The study of pathogenic processes is mostly limited to in vitro assays, cell-culture techniques and post mortem examination of infected animals. A better understanding of the infectious process, efficiency of antimicrobial and antibiotic treatment as well as immunomodulatory effects of different food supplements could be achieved by in vivo real-time monitoring of bacterial colonization in live animals. It was proposed recently to use bacterial pathogens with luminescent or fluorescent phenotypes for photonic detection of bacterial cells in living hosts. 14 It was shown that both bacteria transformed with full cassette of luminescent genes from Xenorhabdus luminescens and with Green Fluorescent Protein (GFP) could be visualized in animal using whole-body luminescent or fluorescent imaging techniques with high sensitivity and in real time. We used this approach to investigate the effect of diet on the time-course of infection in mice orally infected with bioluminescent strain of Salmonella enteritidis.
Bioluminescence methods are gaining increased attention due to their sensitivity, selectivity, and simplicity, along with the fact that bioluminescence can be monitored both in vitro and in vivo. This book introduces bioluminescence and fluorescence systems, along with the principles of their application for in vivo imaging of intracellular processes, and covers recent developments in optical (bioluminescence and fluorescence) imaging in cell biology.
This book is intended for scientists and students involved in basic cell physiology research, as well as industry professionals, engineers, and managers involved in drug discovery and pre-clinical drug development. It discusses the practical aspects of luminescence in vivo imaging for monitoring intracellular processes. While some basic knowledge of biochemistry and biophysics is preferable, the book includes a brief review of fundamental principles to allow those not familiar with these disciplines to grasp basic concepts.
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 bactericidal effect of visible light illumination on bacteria treated with non-toxic photosensitizers (PS) has been shown previously, its effectiveness depended on both cell type and nature of photosensitizer used. The photosensitizer (PS)-mediated bactericidal effect of light against different types of microorganisms including vegetative bacteria (both in planktonic form and in biofilms), bacterial spores, yeasts, viruses was investigated for both cells in liquid media, and on surface. Bactericidal effect was monitored for different photosensitizer such as TBO and derivatives of rosamine at different concentrations. The possibility of using photodynamic treatment for surface sanitation was investigated.
The food industry is increasingly adopting food safety and quality management systems that are more proactive and preventive than those used in the past which have tended to rely on end product testing and visual inspection. The regulatory agencies in many countries are promoting one such management tool, Hazard Analysis Critical Control Point (HACCP), as a way to achieve a safer food supply and as a basis for harmonization of trading standards. Verification that the process is safe must involve microbiological testing but the results need not be generated in real-time. Of all the rapid microbiological tests currently available, the only ones that come close to offering real-time results are bioluminescence-based methods.
Recent developments in application of bioluminescence for food safety issues are presented in the paper. These include the use of genetically engineered microorganisms with bioluminescent and fluorescent phenotypes as a real time indicator of physiological state and survival of food-borne pathogens in food and food processing environments as well as novel bioluminescent-based methods for rapid detection of pathogens in food and environmental samples. Advantages and pitfalls of the methods are discussed.
The problem of bacterial enumeration in different samples is of great importance in many fields of research. Construction of recombinant fluorescent and luminescent bacteria that can be easily detected by nondestructive instrumental methods proves us with an opportunity to monitor bacteria in a wide variety of clinical, environmental and food samples in real time. Three different labels were employed: Green Fluorescent Protein (GFP), Bacterial luciferase (BL) and Firefly Luciferase (FFL). Both plasmid and chromosomal transformants of different strains of E. coli, P. putida and S. enteritidis were used. For the detection of the in vivo GFP the Shimadzu RF 540 spectrofluorimeter, Labsystems FL- 500 plate fluorimeter and Night Owl LB 98 CCD-camera from EG and G Berthold supplied with excitation light source and proper spectral filters both in macroscopic and microscopic mode were used. For the detection of in vivo luminescence of BL and FFL, tube luminometer BG-P from GEM Biomedical Inc., luminometric plate reader from BioOrbit, BIQ Bioview CCD camera from Cambridge Imaging Ltd and Night Owl LB 98 CCD camera both in macroscopic and microscopic mode were used. The expression levels of the labels, their stability, stability of the signal and detection limits of tagged bacteria were investigated. The detection limits for GFP tagged bacteria were 5 X 10<SUP>4</SUP> - 6 X 10<SUP>6</SUP>, for BL tagged bacteria 5 X 10<SUP>2</SUP> - 2 X 10<SUP>5</SUP>, and for FFL tagged bacteria - 4 X 10<SUP>3</SUP> - 10<SUP>6</SUP> CFU/ml, depending on the instrument used. Single bacteria could be detected with the help of the Night Owl in the microscopic mode.
Earlier a comprehensive study of reaction substrate, product and their complexes with the enzyme has been carried out by means of stationary fluorescence spectroscopy. A hypothesis has been suggested according to which protein changes its conformation during the reaction. In this respect it is quite interesting to study both static and dynamic properties of the enzyme molecule. Luckily, according to genetic engineering data, luciferase molecule contains only one tryptophan residue, which makes it a very convenient object for the studies by means of time-resolved fluorescence spectroscopy. The scope of this paper is the study of luciferase tryptophan fluorescence. The methods applied were fluorescence spectrochronography and anisotropy decay analysis.
SC696: Bioluminescence for Food and Environmental Safety
This course will provide with introduction to basics of bioluminescent methods used for rapid analysis of microbiological quality of food and environmental samples. Bioluminescence-based methods currently are the only methods that allow detection of microorganisms in close to real-time format. These methods are found in applications ranging from high-throughput screening of food and environmental samples for microbiological safety and quality to sophisticated methods that provide more detailed information on the bacterial agents present in the sample including their identification, antibiotic and antimicrobial substances resistance, physiological and metabolic state, spatial distribution in the sample, etc.
Basics of bioluminescent systems will be introduced and explained in the course together with principles of their application in microbiological assays. Different formats of bioluminescent methods will be surveyed and discussed in terms of their applicability for the detection of microorganisms. The following tests will be discussed in details: 1) assays for total bacterial counts; 2) hygiene monitoring; 3) identification of microorganisms. Instrumentation and reagents available on the market that are used for these tests will be reviewed beginning with simple hand-held devices intended for field tests followed by automated luminescent plate readers and CCD cameras, pros and cons will be discussed.