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Drugs with Anti-Oxidant Properties Can Interfere with Cell Viability
Laboratory Investigation (2017) 97, 494–497 © 2017 USCAP, Inc All rights reserved 0023-6837/17 $32.00 PATHOBIOLOGY IN FOCUS Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells Niraj Shenoy, Mary Stenson, Joshua Lawson, Jithma Abeykoon, Mrinal Patnaik, Xiaosheng Wu and Thomas Witzig Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays. Laboratory Investigation (2017) 97, 494–497; doi:10.1038/labinvest.2017.18; published online 27 February 2017 INTRODUCTION experiment was repeated, with the same result. -
Indicator Systems for Assessing Public Health Risk in Waters by Daniel
Indicator Systems for Assessing Public Health Risk in Waters by Daniel Oliver Roop A Thesis Submitted to the Faculty of the WORCESTER POLYTECHNIC INSTITUTE in partial fulfillment of the requirements for the Degree of Master of Science in Environmental Engineering by ____________________________________ Daniel Oliver Roop May 2012 APPROVED: ____________________________________ Dr. Jeanine D. Plummer, Major Advisor ____________________________________ Dr. John Bergendahl, Committee Member 1 Abstract For over one hundred years, indicator organisms such as coliforms have been measured as an index of public health risk from transmission of waterborne diseases. Even so, waterborne disease outbreaks have occurred in systems with negative coliform results, many traced to viral or protozoan etiologies. Conversely, no discernible public health outcomes have occurred in systems with positive coliform results. These inconsistencies arise because coliforms, as bacteria, respond differently to environmental stressors and engineered treatment processes than protozoan and viral pathogens. Recent reviews of four decades of indicator and pathogen monitoring indicated that coliphages are more highly correlated to pathogen presence in a variety of waters than coliforms. Therefore, the goal of this research was to re-examine a variety of traditional and novel indicator systems to determine their value as indicators, either singly or as a toolbox. We collected samples of animal feces, wastewaters, source waters and treated drinking waters. Samples were collected from four geographical regions of the United States (Northeast, South, Midwest and West) to assess spatial variability and in all four seasons to assess temporal variability. Samples were monitored for total coliforms, E. coli, male-specific and somatic coliphages, and other physical and chemical water quality parameters including organic carbon, pH and turbidity. -
Nicotinamide Adenine Dinucleotide Is Transported Into Mammalian
RESEARCH ARTICLE Nicotinamide adenine dinucleotide is transported into mammalian mitochondria Antonio Davila1,2†, Ling Liu3†, Karthikeyani Chellappa1, Philip Redpath4, Eiko Nakamaru-Ogiso5, Lauren M Paolella1, Zhigang Zhang6, Marie E Migaud4,7, Joshua D Rabinowitz3, Joseph A Baur1* 1Department of Physiology, Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States; 2PARC, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States; 3Lewis-Sigler Institute for Integrative Genomics, Department of Chemistry, Princeton University, Princeton, United States; 4School of Pharmacy, Queen’s University Belfast, Belfast, United Kingdom; 5Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States; 6College of Veterinary Medicine, Northeast Agricultural University, Harbin, China; 7Mitchell Cancer Institute, University of South Alabama, Mobile, United States Abstract Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells *For correspondence: with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties [email protected] results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with †These authors contributed doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate equally to this work that NAD(H) is the imported species. -
Reactions of Phenoxazine and Some of Its Derivatives Leonard Oro Moore Iowa State College
Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1957 Reactions of phenoxazine and some of its derivatives Leonard Oro Moore Iowa State College Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Organic Chemistry Commons Recommended Citation Moore, Leonard Oro, "Reactions of phenoxazine and some of its derivatives " (1957). Retrospective Theses and Dissertations. 1343. https://lib.dr.iastate.edu/rtd/1343 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. REACTIONS OF PHENOXAZINE AND SOME OF ITS DERIVATIVES by Leonard Oro Moore A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Subject: Organic Chemistry Approved: Signature was redacted for privacy. of Major Work Signature was redacted for privacy. Head of Mkjolr Department Signature was redacted for privacy. Dean of Graduate College Iowa State College 1957 il TABLE OF CONTENTS Page INTRODUCTION 1 HISTORICAL 4 Nomenclature 4 Preparation 5 Aminophenols with hydroxy compounds .... 6 Aminophenols with active halogen compounds. 9 Aminophenols with ortho-qulnones and ortho-hydroxyquinones 9 Phenols with nitroso compounds 12 Oxidative cyclizations 13 Reductive cyclizations 16 N-Substitution Reactions 19 Nuclear Substitution Reactions 21 Oxidation Reactions 23 Other Reactions 27 Physical Properties 29 Uses of Phenoxazine and Its Derivatives 32 Biological staining 33 Treatment of cancer 35 Treatment of tuberculosis 36 Naturally Occurring Phenoxazine Derivatives. -
Resazurin Sodium Salt
Resazurin sodium salt sc-206037 Material Safety Data Sheet Hazard Alert Code Key: EXTREME HIGH MODERATE LOW Section 1 - CHEMICAL PRODUCT AND COMPANY IDENTIFICATION PRODUCT NAME Resazurin sodium salt STATEMENT OF HAZARDOUS NATURE CONSIDERED A HAZARDOUS SUBSTANCE ACCORDING TO OSHA 29 CFR 1910.1200. NFPA FLAMMABILITY1 HEALTH2 HAZARD INSTABILITY0 SUPPLIER Company: Santa Cruz Biotechnology, Inc. Address: 2145 Delaware Ave Santa Cruz, CA 95060 Telephone: 800.457.3801 or 831.457.3800 Emergency Tel: CHEMWATCH: From within the US and Canada: 877-715-9305 Emergency Tel: From outside the US and Canada: +800 2436 2255 (1-800-CHEMCALL) or call +613 9573 3112 PRODUCT USE As indicator: pH range: 3.8, orange; 6.5, dark violet. In the detection of hyposulfite (sulfoxylate). In food research (reductase test). SYNONYMS C12-H7-N-O4.Na, "3H-phenoxazin-3-one, 7-hydroxy-, 10-oxide, sodium", "azoresorcin sodium", "diazoresorcinol sodium", "7-hydroxy- 3H-phenoazin-3-one 10-oxide sodium", "resazoin sodium", "resazurine sodium", "resazurin redox indicator" Section 2 - HAZARDS IDENTIFICATION CHEMWATCH HAZARD RATINGS Min Max Flammability: 1 Toxicity: 1 Body Contact: 2 Min/Nil=0 Low=1 Reactivity: 1 Moderate=2 High=3 Chronic: 0 Extreme=4 CANADIAN WHMIS SYMBOLS 1 of 9 EMERGENCY OVERVIEW RISK Irritating to eyes, respiratory system and skin. POTENTIAL HEALTH EFFECTS ACUTE HEALTH EFFECTS SWALLOWED ! Although ingestion is not thought to produce harmful effects, the material may still be damaging to the health of the individual following ingestion, especially where pre-existing organ (e.g. liver, kidney) damage is evident. Present definitions of harmful or toxic substances are generally based on doses producing mortality (death) rather than those producing morbidity (disease, ill-health). -
Determination of NAD+ and NADH Level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis Wenjun Xie Iowa State University
Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2008 Determination of NAD+ and NADH level in a single cell under H2O2 stress by capillary electrophoresis Wenjun Xie Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Analytical Chemistry Commons Recommended Citation Xie, Wenjun, "Determination of NAD+ and NADH level in a single cell under H2O2 stress by capillary electrophoresis" (2008). Retrospective Theses and Dissertations. 15456. https://lib.dr.iastate.edu/rtd/15456 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. + Determination of NAD and NADH level in a single cell under H2O2 stress by capillary electrophoresis by Wenjun Xie A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Analytical Chemistry Program of Study Committee: Edward S. Yeung, Major Professor Robert S. Houk Srdija Jeftinija Iowa State University Ames, Iowa 2008 Copyright © Wenjun Xie, 2008. All rights reserved. 1454641 1454641 2008 ii To The Four Years iii TABLE OF CONTENTS LIST OF FIGURES iv LIST OF TABLES v ABSTRACT vi CHAPTER 1. INTRODUCTION 1 NAD+, NADH and their biological functions 1 Current approaches of NAD+ and NADH analysis 2 Single cell capillary electrophoresis 4 CE-based enzymatic assay 5 Overview of this work 6 CHAPTER 2. -
The 'Blue Bottle' Experiment
48 The ‘blue bottle’ experiment A colourless solution in a flask is shaken. It turns blue and then gradually back to colourless. The cycle can be repeated many times. Apparatus and chemicals Eye protection • One 1 dm3 conical flask with stopper Access to a nitrogen cylinder (optional) Access to a fume cupboard (optional) The quantities of chemical given are for one demonstration. 8 g of potassium hydroxide (Corrosive) or 6 g of sodium hydroxide (Corrosive) 10 g of glucose (dextrose) 0.05 g of methylene blue (Harmful) 50 cm3 of ethanol (IDA, Industrial Denatured Alcohol) (Highly flammable, Harmful) Technical notes 8 g of potassium hydroxide (Corrosive) or 6 g of sodium hydroxide (Corrosive) Refer to CLEAPSS® Hazcard 91 0.05 g of methylene blue (Harmful) Refer to CLEAPSS® Hazcard 32 Ethanol (IDA, Industrial Denatured Alcohol) (Highly flammable, Harmful) Refer to CLEAPSS® Hazcard 40A Procedure • Health & Safety: Wear safety goggles. Before the demonstration a Make a solution of 0.05 g of methylene blue in 50 cm3 of ethanol (0.1 %). b Weigh 8 g of potassium hydroxide or 6 g of sodium hydroxide into a 1 dm3 conical flask. Add 300 cm3 of water and 10 g of glucose and swirl until the solids are dissolved. Add 5 cm3 of the methylene blue solution. None of the quantities is critical. The resulting blue solution will turn colourless after about one minute. c Stopper the flask. The demonstration Shake the flask vigorously so that air dissolves in the solution. The colour will change to blue. This will fade back to colourless over about 30 seconds. -
Bacterial H2S Oxidation Coupled to the Reduction of Mno2 Studied On
Bacterial H2S oxidation coupled to the reduction of MnO2 studied on new isolated species of the genus Sulfurimonas Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Der Mathematisch-Naturwissenschaftlichen Fakultät Der Universität Rostock Vorgelegt von: Jan Henkel, geb. am 28. Juni 1989 in Fulda aus Rostock Rostock, den ____________________________________ https://doi.org/10.18453/rosdok_id00002580 Gutachter: Prof. Dr. Heide Schulz-Vogt, Leibniz- Institut für Ostseeforschung Warnemünde (IOW), Geomikrobiologie Prof. Dr. Axel Schippers, Bundesanstalt für Geowissenschaften und Rohstoffe (BGR), Geochemie der Rohstoffe Jun.-Prof. Dr. Mirko Basen, Universität Rostock, Mikrobielle Physiologie Jahr der Einreichung: 2019 Jahr der Verteidigung: 2019 Content Summary .................................................................................................................................................. I Zusammenfassung ................................................................................................................................. III 1 Introduction ...................................................................................................................................... 1 2 Material and methods ..................................................................................................................... 11 2.1 Preparation of anoxic cultivation medium ............................................................................ 11 2.2 Preparation of stock solutions -
Special Instructions
Special Instructions Cultivation of Anaerobes The DSMZ holds a large collection of bacteria and archaea that thrive only under anaerobic conditions. In our experience beginners in culturing anaerobes or extremophiles encounter often difficulties in handling these cultures appropriately. This technical information should help everybody who is interested to start working with anaerobes. Please read it carefully, it will answer most frequently asked questions about culturing anaerobes! You will find on this page information to the following topics: General information about anaerobes Recommended vials for culturing strict anaerobes Gassing of media and cultures with oxygen-free gas Preparation of anoxic media buffered with bicarbonate Reducing agents and resazurin Handling of vacuum-dried anaerobic cultures Handling of actively growing anaerobic cultures Literature Notes General information about anaerobes In the broadest sense obligate anaerobes can be defined as microorganisms which are unable to utilize molecular oxygen for growth. A further differentiation is possible based on their relationship to the presence of oxygen. Aerotolerant anaerobes are only slightly inhibited by significant levels of oxygen in the atmosphere. For instance Clostridium intestinale DSM 6191T can grow well on the surface of agar plates incubated in air at atmospheric pressure. The other extreme is represented by strict anaerobes, which die, or immediately stop growing, upon exposure to low levels of oxygen. It is therefore important to retain anoxic conditions during all steps of handling of these microorganisms. Most strict anaerobes require not only the absence of oxygen to initiate growth, but also a redox potential below -300 mV, which can be only achieved by the supplementation of media with reducing agents (see section on Reducing agents and resazurin). -
Microbiological Hazard to the Environment Posed by the Groundwater in the Vicinity of Municipal Waste Landfill Site
ECOLOGICAL CHEMISTRY AND ENGINEERING S Vol. 18, No. 2 2011 1* 1 2 Krzysztof FR ĄCZEK , Jacek GRZYB and Dariusz ROPEK MICROBIOLOGICAL HAZARD TO THE ENVIRONMENT POSED BY THE GROUNDWATER IN THE VICINITY OF MUNICIPAL WASTE LANDFILL SITE ZAGRO ŻENIA MIKROBIOLOGICZNE DLA ŚRODOWISKA POWODOWANE PRZEZ WODY PODZIEMNE W STREFIE ODDZIAŁYWANIA SKŁADOWISKA ODPADÓW KOMUNALNYCH Abstract: In the area and environs of the municipal waste dump Barycz in Krakow, all analyzed groundwater samples, taken from piezometers C-1, P-3, P-6, P-8 and G revealed occurrence of bacteria - indicators of bad sanitary conditions. Even periodical pollution of groundwater results in its bad quality. Presence of fecal Coliform bacteria indicates on anthropogenic origin of the groundwater contamination. The number of cfu of fecal Coliform bacteria in water samples taken from piezometers was decreasing with growing distance from the waste dump borders. However, even in a 1230 m distance north from borders of the waste dump in the piezometer G, in spite of the absence of fecal Coliform bacteria, bad sanitary condition was ascertained, according to other microbiological indicators. Keywords: microbial contamination, groundwater, landfill leachate Landfilling is still the most often used method for solid waste disposal in Poland. However, this method is associated with several problems. Among these are pollution of air, soil and groundwater, high cost associated with landfill operation and than with their closing and remediation. An important issue is leachate which is both chemically and microbiologically contaminated [1, 2]. Landfill leachate consists of the liquid generated by the breakdown of waste, and the infiltrating precipitation [3]. A broad range of xenobiotic compounds occurring in leachate can be linked to household hazardous waste deposited in the landfill [4]. -
Rapid Enumeration of Fecal Coliforms in Water by a Colorimetric /3-Galactosidase Assay L
0099-2240/78/0035-0136$02.00/() APPLIED AND ENVIRONMENIAl MICROBIOLOGY, Jan. 1978, p.136-141 Vol. 35, No. 1 Copyright © 1978 American Society for Microbiology Printed in U.S.A. Rapid Enumeration of Fecal Coliforms in Water by a Colorimetric /3-Galactosidase Assay L. S. WARREN,t R. E. BENOIT,* AND) J. A. JESSEE Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 3 August 1977 The colorimetric f8-galactosidase assay is based upon the enzymatic hydrolysis of the substrate o-nitrophenyl-fl-D-galactoside (ONPG) by fecal coliforms. This technique provides an estimate of the fecal coliform concentration within 8 to 20 h. A 100-ml portion of test sample was passed through a 0.45-j.m membrane filter. This filter was then incubated at 37°C for 1 h in EC medium followed by the addition of filter-sterilized ONPG. The incubation was continued at 44.5°C until a half-maximum absorbance (at 420 nm) was reached. The time between the start of incubation and the half-maximum absorbance was proportional to the concentration of fecal coliforms present. Escherichia coli (K-12) was used to measure the kinetics of substrate hydrolysis and the response time of different cell concentrations. High cell densities produced an immediate response, whereas 1 cell/ml will produce a response in less than 20 h. In field studies in which samples were taken from a range of grossly polluted streams to relatively clean lake water, a linear correlation between ONPG hydrolysis times and fecal coliform most-probable-number values was established. -
Guidelines for the Preservation of Raw Milk by Use of the Lactoperoxidase System
GUIDELINES FOR THE PRESERVATION OF RAW MILK BY USE OF THE LACTOPEROXIDASE SYSTEM CAC/GL 13-1991 INTRODUCTION Milk is an easily perishable raw material. Contaminating bacteria may multiply rapidly and render it unsuitable for processing and/or unfit for human consumption. Bacterial growth can be retarded by refrigeration, thereby slowing down the rate of deterioration. Under certain conditions refrigeration may not be feasible due to economical and/ or technical reasons. Difficulties in applying refrigeration are specially a problem for certain areas in countries setting up or expanding their milk production. In these situations, it would be beneficial to have access to a method, other than refrigeration, for retarding bacterial growth in raw milk during collection and transportation to the dairy processing plant. In 1967 the FAO/WHO Expert Panel on Milk Quality concluded that the use of hydrogen peroxide might be an acceptable alternative in the early stages of development of an organized dairy industry, provided that certain conditions were complied with. However, this method has not achieved any general acceptance as it has several drawbacks, most important of which is the difficulty of controlling its use: it may be misused to disguise milk of basic hygienic quality produced under poor hygienic conditions. The toxicological aspects of the use of relatively high concentrations of hydrogen peroxide in milk have also been questioned. A chemical method for preserving milk would still be of great advantage in certain situations. The search for such a method has therefore continued. Interest has recently been focused on the indigenous antibacterial systems in milk to determine if these could be applied practically to preserve raw milk.