Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence

Rosen, David L.; Sharpless, Charles M.; McGown, Linda B.

doi:10.1021/ac960939w

Cited by 145 publications

(110 citation statements)

References 12 publications

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“…Fluorescence monitoring of DPA release upon heat inactivation of spores is based on the enhancement of the fluorescence emission of the terbium ion (Tb 3ϩ ) upon binding to DPA (37). Recently, buffer conditions and DPA extraction procedures for the fluorescence monitoring assay have been further improved (18,21,33).…”

Section: Methodsmentioning

confidence: 99%

“…An alternative method for the detection of DPA was obtained by the use of the strongly enhanced fluorescence of the lanthanide ion Tb 3ϩ upon DPA binding. This fluorescent DPA assay was initially developed by Rosen et al as a method for the detection of bacterial spores (37). The assay has been optimized by Hindle and Hall to a detection limit of 2 nM DPA, which corresponds to 10 4 spores ml Ϫ1 (18).…”

mentioning

confidence: 99%

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Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Kort

O'Brien

Stokkum

et al. 2005

Appl Environ Microbiol

134

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This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105°C, 120°C, and 131°C, respectively. The estimated Z values were 6.3°C, 6.1°C, and 9.7°C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (T c ), the temperature at which half the DPA content has been released within a fixed incubation time. We found T c values for spores from Bacillus strains 168, A163, and IC4 of 108°C, 121°C, and 131°C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or foodborne illness. They are extremely resistant to heat and other preservation treatments in comparison to vegetative cells. The inactivation of spores requires high temperatures and long heating times, which are costly and detrimental to the nutritional and organoleptic quality of most food products. To minimize the required heat treatment, there is an urgent need in the food industry for tailored preservation procedures, based on models that accurately predict the presence of viable cells at every step of the food production process (6, 41). To assess the required heat inactivation procedure for the most resistant cell type, the bacterial spore, we isolated and classified two food contaminants and developed a rapid and sensitive screening method to determine the heat resistance of their spores.Under nutrient-limited conditions, vegetative cells of Bacillus species undergo the cell differentiation process of sporulation (17, 42). The resulting spores are metabolically dormant and show, besides resistance to heat, resistance to other potentially lethal treatments that include radiati...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Kort

O'Brien

Stokkum

et al. 2005

Appl Environ Microbiol

134

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“…DOPC vesicles with each of these fluorophores were prepared and tested for their usefulness in the assay. The Tb 3+ /DPA system, which has been used in several biological applications such as endospore detection [36], vesicle fusion [37,38], and vesicle leakage [38,39], proved to be superior to the fluo-3 and calcium green 1 systems. The latter fluorophores are sensitive to calcium ions, and there…”

Section: Evaluation Of Fluorescence Systemsmentioning

confidence: 99%

Mechanosensitive channel of large conductance

Kloda

Petrov

Meyer

et al. 2008

The International Journal of Biochemistry & Cell Biology

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The bacterial mechanosensitive channel of large conductance (MscL) is an ideal starting point for understanding the molecular basis of mechanosensation. However, current methods for the characterization of its mutants, patch clamp and bacterial growth analysis, are difficult and time consuming, so a higher throughput method for screening mutants is desired. We have attempted to develop a fluorescence assay for detecting MscL activity in synthetic vesicles. The assay involved the separation of two solutionsone inside and one outside the vesicles-that are separately nonfluorescent but fluorescent when mixed. It was hoped that MscL activity due to downshock of the vesicles would bring about mixing of the solutions, producing fluorescence. The development of the assay required the optimization of several variables: the method for producing a uniform vesicle population containing MscL, the fluorescence system, and the lipid and protein composition of the vesicles. However, no MscL activity was ever detected even after optimization, so the assay was not fully developed. The probable cause of the failure was the inability of current techniques to produce a sufficiently uniform vesicle population.

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“…For the last decade, dipicolinic acid (DPA) has gained considerable popularity as a marker for the detection of bacterial endospores (16,22,25,59,67,83,92,97). Emission enhance-ment assays for DPA and bacterial endospores, utilizing lanthanide ions, were developed at the U.S. Army Research Laboratory at the end of the 20th century (59,67) and were recently improved by a research team at the Jet Propulsion Laboratory (9,10).…”

mentioning

confidence: 99%

“…Emission enhance-ment assays for DPA and bacterial endospores, utilizing lanthanide ions, were developed at the U.S. Army Research Laboratory at the end of the 20th century (59,67) and were recently improved by a research team at the Jet Propulsion Laboratory (9,10). Up to about 20% of the weight of bacterial endospores can be attributed to calcium dipicolinate: i.e., DPA is a natural product observed solely in sporulated bacterial species (75,76).…”

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confidence: 99%

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

et al. 2011

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Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy.

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Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence

Cited by 145 publications

References 12 publications

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

Mechanosensitive channel of large conductance

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

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