Effects of Autoclaving on Bacterial Endospores Studied by Fourier Transform Infrared Microspectroscopy

Perkins, David L.; Lovell, C. R.; Bronk, Burt V.; Setlow, Barbara; Setlow, Peter; Myrick, Michael L.

doi:10.1366/000370204873079

Cited by 35 publications

(29 citation statements)

References 11 publications

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“…On average, the "β/α" ratio is about 0.2 in untreated wild type spores which shifts to 2.4 upon 70% ethanol treatment. We hypothesised that these modifications are the results of a change in the secondary structure of proteins due to denaturation, and are similar to what was previously observed for autoclaved spores [41].…”

Section: Proteins Modifications Of Spores Monitored With Infrared Spesupporting

confidence: 80%

“…3a, ethanol treatment is associated with a loss in DPA content, and the loss is more pronounced upon increase in ethanol concentration from 30 to 70%. Similarly, the intensity of a shoulder band at 1570 cm -1 , (data not shown), also associated with the DPA structure [39][40][41] shows steady decrease upon ethanol treatment. A similar loss of a DPA vibration in the infrared spectrum has also been observed for the treatment of cotE gerE spores with 70%…”

Section: Dpa Loss Followed By Infrared Spectroscopymentioning

confidence: 65%

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Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

Loison

Gervais

Perrier-Cornet

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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In this work, we investigated how a combination of ethanol and high temperature (70 °C), affect the properties of the inner membrane of Bacillus subtilis spores. We observed membrane permeabilization for ethanol concentrations ≥ 50%, as indicated by the staining of the spores' DNA by the cell impermeable dye Propidium Iodide. The loss of membrane integrity was also confirmed by a decrease in the peak corresponding to dipicolinic acid using infrared spectroscopy. Finally, the spore refractivity (as measured by phase contrast microscopy) was decreased after the ethanol-heat treatment, suggesting a partial rehydration of the protoplast. Previously we have used fluorescent lifetime imaging microscopy (FLIM) combined with the fluorescent molecular rotor Bodipy-C12 to study the microscopic viscosity in the inner membrane of Bacillus subtilis spores, and showed that at normal conditions it is characterized by a very high viscosity. Here we demonstrate that the ethanol/high temperature treatment led to a decrease of the viscosity of the inner membrane, from 1000 cP to 860 cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of Bacillus subtilis spores, as well as demonstrates the ability of FLIM -Bodipy-C12 to measure changes in the microviscosity of the spores upon perturbation.

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Section: Proteins Modifications Of Spores Monitored With Infrared Spesupporting

confidence: 80%

Section: Dpa Loss Followed By Infrared Spectroscopymentioning

confidence: 65%

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

Loison

Gervais

Perrier-Cornet

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…The delay in DPA release may result from the slow disintegration of structures within the spore, which may require longer incubation times and higher temperatures than spore inactivation. The disintegration of spore structures has recently been observed by scanning electron microscopy, showing that autoclaved spores have a wrinkled appearance resulting from a loss of internal volume (35). The time points at which 90% of the spores have been inactivated (equal to the D values), as indicated in Fig.…”

Section: Vol 71 2005 Assessment Of Heat Resistance Of Bacterial Spomentioning

confidence: 91%

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

135

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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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“…3A). 18 The most prominent vibrations in mid-infrared spore spectra are due to the amide A at 3300 cm -1 (N-H stretching), amide I at 1650 cm -1 (carbonyl stretching), and the amide II at 1540 cm -1 (a combination of C-N stretching and N-H bending vibrations) protein bands. Other assignments in these typical endospore spectra are lipid C-H stretching in the spectral region from 2850 -2960 cm -1 , and the vibrations at 1443 cm -1 and 1388 cm -1 , assigned as contributions from calcium dipicolinate (CaDPA), as pyridine ring vibrations and symmetric carboxylate stretching respectively.…”

Section: Sem and Ft-ir Reflectance Microspectroscopymentioning

confidence: 97%

“…It was observed that these absorptions appear at the same frequencies as those from B. subtilis spores sampled as dried suspensions on gold-coated microscope slides. 18 Because bacterial spores and other particles scatter light as a function of the wavelengths of light incident upon them, a sloping baseline is observed. A particulate free Anodisc TM does not exhibit this small extinction that diminishes inversely proportional to increasing wavelength.…”

Section: Sem and Ft-ir Reflectance Microspectroscopymentioning

confidence: 99%

Optical Constant Determination of Bacterial Spores in the MIR

Myrick¹

2005

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Effects of Autoclaving on Bacterial Endospores Studied by Fourier Transform Infrared Microspectroscopy

Cited by 35 publications

References 11 publications

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

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

Optical Constant Determination of Bacterial Spores in the MIR

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