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Spie Press Book

Bioluminescense for Food and Environmental Microbiological Safety
Author(s): Lubov Brovko
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Book Description

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.

Book Details

Date Published: 28 March 2007
Pages: 88
ISBN: 9780819466433
Volume: TT74

Table of Contents
SHOW Table of Contents | HIDE Table of Contents
Chapter 1
Basics of Bioluminescence / 1
1.1 Examples of Bioluminescent Reactions / 2
1.1.1 Bioluminescent system of bacteria / 3
1.1.2 Bioluminescent system of insects (fireflies) / 3
1.1.3 Coelenterazine bioluminescent system / 5
1.1.4 Cypridina bioluminescent system / 5
1.1.5 Dinoflagellate bioluminescent system / 6
Chapter 2
Bioluminescent Analysis / 7
2.1 Basics of Kinetic Analysis / 7
2.2 Experimental Approaches for Determining Reaction
Rates / 8
Chapter 3
Enzyme Kinetics and Its AnalyticalApplications / 11
3.1 Experimental Formats for Kinetic-enzymatic Analysis / 12
3.2 Analytical Applications of the Bioluminescent Reaction Catalyzed by Firefly Luciferase / 13
3.3 Bioluminescent ATP Assay (Practical Considerations) / 16
Chapter 4
Basics of Bioluminescence for Microbiological
AssayApplication / 19
4.1 Bioluminescent ATP-based Microbiological Assay: Practical Considerations / 20
4.2 Total-plate Counts, Most Probable Numbers , and Bioluminescent Analysis / 23
Chapter 5
Bioluminescent Microbiological Assay in Food Safety and Quality Control / 29
5.1 Bioluminescent ATP Assay for Testing of Total Bacterial Count (TBC) of Food Products / 29
5.2 Bioluminescent Total Bacterial Count Assays for Several Food Commodities / 33
5.2.1 Raw milk and milk products / 33
5.2.2 Drinking water, brewing, beverages, and fruit juice / 34
5.2.3 Poultry / 36
5.2.4 Meat / 36
Chapter 6
Hygiene Monitoring / 39
Chapter 7
Identification of Microorganisms and Specific Detection of Bacteria Using Bioluminescence / 43
7.1 Antibody-based Bioluminescent Methods for Detection of Bacterial Pathogens / 43
7.2 Bacteriophage-based Bioluminescent Assays for Detection of Pathogens / 47
7.2.1 Bacteriophages as biosorbents / 48
7.2.2 Use of lytic bacteriophages for detection of bacteria / 48
7.2.3 Luciferase reporter bacteriophages as tools for detecting bacteria / 50
Chapter 8
Bioluminescent Toxicity Assays / 53
Chapter 9
Review of Available Instruments and Reagents for Bioluminescent Analysis / 61

References / 65

Recommended Reading / 71

Index / 73


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.

For RT PCR the term "real-time" does not actually mean that detection of bacteria occurs in real time. Instead, this method allows each cycle of DNA amplification to be observed on the computer screen during the sequence of thermal cycling, hence the designation "real-time." In general RT-PCR method comprises the following major steps:

  1. enrichment and/or separation/concentration: 6 to 48 h;
  2. DNA preparation: about one hour;
  3. RT PCR reaction: 0.5 to 1.5 h.

Detection of a single cell of pathogen in 25 g of food by PCR requires, on average, 1-3 days, which is much faster than any available immunoassay (3-5 days), but is still far away from a real- time format.

Bioluminescent microbiological assays are based on the detection of intracellular adenosine-5- triphosphate (ATP) released from the target bacteria, using bioluminescent enzymatic reaction catalyzed by firefly luciferase. Theoretically this method allows for the detection of a single bacterium in the sample. Due to the fact that ATP is present in all live cells, the specificity of an ATP-based assay for certain pathogens relies on the preliminary separation and concentration of the specific bacteria. The following steps are included in the regular experimental protocol:

  1. enrichment and separation of target cells and removal of exogenous ATP (5 min-24 h);
  2. release of intracellular ATP (1-2 min); and
  3. measurement of bioluminescence (1-2 min).

Both real-time PCR and ATP bioluminescence methods have potential for developing rapid microbiological assays on their basis. What is the advantage of ATP BL over RT PCR and why? The most time-consuming step in both assays is the cell separation and concentration. Thus, providing the development of fast and reliable techniques for bacterial cell separation/concentration from large volume samples, the bioluminescent tests can be preferable since it can be performed within single minutes, it is cheaper to run compared with real-time PCR, as well as it can be easily adapted to high-throughput format.

A key objective of this book is to provide readers with information on the current status of bioluminescent assays in microbiology. Both theoretical and practical aspects of the tests are discussed; and strengths and weaknesses of particular techniques are reviewed. Specific experimental protocols are presented to provide a user-friendly reference guide for a wide range of applications. However, bioluminescence techniques have so many various applications in microbiological assays that it is almost impossible to cover all of them in a single book. Rather than describing every single experimental protocol available, general practical considerations of existing methods are provided to help readers develop or improve their own protocols.

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.

Lubov Brovko
February 2007

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