Enhanced spore detection using dipicolinate extraction techniques

Pellegrino, Paul M.; Fell, Nicholas F.; Gillespie, James B.

doi:10.1016/s0003-2670(01)01613-0

Cited by 74 publications

(73 citation statements)

References 15 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). The DPA assay is not sensitive to other cellular material in the spore sample, because DPA is not present in vegetative cells.…”

Section: Methodsmentioning

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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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%

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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“…The ASD employs a rapid chemical test for the detection of bacterial spores based on dipicolinic acid (DPA)-triggered terbium ion (Tb 3þ ) luminescence (Hindle and Hall, 1999;Pellegrino et al, 2002;Rosen, 1999Rosen, , 2006Sacks, 1990), which is closely related to a colorimetric assay first developed in 1958 (Janssen et al, 1958). DPA is present in high concentrations (up to 1 molar, $15% of dry weight) in the core of bacterial spores (Gould and Hurst, 1969).…”

Section: Introductionmentioning

confidence: 99%

An automated front‐end monitor for anthrax surveillance systems based on the rapid detection of airborne endospores

Yung

Lester

Bearman

et al. 2007

Biotech & Bioengineering

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A fully automated anthrax smoke detector (ASD) has been developed and tested. The ASD is intended to serve as a cost effective front-end monitor for anthrax surveillance systems. The principle of operation is based on measuring airborne endospore concentrations, where a sharp concentration increase signals an anthrax attack. The ASD features an air sampler, a thermal lysis unit, a syringe pump, a time-gated spectrometer, and endospore detection chemistry comprised of dipicolinic acid (DPA)-triggered terbium ion (Tb(3+)) luminescence. Anthrax attacks were simulated using aerosolized Bacillus atrophaeus spores in fumed silica, and corresponding Tb-DPA intensities were monitored as a function of time and correlated to the number of airborne endospores collected. A concentration dependence of 10(2)-10(6) spores/mg of fumed silica yielded a dynamic range of 4 orders of magnitude and a limit of detection of 16 spores/L when 250 L of air were sampled. Simulated attacks were detected in less than 15 min.

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“…While, PCR and immunoassays continue to be developed, other methods are also being developed that rapidly assess the extent of contamination in the event of an attack, such as fluorescence, luminescence, mass spectrometry, and Raman spectroscopy (Nudelman et al, 2000;Pellegrino et al, 2002;Hathout et al, 2003;Farquharson et al, 2004, respectively). These methods focus on portability and sensitivity, rather than on specificity.…”

mentioning

confidence: 99%

“…Relatively fast methods have been developed to chemically extract the acid of CaDPA, dipicolinic acid (DPA; Pellegrino et al, 2002), and then to detect it directly by mass spectrometry (Beverly et al, 1996;Hathout et al, 2003), fluorescence (Nudelman et al, 2000), or indirectly by luminescence (Pellegrino et al, 2002;Rosen et al, 1997). Although mass spectrometry provides a relatively high degree of discrimination and sensitivity, it still requires significant time due to sample preparation.…”

mentioning

confidence: 99%

“…Although mass spectrometry provides a relatively high degree of discrimination and sensitivity, it still requires significant time due to sample preparation. Hot dodecylamine (DDA) has been used to extract DPA and form a highly luminescent complex with terbium (Pellegrino et al, 2002). Although measurements have been performed in as little as five minutes, it was found that as many as three concentration-dependent complexes can form, each with different lifetimes.…”

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confidence: 99%

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