Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers

2010 ◽  
Vol 82 (9) ◽  
pp. 3840-3847 ◽  
Author(s):  
Lingbo Kong ◽  
Pengfei Zhang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Raman Spectroscopy ◽  
Optical Tweezers ◽  
Spore Germination ◽  
Phase Contrast ◽  
Bacterial Spore ◽  
Phase Contrast Microscopy ◽  
Contrast Microscopy

Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers

2011 ◽  
Vol 6 (5) ◽  
pp. 625-639 ◽  
Author(s):  
Lingbo Kong ◽  
Pengfei Zhang ◽  
Guiwen Wang ◽  
Jing Yu ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Fluorescence Microscopy ◽  
Optical Tweezers ◽  
Spore Germination ◽  
Phase Contrast ◽  
Bacterial Spore

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.

Sign in / Sign up

Export Citation Format

Share Document