This tutorial text provides an introduction to basics of bioluminescent methods used for rapid analysis of microbiological safety and quality of food and environmental samples. This book is intended for engineers, scientists, students, and managers involved in the design and/or use of biosafety assays. It discusses the practical aspects of bioluminescent microbiological analysis. Some basic knowledge of biochemistry, microbiology, and biophysics is preferable; however, a brief review of fundamental principles are included that will allow people who are unfamiliar with these disciplines to grasp their basic concepts.
The threat of biological warfare and the demand for safer food and water are an increasing concern. The best defense against bacterial infectious agents is their early detection and/or identification. Time and detection sensitivity are crucial for this type of microbiological analysis.
Besides the social aspects, successful competitiveness in international food markets increasingly depends on the provision of fresh, wholesome, and safe food. The accepted strategy worldwide is to prevent food and environmental contamination at the early stages of the food chain rather than to test the final product and, in case of failure, recall it. The control program used by most food producers worldwide and required by food inspection agencies in most of developed countries is the Hazard Analysis and Critical Control Point (HACCP) system. HACCP is a management system with which food safety is addressed through the analysis and control of biological, chemical, and physical hazards from raw material production, procurement, and handling to manufacturing, distribution, and consumption of the finished product. Monitoring usually relies on surveillance of physical and/or chemical parameters such as time and temperature of heating or pH, whereas validation of HACCP performance requires testing for the absence of specific food-related pathogens such as Salmonella, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, and others. There is some controversy as to whether microbiological tests can be used to monitor critical control points (CCPs) because of the length of the time needed to generate results and the sampling strategy required to obtain meaningful results. However, considerable advantages may accrue if microbiological control can be performed quickly and implemented into HACCP system.
The problem of rapid microbiological analysis for food and environmental
samples is further complicated by necessary sensitivity (“zero tolerance”), and by the fact that bacteria in both environmental and food samples are not distributed evenly most of the time, resulting in statistically sound sampling requirements. According to requirements of food inspection agencies worldwide, “zero-bacteria” in food and/or environmental samples mean that there is not a single cell of target pathogens in 25 g of sample. In practice, for most nonliquid materials, 25 g of the substance is homogenized in 225 ml of media (or buffer) and is analyzed as a whole. Therefore, the required sensitivity is 1 cell per 250 ml of sample. Currently, there is no method available that is capable of reaching such levels of sensitivity without an extended enrichment step. Two methods that are currently the closest to a real-time format for detection are real-time polymerase chain reaction (real-time PCR) and ATP-bioluminescence.
The number of companies that produce instruments and kits for bioluminescent analysis has increased dramatically during the last decade. The main area of industrial bioluminescence application is hygiene monitoring for food processing plants and establishments. These applications allow for fast and reliable monitoring of the cleanliness of surfaces and equipment. There is no need for expensive complicated equipment and highly trained personnel. These methods can be performed in “field” conditions, using hand-held devices. More specific bioluminescent methods for the detection of certain pathogens in food and environmental samples were developed recently. Most of them are not yet available commercially. It is intended through this book to encourage those in need of rapid, specific, and sensitive pathogen tests to become familiar with novel bioluminescent techniques and implement them in the routine diagnostic and control systems.
The book has three main sections. The first deals with the basics of bioluminescence. In this section, general mechanisms of bioluminescent reactions are discussed and major bioluminescent systems most relevant to analytical applications are reviewed. The second part of this book describes principles of bioluminescent analysis and their use for bacteria detection. The third section provides experimental protocols available for bioluminescent detection of bacteria in different food and environmental samples.