scholarly journals Use of Raman Spectroscopy and Phase-Contrast Microscopy To Characterize Cold Atmospheric Plasma Inactivation of Individual Bacterial Spores

2016 ◽  
Vol 82 (19) ◽  
pp. 5775-5784 ◽  
Author(s):  
Shiwei Wang ◽  
Christopher J. Doona ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Raman Spectroscopy ◽  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Atmospheric Plasma ◽  
Phase Contrast Microscopy ◽  
Content Type ◽  
Cold Atmospheric Plasma ◽  
Promising Tool ◽  
Contrast Microscopy ◽  
Plastic Bag

ABSTRACTRaman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individualBacillus subtilisspores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated withl-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spore's inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly.IMPORTANCEMuch research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this study, Raman spectroscopy and phase-contrast microscopy were used to analyze CaDPA levels and kinetics of nutrient- and non-nutrient-germinant-induced germination of multiple individual spores ofBacillus subtilisthat were treated by a planar CAP device. The roles of different plasma species involved in spore inactivation were also investigated. The knowledge obtained in this study will aid in understanding the mechanism(s) of spore inactivation by CAP and potentially facilitate the development of more effective and efficient plasma sterilization techniques in various applications.

scholarly journals Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

2013 ◽  
Vol 80 (1) ◽  
pp. 345-353 ◽  
Author(s):  
Lingbo Kong ◽  
Christopher J. Doona ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
High Pressure ◽  
Spore Germination ◽  
Phase Contrast ◽  
Constant Pressure ◽  
Ambient Pressure ◽  
Lytic Enzyme ◽  
Phase Contrast Microscopy ◽  
Wild Type ◽  
Content Type ◽  
Contrast Microscopy

ABSTRACTGermination ofBacillusspores with a high pressure (HP) of ∼150 MPa is via activation of spores' germinant receptors (GRs). The HP germination of multiple individualBacillus subtilisspores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores' germination kinetics were heterogeneous; (ii) individual spores' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (Tlag) prior to a period of the rapid release (ΔTrelease) of the spores' dipicolinic acid in a 1:1 chelate with Ca2+(CaDPA); (iii) spore germination at 50 MPa had longer averageTlagvalues than that at ∼150 MPa, but the ΔTreleasevalues at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv)B. subtilisspores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔTreleasevalues ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar averageTlagvalues; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.

scholarly journals Contact-Free Inactivation of Candida albicans Biofilms by Cold Atmospheric Air Plasma

2012 ◽  
Vol 78 (12) ◽  
pp. 4242-4247 ◽  
Author(s):  
Tim Maisch ◽  
Tetsuji Shimizu ◽  
Georg Isbary ◽  
Julia Heinlin ◽  
Sigrid Karrer ◽  
...  
Keyword(s):  
Health Care ◽  
Candida Albicans ◽  
Treatment Time ◽  
Atmospheric Plasma ◽  
Air Plasma ◽  
Content Type ◽  
Cold Atmospheric Plasma ◽  
Promising Tool ◽  
Plasma Device ◽  
Contact Free

ABSTRACTCandida albicansis one of the main species able to form a biofilm on almost any surface, causing both skin and superficial mucosal infections. The worldwide increase in antifungal resistance has led to a decrease in the efficacy of standard therapies, prolonging treatment time and increasing health care costs. Therefore, the aim of this work was to demonstrate the applicability of atmospheric plasma at room temperature for inactivatingC. albicansgrowing in biofilms without thermally damaging heat-sensitive materials. This so-called cold atmospheric plasma is produced by applying high voltage to accelerate electrons, which ionize the surrounding air, leading to the production of charged particles, reactive species, and photons. A newly developed plasma device was used, which exhibits a large plasma-generating surface area of 9 by 13 cm (117 cm2). Different time points were selected to achieve an optimum inactivation efficacy range of ≥3 log10to 5 log10reduction in CFU per milliliter, and the results were compared with those of 70% ethanol. The results obtained show that contact-free antifungal inactivation ofCandidabiofilms by cold atmospheric plasma is a promising tool for disinfection of surfaces (and items) in both health care settings and the food industry, where ethanol disinfection should be avoided.

scholarly journals Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation

2003 ◽  
Vol 185 (17) ◽  
pp. 5306-5309 ◽  
Author(s):  
Samuel Amiteye ◽  
Kazuo Kobayashi ◽  
Daisuke Imamura ◽  
Shigeo Hosoya ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Dipicolinic Acid ◽  
Diacylglycerol Kinase ◽  
Phase Contrast Microscopy ◽  
Electron Microscopic ◽  
Contrast Microscopy ◽  
Microscopic Studies ◽  
Essential Function ◽  
Kinase Homologue

ABSTRACT The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

scholarly journals Rough Dental Implant Surfaces and Peri-Implantitis: Role of Phase-Contrast Microscopy, Laser Protocols, and Modified Home Oral Hygiene in Maintenance. A 10-Year Retrospective Study

2021 ◽  
Vol 11 (11) ◽  
pp. 4985
Author(s):  
Gianluigi Caccianiga ◽  
Gérard Rey ◽  
Paolo Caccianiga ◽  
Alessandro Leonida ◽  
Marco Baldoni ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Phase Contrast ◽  
Vertical Movement ◽  
Subgingival Plaque ◽  
Phase Contrast Microscopy ◽  
Implant Surface ◽  
Total Percentage ◽  
Contrast Microscopy

The aim of this study was to evaluate two different kinds of rough implant surface and to assess their tendency to peri-implantitis disease, with a follow-up of more than 10 years. Data were obtained from a cluster of 500 implants with Ti-Unite surface and 1000 implants with Ossean surface, with a minimum follow-up of 10 years. Implants had been inserted both in pristine bone and regenerated bone. We registered incidence of peri-implantitis and other causes of implant loss. All patients agreed with the following maintenance protocol: sonic brush with vertical movement (Broxo), interdental brushes, and oral irrigators (Broxo) at least two times every day. For all patients with implants, we evaluated subgingival plaque samples by phase-contrast microscopy every 4 months for a period of more than 10-years. Ti-Unite surface implants underwent peri-implantitis in 1.6% of the total number of implants inserted and Ossean surface implants showed peri-implantitis in 1.5% of the total number of implants. The total percentage of implant lost was 4% for Ti-Unite surfaces and 3.6% for Ossean surfaces. Strict control of implants leads to low percentage of peri-implantitis even for rough surfaces dental implants.

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