acid Formed Double Amounts of Purines and Vitamins prior to Depletion Turb i d i t y A d d Formed Uninoeulated control Basal medium Basal Basal Basal Basal Basal Basal Basal Basal Basal Basal Basal Mn ** Fe** Mg +* Ha* Mh*+, MC> Mn* , Mg**, ffin Mn* +J N a \" Mg Fe** Fe** Mg** Na* Na* Fe** Mn Na*, ,Mg~ Fe 100 96.0 90.0 98.0 93.5 98.0 88.5 93.2 62.5 88.0 93.0 63.5 69.0 85.0 0.1 2.2 5.5 2.1 3.1 2.2 5.4 3.1 6.4 5.4 2.9 6.4 6.0 5.6 100 98.5 91.0 99.0 96.5 99.0 94.5 69T5 92.0 68.0 71.0 88.0 1.5 2.3 4.4 2.4 3.5 2.3 4.5 6 \" a 4.8 f.3 6.0 5.0 100 97.5 93.0 97.5 96.5 99.0 93.0 73.0 89.5 73.0 70.0 86.5 2.8 5.2 2.5 3.2 2.0 5.2 6~1 5.1 6.2 6.2 5.4 *Nepholometer reading, percent l i g h t transmitted. *ml. of N\/lfr NaOH required to neutralize 10 ml. medium. Results reported are the average of duplicates. 47 At 24 hours there was growth only In the tubes to which both manganese and magnesium had been added. However at 48 hours there was growth i n a l l tubes. In a l l three media i t was possible to demonstrate a need for magnesium. However manganese has to be present before the magnesium has any significant influence. Even with mould depletion there i s no evidence of a need for iron or sodium. There i s the possibility that the mould produced traces of c i t r i c acid which bound magnesium as a complex. However, analysis failed to reveal the presence of any citrate. It can be seen that the addition of extra amounts of vitamins and purines has no advantageous effect on either growth or acid production. An experiment was then set up combining the two preced-ing methods of depletion. The basal medium was depleted for 12 days with A. nieer. the growth eentrifuged off, the pH brought back to 7.0, and the medium reautoclaved. It was then depleted with L. mesenteroldes for 48 hours. It would appear from Table XII that this additional depletion has no significant advantage over the depletion by the mould alone. In another experiment calcium, zinc and cobalt at con-centrations of 1 ppm; copper at a concentration of .05 ppm, and molybdenum at a concentration of .02 ppm were added to a mould depleted medium singly and i n various combinations. However, no stimulatory effect was observed with any of these trace minerals; molybdenum and zinc showed a slight inhibitory - 48 -TABLE XII Influence of Variations i n the Depletion Technique on Demonstrable Inorganic Requirements of Leuconostoc Mesenteroldes f Mould Depletion 12 days Mould Depletion 12 days Leuconostoc 2 days Turbidity \u00bb Acid formed Turbi d i t y Acid formed Uninoculated control 100 0.5 100 ' 0.5 Basal medium 99.0 1.2 98.0 1.3 Basal \u2022 Mn*** 88.7 3.6 83.5 4.6 Basal + Mg*\"* 96.0 2.5 98.0 2.1 Basal * Fe*+ 97.0 1.4 98.2 1.5 Basal + Mn**, Fe** 87.0 3.5 80.0 4.5 Basal + Mg**, Fe** 93.0 2.6 95.0 2.4 Basal + Mn**, Mg** 69.7 5.4 66.7 5.4 Basal * Mn**,Mg+*,Fe4+ 68.5 5.5 67.7 5.5 *Nepholometer reading, percent l i g h t transmitted *ml. .IN NaOH required to neutrali z e 10 ml. medium Results are average of duplicates. 49 -e f f e c t . In the r e s u l t s obtained from these studies, i r o n , the mineral of prime i n t e r e s t , has not been found to be necessary or even stimulatory f o r the growth of L. mesenteroldes. Waring and Workman (194-7) have found that at pH's ranging from s i x to eight repeated extractions with 8-hydroxyquinoline and chloroform w i l l remove trace amounts of Cu, Fe, Mn and Zn. Although the method undoubtedly i s more e f f i c i e n t i n a simple mineral medium, i t was used i n t h i s instance f o r extraction purposes on the basal medium. Both undepleted and mould depleted medium were extracted i n accordance with Waring and Workman's method. The r e s u l t i n g media were dispensed, the desired additions made, autoclaved and f i n a l l y inoculated with L. mesenteroldes. Employing t h i s accepted procedure of depletion the stimulatory effect of i r o n was found to be pronounced, Sable X I I I . There i s evidence that a factor other than manganese, magnesium and i r o n i s the l i m i t i n g agent i n the extracted, depleted medium. The concentration of i r o n i n the previous experiment was such that the p o s s i b i l i t y that trace minerals, occurring as contaminents i n the added i r o n , are causing the stimula-t i o n , cannot be overlooked. To check t h i s p o s s i b i l i t y , a further experiment was set up varying the amounts of i r o n present from 0.01 Yto 0.1 mg. FeSO^TB^O\/lO ml. medium. In t h i s series of experiments the procedure was not l i m i t e d to three extractions with 8-hydroxyquinoline,but whs c a r r i e d out TABLE X I I I Influence of Added Minerals on the Growth \u00a9f Leuconostoc mesenteroldes i n Media Extracted with 8-hydroxyquinoline Undepleted medium Depleted medium Extracted with Extracted with Unextraeted 8-hydroxy- Unextraeted 8\u2014hydroxy-aulnoline auinoHne > Turbidity K A e i d * Turbidits Acid Turbidity Acid T u r b i d i t j Acid formed formed formed formed Uninoculated control 100 0.9 100 0.7 10\u00a9 0.5 100 0.3 Basal medium 87 3.2 89 2.9 89 3.0 93 1.1 -Basal 4 Mn** 52 7.6 76 4.9 58 6.8 78 3.7 Basal \u2022 Mn*\"*, Mg** 5\u00a9 6.5 74 5.1 57 6.7 79 3.9 Basal \u2022 Fe*1\"*\" 87 3.3 92 2.1 86 3.1 98 0.7 Basal \u2022 Mn**, Fe** 56 6.4 54 7.8 58 6.8 63 5.0 Basal * Hn**,Mg**,Fe** 50 7.3 62 6.0 58 6.8 65.5 5.3 Nepholometer reading, percent l i g h t transmitted. *ml. of N\/1\u00a9 NaOH required to neutralize 10 ml. medium. Results reported are the average of duplicates. u n t i l no green colouration appeared i n the chloroform layer. Six repeated extractions with the 8-hydroxyquinoline and chloroform were required. After the last extraction the procedure for washing the medium was altered to six washings with 15 ml. of chloroform and two with 25 ml. quantities, with a lapse of thirty minutes between the last two washings. One half of the extracted medium was then hydrolyzed i n a boiling water bath for thirty minutes with HCI ( f i n a l concentration of 0*15), cooled, washed twice with 25 ml. chloroform, neutralized, and dispensed along with the portion extracted by the usual procedure. This technique was adopted on the basis of the evidence adduced by F. W. Tanner, J r . (1950) who showed that 8-hydroxyquinoline w i l l not completely extract a l l the cations i n the presence of amino acids unless a mild hydrolysis i s carried out. It would appear from the results with either the extracted or the extracted and hydrolyzed media that iron i s definitely stimulatory, Table XIV. However, this occurs only i f manganese i s also present. I f the addition of iron was acting only as a factor to overcome the inhibitory effect of 8-hydroxyquinoline, the tubes containing iron i n a concentra-tion of 0.01 r\/10 ml. medium would probably show no reasonable effect. The fact that 0.01 y of FeS04 with manganese or with manganese and magnesium i s as in f l u e n t i a l as .001 mg. or .01 mg. FeS0 4 would seem to indicate that iron i s needed for optimum growth and i t i s not to be attributed to the overcom-ing of an inhibitory influence. The stimulatory effect i s - 52 -TABLE X I V Influence on Leuconostoc mesenteroldes of Variations i n Concentration of Iron i n 8-hydroxyquinoline Extracted Medium Extracted Extracted -Hydrolyzed Turb-i d i t y Acid formed Turb-i d i t y Acid formed Uninoeulated control 100 0 . 4 100 0.5 Basal medium 99.7 0.7 99.5 0.9 Basal \u2022 Mn** 91.2 5.9 91.5 3.2 Basal 4 Mg** 98.2 0.95 98.0 1.05 Basal 4 Fe** .1 mg. 100 0.5 97.5 1.3 Basal + Fe** \u202201 mg. 100 0.5 97.5 1.1 Basal Fe** \u2022001 mg. 100 0.6 98.0 1.0 Basal + Fe** .01 r 100 0.6 97.7 1.1 Basal 4 Mn**, ,Mg** 68.7 6 . 4 90.00 3 . 4 Basal 4 Mn**, \u201e 44 , ,Fe fl mg. 67.7 6.8 90.00 4 . 2 Basal + Mn**. ,Fe**.01 mg. 67.7 6.5 90.00 3.6 Basal 4 Mn*4, ,Fe + 4.001 mg. 66.5 6.6 91.0 3 . 4 Basal 4 Mn**, ,Fe**.01 * 70.0 6.5 89.5 3.6 Basal 4 Mg*4, , F e 4 4 . l mg. 97.0 1 . 4 97.0 1.5 Basal + Mg4*, ,Fe*4.01 mg. 97.5 1.1 98.0 1.6 Basal 4 Mg44, ,Fe 4 4.001 mg. 98.0 1.1 97.0 1.3 Basal + Mg**, ,Fe**.01 v 97.0 1.1 97.5 1.3 Basal 4 Mn**, ,ffig**,Fe44.lmg. 68,5 7.3 89.0 4 . 1 Basal 4 Mn**, ,Mg**,Fe44.01mg. 69.0 6.6 90.5 3.5 Basal 4 Mn , 4+ 4+ Mg ,Fe .OOlmg 72.5 6.0 92.5 3.2 Basal Mn**, Mg**,Fe4+.01y 68.0 6.5 91.0 3.3 Iron concentrations are the amount of FeSO4.7H.2O i n 10 ml. medium 53 also shown not to he due to trace elements added as contaminants i n the i r o n . Although the same general picture holds f o r both the extracted and extracted-hydrolyzed medium with regard to the stimulatory e f f e c t of manganese and i r o n , the a d d i t i o n a l procedure of hydrolysis apparently removes some fa c t o r e s s e n t i a l f o r optimum growth other than manganese, magnesium and iron * The amino acid, tryptophane i s destroyed by acid hydrolysis, and i t was, therefore, thought that t h i s could possibly be the e s s e n t i a l missing factor* An experiment was set up i n which tryptophane, and also zinc and copper i n concentrations of ,5 mg\/10 ml* medium; 1 ppm and *05 ppm, respectively, were added to the medium singly and i n combina-t i o n with manganese, magnesium and iron * However, these additions showed no apparent influence on growth or acid production* This influence of i r o n and manganese has been shown f o r only one s t r a i n of L. mesenteroldes* To determine whether th i s e f f e c t was general f o r heterofermentative bacteria or merely s p e c i f i c f o r just the one s t r a i n , and to observe the e f f e c t on c e r t a i n homofermentative species, an experiment was set up employing three heterofermentative and two homoferment-ative bacteria* Two strains of L. mesenteroldes were used; T7, the culture under study, and T4, i s o l a t e d from ensilage* Cultures of E. c o l i . S. f a e c a l l s . and S. l a c t i s were also employed. - 5\"4 -Ferrous chloride which had been purified by the ion exchange method was used i n this experiment as the source of- iron. Both Leuconostoc cultures exhibited a stimulation on the addition of iron, although with Leuconostoc T 7 the effect was much more pronounced, Table XV. The fact that Leuconostoc T4 produced much better growth than did T7 would seem to Indicate that the former i s much less fastidious with respect to i t s growth requirements. Thj\u00a9 medium, even with addition of manganese, magnesium and iron appears to be unfavourable for the development of E. c o l l . S. l a c t i s and S. faecalls. TABLE XV The Influence of Iron on Microorganisms i n Medium Extracted with 8-hydroxyquinoline L. mesenteroldes T7 L. mesenteroldes T4 E. c o l i S.faecalls S . l a c t i s + Turb-i d i t y Acid* formed Turb-i d i t y Acid formed Turb-i d i t y Acid formed Turb-i d i t y Acid formed Turb-i d i t y Acid formed Uninoculated control 100 0.6 100 0.6 100 0.7 100 0.7 100 0.7 Basal medium 99 1.3 68 2.0 92 1.3 94 1.2 98 0.9 Basal + Mn* 93 2.0 59 4.1 90 1.5 93 1.3 98 1.0 Basal * MS*,Mg* 95 1.9 60 4.0 87 1.7 93 1.3 98 1.1 Basal \u2022 F$*2ppm 97 1.5 63 2.6 91 1.4 94 1.2 100 1.0 Basal * Fe*20ppb 98 1.4 65 2.1 89 1.5 95 1.2 99 1.0 Basal *\u2022 Mn*\",Fe*2ppm 81.5 3.6 57 4.6 86 1.9 93 1.4 97 1.1 Basal + Mn*,FS*20 ppb 86 2.6 58 4.3 86 1.8 94 1.3 97 1.1 Basal+Mn* ,Mg*Fe*2ppm 75.5 4.3 56 4.9 87 1.6 93 1.4 97 1.2 Basal*Mll*M|fFe*20pph 87 2.2 56 4.8 87 1.5 93 1.3 98 1.2 Ion exchanged ferrous chloride was employed. The concentrations are the amoun \"^Nepholometer reading, percent l i g h t transmitted. *ml. N\/10 NaOH required to neutralize 10 ml. medium. present. - 56 -DISCUSSION The experimental r e s u l t s presented demonstrate that with the depletion technique employed, magnesium and manganese are ess e n t i a l f o r the growth of Leuconostoc mesteroides. D e f i n i t e evidence has not been obtained that i r o n i s e s s e n t i a l f o r t h i s microorganism but i t has been shown, however, to be stimulatory i n media extracted with 8-hydroxyquinoline and o chloroform* The observation that manganese i s e s s e n t i a l f o r t h i s p a r t i c u l a r s t r a i n of L. mesenteroldes confirms the work of McLeod and S n e l l (194-7) on the two strains of t h i s species used by them. I t i s int e r e s t i n g to note that while they were able to show the e s s e n t i a l i t y f o r manganese by employing L. arabinosus as the organism f o r depletion, any attempt to use the same organism i n t h i s work resulted i n f a i l u r e * Reasons f o r the f a i l u r e to obtain growth of t h i s p a r t i c u l a r s t r a i n of L. mesenteroldes on such a medium could not be adduced. As f a r as i s known t h i s i s the f i r s t time magnesium has been shown to be e s s e n t i a l or even stimulatory f o r Leuconostoc mesenteroldes. McLeod & S n e l l , by the addition of c i t r a t e to the medium, showed such a small increase i n growth with the addition of 1000 ppm. Mg** plus 100 ppm. Mn** as to be almost i n s i g n i f i c a n t . . However, with the mould depletion technique employed i n t h i s work a vigorous stimulation could be observed with only 19.7 ppm Mg** plus 2.4 ppm. Mn**. One major d i f f i c u l t y encountered In the use of the depletion technique i s the p o s s i b i l i t y that, during the growth period of the depletion organism, substances which w i l l subsequently i n h i b i t the bacteria under i n v e s t i g a t i o n , w i l l be introduced i n t o the medium. This i s a possible explanation of the lack of growth when L. arabinosus was employed. In c e r t a i n cases i t i s possible that substances formed during depletion w i l l make no difference to the o v e r a l l picture. An example of t h i s i s c i t r i c acid. Campbell and Gunsalus (1944), showed that c i t r i c acid forms complexes with c e r t a i n m e t a l l i c ions thus i n h i b i t i n g growth. McLeod and S n e l l made us of t h i s fact to show that magnesium was stimulatory for c e r t a i n organisms. Another example i s that of 8-hydroxyquinoline which has been shown to exert an i n h i b i t o r y influence on organisms, (Albert et a l , 1947). This compound acts as a chelating agent, thus removing e s s e n t i a l trace minerals from the medium. Amino acids and 8-hydroxyquinoline have c e r t a i n degrees of a f f i n i t y f o r various cations and although i n c e r t a i n instances the s t a b i l i t y constant of oxine i s l e s s than that of the amino acids, i t i s able to compete f o r the cations due to i t s high i o n i z a t i o n constant at an a l k a l i n e pH (Albert, 1950). Thus i n the depletion technique with 8-hydroxyquinoline, although there w i l l be competition between the oxine and amino acids present i n the medium for the trace minerals present, oxine should produce the greater quantity - 58 -of metal complex. These complexes formed are presumably removed from the medium by treatment with chloroform. Gale & M i t c h e l l (1949) demonstrated that by using a casein digest medium, 15 oxine\/ml. would i n h i b i t growth of Staph, aureus f o r 48 hours; the i n h i b i t i o n being caused by the complexes formed. I t i s possible that t h i s amount could remain i n the medium af t e r chloroform treatment, thus i f ions such as i r o n and manganese, as i s the case i n the findings recorded herein, are able to reverse t h i s i n h i b i t i o n , i t would indicate that these elements were es s e n t i a l f o r the metabolism of the organism. This i s borne out further by the fact that no influence was observed i n the case of copper or z i n c . The r e s u l t s obtained with i r o n are of prime i n t e r e s t due to the possible significance they may have on the r e l a t i o n s h i p between the metabolism of homofermentative and heterofermenta-t i v e l a c t i c acid bacteria. I t i s an accepted fact that anaerobes have no cytochrome oxidase, peroxidase or catalase, and therefore no system to take intermediate compounds to oxygen without the formation of peroxide. Because i r o n i s i n t r i c a t e l y related t o t h i s scheme i t has been generally assumed that anaerobes have no need f o r i r o n . Kubowitz (1934) working on the effect of earbon monoxide on CI. butylicum fermentation and l a t e r Pappenheimer and Shaskan (1944) working on the re l a t i o n s h i p of carbon monoxide and i r o n on CI. w e l c h i i and CI. acetobutylicum disproved t h i s assumption. Waring & Workman (1944) were able to show s i m i l a r r e s u l t s f or A . indologenes. I n a l l cases the - 59 -organisms studied, when grown on an iron deficient medium or i n the presence of carbon monoxide reverted from the normal heterofermentative system to a pure l a c t i c acid type. Obviously an iron enzyme - not cytochrome - i s involved i n the normal fermentative pathway of these heterofermentative organisms. Home-and hetero-fermentatlve l a c t i c acid bacteria differ mainly i n that the former produces 90-9% l a c t i c acid (Orla Jensen, 1919 and Hucker, 1928) while the latter produce not more than 50$ l a c t i c acid; the other 50% being composed of aleohol, volatile acid and carbon dioxide (Hucker and Pederson). Therefore, i t seems reasonable that L. mesenteroldes could possibly have an enzyme requiring iron, either i n i t s structure or as a coenzyme. This supposition i s supported by the fact that while the two Leuconostoc cultures showed stimulation on the addition of iron, S. faecalls and S. l a c t i s were unaffected. - 60 -SUMMARY 1 . The influence of manganese, magnesium and i r o n on the growth of Leuconostoc mesenteroldes T7 has been determined, 2. Regardless of the method of depletion of the medium the e s s e n t i a l nature of manganese was demonstrated. 3. Magnesium was only stimulatory i n a medium which had been depleted f o r twelve days by A. nieer. and then only i n the presence of manganese. 4 . The readdition of vitamins and purines to media a f t e r depletion, although increasing growth and acid production, had no great influence on the o v e r a l l picture. 5. In a medium extracted with 8-hydroxyquinoline and chloroform the stimulatory e f f e c t of i r o n i n concentrations from . 0 1 1 - . 1 mg.FeSO^.7H 2 0\/10 ml. was demonstrated. Further extraction of t h i s medium by a mild hydrolysis gave e s s e n t i a l l y the same r e s u l t s . Apparently an unknown f a c t o r was removed by t h i s a d d i t i o n a l treatment. Experiments showed that t h i s factor was not tryptophane, zinc or copper. - 61 -PART I I I - RESPIRATION STUDIES With the demonstration that manganese was e s s e n t i a l f o r the growth of Leuconostoc mesenteroldes i t was hoped that by a consideration of the r e l a t i v e quantities of carbon dioxide and acid produced on the addition of increasing amounts of th i s mineral to the growth medium., the ro l e of t h i s i o n i n the metabolic system of Leuconostoc could be determined. The greater part of the work on enzymes and t h e i r r e l a -t i o n to metabolic systems has been car r i e d out on tissues and yeast extracts and on is o l a t e d enzymes from these extracts. Although what has been shown to be true f o r such extracts does not necessarily hold f o r b a c t e r i a l preparations, i t i s now an accepted f a c t that anaerobic bacteria d i s s i m i l a t e carbohydrates by way.of the Embden-Meyerhof system. The l i t e r a t u r e shows that magnesium exerts a stimulatory e f f e c t on c e r t a i n enzymes i n t h i s system, nearly a l l of which are concerned with phosphorylation processes. Erdtman ( 1 9 2 7 ) , (1928) , found that f o r dialyzed kidney magnesium was stimulat-ory i n r e l a t i v e l y low concentrations, calcium was not i n f l u e n -t i a l , and zinc was i n h i b i t o r y . The opposite e f f e c t of magnes-ium was demonstrated i n bone enzyme phosphatase by Hammerberg (1929) . According to data obtained by Jenner and Kay (193D a l l animal phosphatases - kidney, bone, l i v e r , blood c e l l s , etc., are activated by magnesium. Phosphorylase, the enzyme catalyzing the reaction glycogen to glucose - 1 - phosphate, i s apparently a protein-Mg-adenylic a c i d . This f a c t was brought out by Cor i and Cori(1936) - 6 2 -and l a t e r by Green et a l (1942) working with minced frog muscle and an aqueous extract of rabbit muscle, respectively. Magnesium was also shown to be associated with phospho-glucomutase by Gori and Cori (1938). Najjar (1948) i s o l a t e d t h i s enzyme from minced rabbit muscle and confirmed the former workers 1 observation that magnesium at a concentration of \u00bbG05 - .0025 M. and cysteine were stimulatory f o r optimum a c t i v i t y . Manganese and cobalt could replace the magnesium. K i e l y and Meyerhof (1948) observed that adenosine-triphosphatase i s o l a t e d from the leg muscle of rate, was ' strongly activated by magnesium, s l i g h t l y by manganese and not at a l l by calcium, barium or cobalt. Erythrocyte pyrophosphatase was found to be i n a c t i v e without magnesium and Inhibited by calcium, copper, zinc and other metals (Naganna and Menon, 1948). Work on the b a c t e r i a l phosphatases of Bact. a c l d l  propionic! was carried out by Pett and Wynne (1933). Of the numerous metals t r i e d , magnesium was the only one which accelerated the a c t i v i t y of the enzyme. Thus magnesium i s apparently a stimulatory factor i n most phosphorylation processes, at least i n animal tissues and i n yeast extracts. On the other hand, the l i t e r a t u r e contains only scanty evidence f o r the need of manganese as an activator i n the anaerobic d i s s i m i l a t i o n of carbohydrates. Warburg and C h r i s t i a n (1941) Isolated enolase from yeast and animal tissue and found that the protein alone did not become active u n t i l magnesium, manganese, or zinc were added. - 63 -In order to determine the influence of manganese on the r e s p i r a t i o n of Leuconostoc mesenteroldes experiments employing the Warburg technique were carr i e d out, METHODS A 24 hour culture of Leuconostoc mesenteroldes i n 10 ml, of Yeast Tryptic Digest Broth was centrifuged down, washed twice with phy s i o l o g i c a l s a l i n e , and resuspended i n 5 ml* of s a l i n e , 2*5 ml* of t h i s suspension was added to a f l a s k con-tai n i n g 250 ml, of growth medium. The growth medium employed was mould depleted basal medium. The f l a s k s were Incubated at 30\u00b0C f o r 48 hours, a f t e r which time 10 ml, of the culture was removed and the amount of acid produced during incubation determined. The remainder was centrifuged f o r 12 minutes. The c e l l s were then washed twice with h a l f the o r i g i n a l volume of physiological s a l i n e . A f t e r the f i n a l washing the c e l l s were suspended i n enough sa l i n e so that 0.2 ml. of the c e l l suspension, made up to 10 ml., gave a reading of 70-73% on the nepholometer. The suspensions were used immediately a f t e r being prepared. A conventional Warburg apparatus was employed. In a l l experiments the volume of each f l a s k was 3*3 ml. Each f l a s k contained 0.5 ml. NaHCO^ buffer, 1 ml. c e l l suspension and phys i o l o g i c a l saline to make the t o t a l volume to 3*3 ml. One sidearm held 0.3 ml. of .3N HCI, while the other (except those of the endogenous fla s k s ) held 0.4 ml. sucrose. White phosphorous, to take up oxygen, was placed i n the centre w e l l . -64 -The substrate, sucrose, was made up so that 0*2 ml. was equivalent to l8uM 0 2\u00bb In order to obtain an environment of pH 7\u00ab8 with an atmosphere of 2.5$ carbon dioxide i n nitrogen, \u20222117 mg. NaHCO^ made up to 100 ml. was used (Umbreit,1948). A l l glassware employed, including the Warburg flasks, was chemically cleaned with 10$ n i t r i c acid. A l l solutions \u2022 were made up with glass d i s t i l l e d water. - 65 -EXPERIMENTAT. fiwp DISCUSSION Preliminary experiments were carr i e d out with Leuconostoc mesenteroldes\" to determine the optimum conditions f o r a c t i v i t y . From these experiments i t was found that by using sucrose rather than glucose as substrate, a much higher production of both acid and carbon dioxide resulted over the course of one hour. The optimum pH f o r maximum a c t i v i t y was between 7.4 and 7 * 8 , increasingly lower pH's allowing the l i b e r a t i o n of COgbefore the reaction was complete. Apparently no great difference r e s u l t s i n a c t i v i t y with increasing age of culture; c e l l s grown f o r 48 hours were equally as active as those grown f o r 24 hours. Therefore, using sucrose as substrate, 48 hour c e l l s and a rea c t i o n medium of pH 7 * 8 , Warburg runs were carr i e d out i n order to determine whether or not manganese was concerned i n the breakdown of sucrose to l a c t i c acid and other products of the Embden-Meyerhof system. An experiment was carried out to f i n d i f the amount of growth and acid production varied according to the amount of manganese present. Manganese i n concentrations from 0 to 2,4 ppm. Mn*\"* was added to tubes of mould depleted basal medium. As can be seen i n Table XVI, with an increase i n manganese, the aci d production increased accordingly. Thus i t was possible to prepare the following f l a s k s f o r inocula-t i o n : - 66 -TABLE XVI The Influence of Varying the Amount of Manganese on the Acid Production of Leuconostoc mesenteroldes Varying Amounts of Mn** 0 .05 ppm. 0.1 ppm. lppm 2 .4 ppm Basal medium 3.6 5.1 5.8 7.1 7.1 Depleted basal medium 0 . 4 0 . 4 0.6 1.6 3.6 Depleted basal medium \u2022 Mg** 19.7 ppm. 0 . 4 2 .4 2.8 3.9 4 .0 Figures are the ml. .IN NaOH required to neu t r a l i z e 10 ml .medium - 67 -1. Depleted haaal 4 .05 ppm Mn** 2 . Depleted basal 4 \u20221 ppm Mn** 3 . Depleted basal 4 2 . 4 ppm Mn*+ 4 . Depleted basal 4 .05 ppm Mn** +19.7 ppm Mg** 5. Depleted basal 4 .1 ppm Mn** 419 .7 ppm Mg4\"* 6 . Depleted basal 4 2 . 4 ppm Mn** 419 .7 ppm Mg** 7 . Depleted basal 4 2 . 4 ppm Mn** \u2022Mineral s o l u t i o n 8 . Depleted basal 4 2 . 4 ppm Mn** \u2022Mineral s o l u t i o n * 1 9 . 7 Ppm Mg++ The mineral s o l u t i o n consisted of calcium, zinc, cobalt i n concentrations of 0.05 ppm. and copper and molybdenum i n concentrations of .05 ppm. and .02 ppm., re s p e c t i v e l y . 5 ml. of a 20$ sucrose solution was added a s e p t i c a l l y to each f l a s k a f t e r s t e r i l i z a t i o n . In order to measure carbon dioxide evolution and acid production four Warburg f l a s k s are required. Flask I and I I both contain NaNCO^, c e l l s and sa l i n e ; and Flask I I contains i n addition, sucrose. These two were tipped immediately a f t e r equilibrium had been reached. This was done i n order to determine the amount of carbon dioxide present as b i c a r -bonate (Flask I) and the amount present i n sucrose (Flask I I ) . Flaste I I I and IV contained the same substances as I and I I , r e s p e c t i v e l y . A f t e r equilibrium had been reached the sucrose i n Flask IV was tipped i n and both were shaken i n the water bath u n t i l the reaction was considered complete. At t h i s time the acid was tipped i n . - 68 -At the same time another four f l a s k s which were r e p l i c a t e s of the f i r s t four, except that manganese to a f i n a l concentration of 2 ppm. was added to the main f l a s k , were prepared. This was done i n order to determine whether or not the addition of manganese would influence the carbon dioxide evolution and acid production of the c e l l s . The endogenous C0 2 evolution i s the difference between Flasks I and I I I . The C0 2 produced by L. mesenteroldes i s the difference between endogenous C0 2 evolution and Flask IV. I f any a c i d i s formed during the reaction i t combines with the ions of the bicarbonate and releases C0 2. Therefore, a c i d produced can be measured by subtracting the amount of C0 2 retained i n the buffer of Flask IV just p r i o r to tipping from the amount i n the o r i g i n a l bicarbonate. I f i t can be shown there i s no difference i n a c i d pro-duction or carbon dioxide evolution per c e l l with varying amounts of manganese, then i t can be said that the manganese i s apparently not concerned with the enzyme system Involved i n the d i s s i m i l a t i o n of sucrose to l a c t i c a c i d . This i s thus the case with a l l data compiled from the above experi-ments. At no time, even with the addition of the mineral s o l u t i o n to the growth medium or the manganese to the Warburg f l a s k , was there any s i g n i f i c a n t difference i n the a c i d production or carbon dioxide evolution per c e l l ; approximately 28 mm3 C O 2 and 40 mm3 of acid being formed i n a l l cases. However, i n that manganese i s needed f o r growth, i t w i l l be incorporated i n t o the protein constituents of the b a c t e r i a l c e l l . Now, i f the acid production and carbon dioxide evolution need only one-tenth t h i s amount, the quantity necessary w i l l already be present and therefore, no e f f e c t w i l l be apparent. Therefore, although i t cannot be d e f i n i t e -l y concluded that manganese i s not needed i n the d i s s i m i l a t i o n of sucrose by Leuconostoc mesenteroldes, there i s an i n d i c a -t i o n that i t i s not e s s e n t i a l or stimulatory. This i s supported by the f a c t that, thus f a r , no report has been made i n the l i t e r a t u r e on the stimulatory e f f e c t of manganese on the Bmbden-Meyerhof system. - 70 -ADDENDUM Yeast Tryptic Digest Agar Tryptic casein digest Yeast extract e Agar Tomato Juice Agar Yeast extract Tomato j u i c e Tryptone Agar l.< 10.( l.< 1.! Yeast Tryptic Digest G e l a t i n Trypt i c casein digest Yeast extract KpHPO^, Glucose Gelatin Peptone Milk Bacto peptone Skim milk Yeast Sucrose Broth Tryptone K 0 H P O 4 Sucrose Yeast extract Sucrose Gelatin Agar Tryptone Yeast extract K 9 H P O 4 Sucrose Gelatin Agar Cabbage Broth Tryptone Cabbage juice KpHP04 Glucose 1.00 10.0% 0.2$ 1.0% by volume by volume by volume 33.3$ 1.0% 0.2$ 0 .55 12.05 0.% 11 gm. to 100 ml.water 0.2$ 2. \" 0, 0.% 0.2% 1.0% by volume - 71 -BIBLIOGRAPHY Al b e r t , A.. Rubbo, A. A., Goldacre, R. J . , and Balfour, G. B., (1947). Influence of chemical c o n s t i t u t i o n on a n t i -b a c t e r i a l a c t i v i t y . Part I I I A Study of 8-hydroxyquino-l i n e and related compounds, B r i t . Jour. Exp. Path.28.69. Albert, A., (1950), A v i d i t y of f o l i c acid and other pteridines fo r ions of heavy metals, Comm. Biochem. Soc. A p r i l . Barton-Wright, E. 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I., (1944), The inorganic nutrient requirements of E. c o l l . ? Arch. Biochem. jjfj. 121. Zobell, C. E, and Meyer, K. F., (1932), Metabolism studies on the Brucella group, nutrient requirements i n synthetic media. Jour. Infect, Dis. 51. 344. ","@language":"en"}],"Genre":[{"@value":"Thesis\/Dissertation","@language":"en"}],"IsShownAt":[{"@value":"10.14288\/1.0106731","@language":"en"}],"Language":[{"@value":"eng","@language":"en"}],"Program":[{"@value":"Animal Science","@language":"en"}],"Provider":[{"@value":"Vancouver : University of British Columbia Library","@language":"en"}],"Publisher":[{"@value":"University of British Columbia","@language":"en"}],"Rights":[{"@value":"For non-commercial purposes only, such as research, private study and education. 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