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STUDIES with RESAZURIN REDUCTION by DICKSON L. S. LIU

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STUDIES with RESAZURIN REDUCTION by DICKSON L. S. LIU STUDIES WITH RESAZURIN REDUCTION by DICKSON L. S. LIU B. S., Taiwan Provincial Chung-Hsing University, 1962 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in Agricultural Microbiology in the Division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1966 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for ex• tensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives! It is understood that copying or publication of this thesis for finan• cial gain shall not be allowed without my written permission. Department of a'Y-J n.%. The University of British Columbia Vancouver 8, Canada Date /I >• ' 9fS / — ii ABSTRACT A modification of Mover's method has been found satisfactory for the quantitative measurement of resazurin reduction in biological systems. For convenience, the reducing systems were studied separately and the con• tribution of each system to total dye reduction was determined. The rapid reduction of resazurin in milk due to sunLlight was found to be a photo• chemical reaction initiated by the excitation of the dye by light of a wavelength of approximately 600 mu. It was found that the main reducing system in normal raw milk was ascorbic acid dependent, whereas in mastitic milk the main reducing system was leucocytes. Physical fractionation of mastitic milk, sonic treatment, and the use of metabolic inhibitors strongly indicated that there was an intimate relationship between the presence of leucocytes and the reducing activities of mastitic milk. Studies with cell-free extracts of leucocytes and bacteria showed that the reducing systems were essentially the same, and involved reduced pyridine nucleotides as electron donors. The mechanisms of resazurin reduction have been confirmed by studies involving the use of metabolic inhibitors, respirometry, variation of the energy source and kinetic studies. Finally, the successful reconstruction of several artificial resazurin reducing systems further confirmed the validity of this mechanism. iii Table of Contents Page INTRODUCTION . viii LITERATURE REVIEW ..... 1 MATERIALS AND METHODS 'V7 I. Milk samples. 7 II. Isolation of Leucocytes 7 III. Isolation of thermoduric micrococci 8 VI. Analytical methods 8 1. Resazurin reduction 8 i. Spectrophotometric assay 8 ii. Free radical reaction. .......... 10 2. Routine analytical determinations. ...... 10 EXPERIMENTAL RESULTS 14 I. Chemical and physical properties of resazurin ... 14 1. Spectra 14 2. Light sensitivity 17 3. Effect of ascorbic acid oxidase on light indu• ced resazurin reduction in milk 19 II. Reducing system in leucocytes and its role in the .. reduction of resazurin in mastitic milk ...... 21 1. Physical fraction of mastitic milk ...... 21 2. Influence of ascorbic acid oxidase on the rate of resazurin reduction by leucocytes . 23 3. The effect of sonic treatment on the reducing system in leucocytes and in mastitic milk. 23 4. Influence of iodoacetamide on the rate of resa• zurin reduction of leucocytes and of mastitic milk 26 iv Table of Contents (continued) Page 5. Production of lactic acid from leucocytes. ... 28 6. Influence of iodoacetamide on the production of lactic acid in leucocytes 28 7. The effect on incubation time on the rate of resazurin reduction by leucocytes 28 8. Studies with cell-free extracts of leucocytes. 32 III. Resazurin reducing system in normal raw milk 35 1. Ascorbic acid 35 2. Leucocytes ................... 37 3. Bacteria 37 4. Glucose 39 IV. Resazurin reducing system of bacteria 42 1. Influence of carbohydrate sources on the rate of resazurin reduction 42 i. Level of glucose affects on the rate of resazurin reduction by thermoduric Micro• coccus No. H 42 ii. Glucose, galactose and lactose as energy sources. .................. 42 2. Influence of various amounts of milk on the rate of resazurin reduction ............. 44 3. Comparison of the reducing ability of bacteria . 44 4. Oxygen uptake measurement. 44 5. Studies with cell-free extracts of bacteria. 49 i. Pseudomonas aeruginosa ATCC 9027 49 ii. Thermoduric Micrococcus No. H. 50 iii. Streptococcus lactis 53 V Table of Contents (continued) Page 6. Determination of level of pyridine nucleotides in bacteria 58 i. Pseudomonas aeruginosa ATCC 9027 58 ii. Streptococcus lactis 58 iii. Thermoduric Micrococcus No. H. 60 7. Kinetic study of resazurin reduction ...... 60 8. Level of NADH oxidase and its relation to the rate of resazurin reduction 61 V. The construction of an artificial resazurin reducing system. .......... , 64 1. Lactic acid dehydrogenase and alcohol dehydrogen• ase. .......... ............ 64 2. Glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. ................. 65 GENERAL DISCUSSION 70 SUMMARY . 76 BIBLIOGRAPHY 77 vi List of Figures Figure Page 1. Spectra of resazurin and resorufin in n-butanol saturated with sodium bicarbonate. ....... 15 2a. Spectrum of resazurin in 0.05 M Tris buffer (pH 7.4). 16 2b. Spectrum of ascorbic acid in distilled water. .... 16 3a. The time course of resazurin reduction induced by light at different wavelength 18 3b. The effect of wavelength of light on the rate of resazurin reduction 18 4. The time course of lactic acid formation from leucocytes 29 5. The effect of incubation time on the rate of resazurin reduction by leucocytes. 31 6. The time course of the oxidation of reduced pyridine nucleotides by a cell-free extract of leucocytes. 33 7. The time course of resazurin reduction by pyridine nucleotides in the presence of a cell-free extract of leucocytes 34 8. The effect of addition of ascorbic acid to raw milk on the rate of resazurin reduction .' 36 9. The effect of addiding washed leucocytes to raw milk on the rate of resazurin reduction 38 10. The effect of addition of washed cells of Strepto• coccus lactis to raw milk on the rate of resazurin reduction 40 11. Influence of glucose level on the rate of resazurin reduction by thermoduric Micrococcus No. H ... 43 12. Influence of varying amounts of milk on the rate of resazurin reduction by bacteria 46 13. The time course of resazurin reduction in milk by added \ bacteria 47 vii List of Figures (continued) Figure Page 14. Oxygen uptake by Streptococcus lactis and thermo- duric Micrococcus No. H with 5 )M of glucose . 48 15. The time course of the oxidation of reduced pyridine nucleotides by a cell-free extract of P. aeruginosa .... .......... 51 16. The time course of resazurin reduction by pyridine nucleotides in the presence of a cell-free extract of P. aeruginosa 52 17. The time course of the oxidation of reduced pyridine nucleotides by a cell-free extract of thermo- duric Micrococcus No. H 54 18. The time course of resazurin reduction by pyridine nucleotides in the presence of a cell-free extract of thermoduric Micrococcus No. H ........ 55 19. The time course of the oxidation of reduced pyridine nucleotides by a cell-free extract of Strepto• coccus lactis 56 20. The time course of resazurin reduction by pyridine nucleotides in the presence of a cell-free extract of Streptococcus lactis 57 21. Kinetic studies of resazurin reduction by cell-free extract of Streptococcus lactis and thermoduric Micrococcus No. H ........ 62 22. Artificial resazurin reducing system (lactic acid dehydrogenase and alcohol dehydrogenase). ... 66 23. Artificial resazurin reducing system (glucose-6- phosphate dehydrogenase and glutamate dehydro• genase) 68 ACKNOWLEDGEMENT I would like to express my sincere gratitude to Dr. J. J. R. Campbell for his guidance and encourage• ment during the course of this study. I would also like to express my gratitude to Dr. A. Gronlund and Dr. W. E. Razzell for their welcome suggestions and criticisms, and to the Canada Department of Agriculture for the financial assistance in the Form of an E.M.R. grant. viii INTRODUCTION Previous studies on the nature of resazurin reduction in milk have been restricted by the concept that the dye reduction was due to the change of potential in milk (Nilsson, 1959). The absence of an accurate method for quantitative measurement of dye reduction and the lack of biochemical training of the workers were the main reasons for the reports that the reducing system in leucocytes was not enzymatic in nature. Nilsson (1955) repeatedly washed leuco• cytes with sterile water and reported that there was no reducing system in leucocytes. The problem remained unsolved until Moyer (1963) introduced an accurate spectrophotometry method for quantitative measurement of the dye in milk. The present investigation was concerned mainly with the mechanism of resazurin reduction by normal milk, mastitic milk, leucocytes and bacteria. It was felt that elucidation of the mechanism of resazurin reduction would facilitate improvements in this technique. LITERATURE REVIEW The present investigation was concerned mainly with the mechanism of resazurin reduction by normal fluid milk, mastitic milk, leucocytes and bacteria. It is felt that elucidation of the mechanism of resazurin reduction will facilitate further improvements in the application of this technique. In the present work no effort was made to compare the resazurin reduction method with other milk quality tests, such as plate count, catalase and potentiometric measurements. These methods have been compared in a review by Johns (1959). Resazurin was discovered by Weselsky and was first used by Pesch and Simmert in 1929 for estimating the bacterial content of milk. The resazurin test was introduced to this continent in the form of a "one hour" method by Ramsdell in 1935. It has since received increasing attention. Johns (1942) observed that not only was resazurin a powerful tool for assessing the numbers of bacteria in milk, but also it was sensitive to non-bacterial factors such as mastitis and late lactation which are associated with abnormal milk.
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