Abstract

Heat activation of dormant bacterial spores is a short treatment at a sublethal temperature that potentiates and synchronizes spore germination. In this paper, laser tweezers Raman spectroscopy (LTRS) was used to study the heat activation of single spores of Bacillus cereus and Bacillus subtilis. We measured the Raman spectra of single spores without treatment, during heat activation at 65 °C (B. cereus) or 70 °C (B. subtilis), and following heat activation and cooling to 25 °C. Principle component analysis (PCA) was applied to discriminate among the three groups of spores based on their Raman spectra. The results indicated that: (1) there are large changes in the Raman bands of Ca-DPA and protein for both B. cereus and B. subtilis spores during heat activation, indicative of changes in spore core state and partial protein denaturation at the heat activation temperatures; (2) these spectral changes become smaller once the heated spores are cooled, consistent with heat activation being reversible; (3) minor spectral differences between untreated and heat-activated and cooled spores can be discriminated by PCA based on non-polarized and polarized Raman spectra; and (4) analysis based on polarized Raman spectra reveals that partial denaturation of protein during heat activation is mainly observed in the vertically polarized component.

© 2009 OSA

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2009 (2)

L. Peng, D. Chen, P. Setlow, and Y. Q. Li, “Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics,” Anal. Chem. 81(10), 4035–4042 (2009).
[CrossRef] [PubMed]

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

2008 (1)

2007 (2)

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

2006 (4)

D. Chen, S. S. Huang, and Y. Q. Li, “Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy,” Anal. Chem. 78(19), 6936–6941 (2006).
[CrossRef] [PubMed]

P. Setlow, “Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals,” J. Appl. Microbiol. 101(3), 514–525 (2006).
[CrossRef] [PubMed]

A. C.-T. Ko, L.-P. Choo-Smith, M. Hewko, M. G. Sowa, C. C. Dong, and B. Cleghorn, “Detection of early dental caries using polarized Raman spectroscopy,” Opt. Express 14(1), 203–215 (2006).
[CrossRef] [PubMed]

A. Alimova, A. Katz, P. Gottlieb, and R. R. Alfano, “Proteins and dipicolinic acid released during heat shock activation of Bacillus subtilis spores probed by optical spectroscopy,” Appl. Opt. 45(3), 445–450 (2006).
[CrossRef] [PubMed]

2005 (3)

C. Xie, D. Chen, and Y. Q. Li, “Raman sorting and identification of single living micro-organisms with optical tweezers,” Opt. Lett. 30(14), 1800–1802 (2005).
[CrossRef] [PubMed]

C. Creely, G. Singh, and D. Petrov, “Dual wavelength optical tweezers for confocal Raman spectroscopy,” Opt. Commun. 245(1-6), 465–470 (2005).
[CrossRef]

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

2004 (2)

H. Liem, J. Cabanillas-Gonzalez, P. Etchegoin, and D. D. C. Bradley, “Glass transition temperatures of polymer thin films monitored by Raman scattering,” J. Phys. Condens. Matter 16(6), 721–728 (2004).
[CrossRef]

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

2003 (2)

R. G. K. Leuschner and P. J. Lillford, “Thermal properties of bacterial spores and biopolymers,” Int. J. Food Microbiol. 80(2), 131–143 (2003).
[CrossRef]

P. Setlow, “Spore germination,” Curr. Opin. Microbiol. 6(6), 550–556 (2003).
[CrossRef] [PubMed]

2002 (1)

2000 (1)

M. Paidhungat, B. Setlow, A. Driks, and P. Setlow, “Characterization of spores of Bacillus subtilis which lack dipicolinic acid,” J. Bacteriol. 182(19), 5505–5512 (2000).
[CrossRef] [PubMed]

1999 (1)

S. Ablett, A. H. Darke, P. J. Lillford, and D. R. Martin, “Glass formation and dormancy in bacterial spores,” Int. J. Food Sci. Technol. 34(1), 59–69 (1999).
[CrossRef]

1997 (1)

J. M. Benevides, M. Tsuboi, J. K. H. Bamford, and G. J. Thomas., “Polarized Raman spectroscopy of double-stranded RNA from bacteriophage 46: local Raman tensors of base and backbone vibrations,” Biophys. J. 72(6), 2748–2762 (1997).
[CrossRef] [PubMed]

1996 (1)

A. Mahadevan-Jansen and R. Richards-Kortum, “Raman spectroscopy for the detection of cancers and precancers,” J. Biomed. Opt. 1(1), 31–70 (1996).
[CrossRef]

1993 (1)

V. Sapru and T. P. Labuza, “Glassy state in bacterial spores predicted by polymer glass-transition theory,” J. Food Sci. 58(2), 445–448 (1993).
[CrossRef]

1976 (1)

V. J. C. Lin and J. L. Koenig, "Raman studies of bovine serum albumin," Biopoly. 15(1), 203–218 (1976).
[CrossRef]

1971 (1)

J. H. Martin and P. W. Blackwood, “Effects of sub-lethal heat-shock, β-alanine, and L-alanine on germination and subsequent destruction of Bacillus spores by pasteurization,” J. Dairy Sci. 55, 577–580 (1971).
[CrossRef]

1965 (1)

A. M. Cook and M. R. W. Brown, “Relationship between heat activation and percentage colony formation for Bacillus stearothermophilus spores: effects of storage and pH of the recovery medium,” J. Appl. Bacteriol. 28(3), 361–364 (1965).
[CrossRef] [PubMed]

1962 (1)

G. W. Gould and A. D. Hitchins, “Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bonds,” J. Gen. Microbiol. 33, 413–423 (1962).

1943 (1)

F. R. Evans and H. R. Curran, “The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria,” J. Bacteriol. 46(6), 513–523 (1943).
[PubMed]

Ablett, S.

S. Ablett, A. H. Darke, P. J. Lillford, and D. R. Martin, “Glass formation and dormancy in bacterial spores,” Int. J. Food Sci. Technol. 34(1), 59–69 (1999).
[CrossRef]

Alfano, R. R.

Alimova, A.

Bailey, M. J.

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

Bamford, J. K. H.

J. M. Benevides, M. Tsuboi, J. K. H. Bamford, and G. J. Thomas., “Polarized Raman spectroscopy of double-stranded RNA from bacteriophage 46: local Raman tensors of base and backbone vibrations,” Biophys. J. 72(6), 2748–2762 (1997).
[CrossRef] [PubMed]

Benevides, J. M.

J. M. Benevides, M. Tsuboi, J. K. H. Bamford, and G. J. Thomas., “Polarized Raman spectroscopy of double-stranded RNA from bacteriophage 46: local Raman tensors of base and backbone vibrations,” Biophys. J. 72(6), 2748–2762 (1997).
[CrossRef] [PubMed]

Blackwood, P. W.

J. H. Martin and P. W. Blackwood, “Effects of sub-lethal heat-shock, β-alanine, and L-alanine on germination and subsequent destruction of Bacillus spores by pasteurization,” J. Dairy Sci. 55, 577–580 (1971).
[CrossRef]

Bradley, D. D. C.

H. Liem, J. Cabanillas-Gonzalez, P. Etchegoin, and D. D. C. Bradley, “Glass transition temperatures of polymer thin films monitored by Raman scattering,” J. Phys. Condens. Matter 16(6), 721–728 (2004).
[CrossRef]

Brown, M. R. W.

A. M. Cook and M. R. W. Brown, “Relationship between heat activation and percentage colony formation for Bacillus stearothermophilus spores: effects of storage and pH of the recovery medium,” J. Appl. Bacteriol. 28(3), 361–364 (1965).
[CrossRef] [PubMed]

Cabanillas-Gonzalez, J.

H. Liem, J. Cabanillas-Gonzalez, P. Etchegoin, and D. D. C. Bradley, “Glass transition temperatures of polymer thin films monitored by Raman scattering,” J. Phys. Condens. Matter 16(6), 721–728 (2004).
[CrossRef]

Chen, D.

L. Peng, D. Chen, P. Setlow, and Y. Q. Li, “Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics,” Anal. Chem. 81(10), 4035–4042 (2009).
[CrossRef] [PubMed]

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

D. Chen, S. S. Huang, and Y. Q. Li, “Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy,” Anal. Chem. 78(19), 6936–6941 (2006).
[CrossRef] [PubMed]

C. Xie, D. Chen, and Y. Q. Li, “Raman sorting and identification of single living micro-organisms with optical tweezers,” Opt. Lett. 30(14), 1800–1802 (2005).
[CrossRef] [PubMed]

Choo-Smith, L.-P.

Ciancia, R.

Cleghorn, B.

Coleman, W. H.

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

Cook, A. M.

A. M. Cook and M. R. W. Brown, “Relationship between heat activation and percentage colony formation for Bacillus stearothermophilus spores: effects of storage and pH of the recovery medium,” J. Appl. Bacteriol. 28(3), 361–364 (1965).
[CrossRef] [PubMed]

Cowan, A. E.

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

Creely, C.

C. Creely, G. Singh, and D. Petrov, “Dual wavelength optical tweezers for confocal Raman spectroscopy,” Opt. Commun. 245(1-6), 465–470 (2005).
[CrossRef]

Curran, H. R.

F. R. Evans and H. R. Curran, “The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria,” J. Bacteriol. 46(6), 513–523 (1943).
[PubMed]

Darke, A. H.

S. Ablett, A. H. Darke, P. J. Lillford, and D. R. Martin, “Glass formation and dormancy in bacterial spores,” Int. J. Food Sci. Technol. 34(1), 59–69 (1999).
[CrossRef]

De Luca, A. C.

Dinno, M. A.

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

C. G. Xie, M. A. Dinno, and Y. Q. Li, “Near-infrared Raman spectroscopy of single optically trapped biological cells,” Opt. Lett. 27(4), 249–251 (2002).
[CrossRef]

Dong, C. C.

Driks, A.

M. Paidhungat, B. Setlow, A. Driks, and P. Setlow, “Characterization of spores of Bacillus subtilis which lack dipicolinic acid,” J. Bacteriol. 182(19), 5505–5512 (2000).
[CrossRef] [PubMed]

Etchegoin, P.

H. Liem, J. Cabanillas-Gonzalez, P. Etchegoin, and D. D. C. Bradley, “Glass transition temperatures of polymer thin films monitored by Raman scattering,” J. Phys. Condens. Matter 16(6), 721–728 (2004).
[CrossRef]

Evans, F. R.

F. R. Evans and H. R. Curran, “The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria,” J. Bacteriol. 46(6), 513–523 (1943).
[PubMed]

Gemperline, P. J.

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

Ghosh, S.

S. Ghosh and P. Setlow, “The preparation, germination properties and stability of superdormant spores of Bacillus cereus,” J. Appl. Microbiol. (to be published).
[PubMed]

Gottlieb, P.

Gould, G. W.

G. W. Gould and A. D. Hitchins, “Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bonds,” J. Gen. Microbiol. 33, 413–423 (1962).

Griffiths, R. I.

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

Hewko, M.

Hitchins, A. D.

G. W. Gould and A. D. Hitchins, “Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bonds,” J. Gen. Microbiol. 33, 413–423 (1962).

Huang, S. S.

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

D. Chen, S. S. Huang, and Y. Q. Li, “Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy,” Anal. Chem. 78(19), 6936–6941 (2006).
[CrossRef] [PubMed]

Huang, W. E.

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

Katz, A.

Kempf, C. R.

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

Ko, A. C.-T.

Koenig, J. L.

V. J. C. Lin and J. L. Koenig, "Raman studies of bovine serum albumin," Biopoly. 15(1), 203–218 (1976).
[CrossRef]

Labuza, T. P.

V. Sapru and T. P. Labuza, “Glassy state in bacterial spores predicted by polymer glass-transition theory,” J. Food Sci. 58(2), 445–448 (1993).
[CrossRef]

Leuschner, R. G. K.

R. G. K. Leuschner and P. J. Lillford, “Thermal properties of bacterial spores and biopolymers,” Int. J. Food Microbiol. 80(2), 131–143 (2003).
[CrossRef]

Li, Y. Q.

L. Peng, D. Chen, P. Setlow, and Y. Q. Li, “Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics,” Anal. Chem. 81(10), 4035–4042 (2009).
[CrossRef] [PubMed]

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

D. Chen, S. S. Huang, and Y. Q. Li, “Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy,” Anal. Chem. 78(19), 6936–6941 (2006).
[CrossRef] [PubMed]

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

C. Xie, D. Chen, and Y. Q. Li, “Raman sorting and identification of single living micro-organisms with optical tweezers,” Opt. Lett. 30(14), 1800–1802 (2005).
[CrossRef] [PubMed]

C. G. Xie, M. A. Dinno, and Y. Q. Li, “Near-infrared Raman spectroscopy of single optically trapped biological cells,” Opt. Lett. 27(4), 249–251 (2002).
[CrossRef]

Liem, H.

H. Liem, J. Cabanillas-Gonzalez, P. Etchegoin, and D. D. C. Bradley, “Glass transition temperatures of polymer thin films monitored by Raman scattering,” J. Phys. Condens. Matter 16(6), 721–728 (2004).
[CrossRef]

Lillford, P. J.

R. G. K. Leuschner and P. J. Lillford, “Thermal properties of bacterial spores and biopolymers,” Int. J. Food Microbiol. 80(2), 131–143 (2003).
[CrossRef]

S. Ablett, A. H. Darke, P. J. Lillford, and D. R. Martin, “Glass formation and dormancy in bacterial spores,” Int. J. Food Sci. Technol. 34(1), 59–69 (1999).
[CrossRef]

Lin, V. J. C.

V. J. C. Lin and J. L. Koenig, "Raman studies of bovine serum albumin," Biopoly. 15(1), 203–218 (1976).
[CrossRef]

Mace, J.

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

Mahadevan-Jansen, A.

A. Mahadevan-Jansen and R. Richards-Kortum, “Raman spectroscopy for the detection of cancers and precancers,” J. Biomed. Opt. 1(1), 31–70 (1996).
[CrossRef]

Martin, D. R.

S. Ablett, A. H. Darke, P. J. Lillford, and D. R. Martin, “Glass formation and dormancy in bacterial spores,” Int. J. Food Sci. Technol. 34(1), 59–69 (1999).
[CrossRef]

Martin, J. H.

J. H. Martin and P. W. Blackwood, “Effects of sub-lethal heat-shock, β-alanine, and L-alanine on germination and subsequent destruction of Bacillus spores by pasteurization,” J. Dairy Sci. 55, 577–580 (1971).
[CrossRef]

Martinelli, V.

Newton, R. J.

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

Paidhungat, M.

M. Paidhungat, B. Setlow, A. Driks, and P. Setlow, “Characterization of spores of Bacillus subtilis which lack dipicolinic acid,” J. Bacteriol. 182(19), 5505–5512 (2000).
[CrossRef] [PubMed]

Pelczar, P. L.

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

Peng, L.

L. Peng, D. Chen, P. Setlow, and Y. Q. Li, “Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics,” Anal. Chem. 81(10), 4035–4042 (2009).
[CrossRef] [PubMed]

Pesce, G.

Petrov, D.

C. Creely, G. Singh, and D. Petrov, “Dual wavelength optical tweezers for confocal Raman spectroscopy,” Opt. Commun. 245(1-6), 465–470 (2005).
[CrossRef]

Richards-Kortum, R.

A. Mahadevan-Jansen and R. Richards-Kortum, “Raman spectroscopy for the detection of cancers and precancers,” J. Biomed. Opt. 1(1), 31–70 (1996).
[CrossRef]

Rotoli, B.

Rusciano, G.

Sabelnikov, A.

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

Sapru, V.

V. Sapru and T. P. Labuza, “Glassy state in bacterial spores predicted by polymer glass-transition theory,” J. Food Sci. 58(2), 445–448 (1993).
[CrossRef]

Sasso, A.

Selvaggi, L.

Setlow, B.

M. Paidhungat, B. Setlow, A. Driks, and P. Setlow, “Characterization of spores of Bacillus subtilis which lack dipicolinic acid,” J. Bacteriol. 182(19), 5505–5512 (2000).
[CrossRef] [PubMed]

Setlow, P.

L. Peng, D. Chen, P. Setlow, and Y. Q. Li, “Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics,” Anal. Chem. 81(10), 4035–4042 (2009).
[CrossRef] [PubMed]

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

P. Setlow, “Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals,” J. Appl. Microbiol. 101(3), 514–525 (2006).
[CrossRef] [PubMed]

P. Setlow, “Spore germination,” Curr. Opin. Microbiol. 6(6), 550–556 (2003).
[CrossRef] [PubMed]

M. Paidhungat, B. Setlow, A. Driks, and P. Setlow, “Characterization of spores of Bacillus subtilis which lack dipicolinic acid,” J. Bacteriol. 182(19), 5505–5512 (2000).
[CrossRef] [PubMed]

S. Ghosh and P. Setlow, “The preparation, germination properties and stability of superdormant spores of Bacillus cereus,” J. Appl. Microbiol. (to be published).
[PubMed]

Shelenkova, L.

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

Singh, G.

C. Creely, G. Singh, and D. Petrov, “Dual wavelength optical tweezers for confocal Raman spectroscopy,” Opt. Commun. 245(1-6), 465–470 (2005).
[CrossRef]

Sowa, M. G.

Tang, W.

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

Thomas, G. J.

J. M. Benevides, M. Tsuboi, J. K. H. Bamford, and G. J. Thomas., “Polarized Raman spectroscopy of double-stranded RNA from bacteriophage 46: local Raman tensors of base and backbone vibrations,” Biophys. J. 72(6), 2748–2762 (1997).
[CrossRef] [PubMed]

Thompson, I. P.

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

Tsuboi, M.

J. M. Benevides, M. Tsuboi, J. K. H. Bamford, and G. J. Thomas., “Polarized Raman spectroscopy of double-stranded RNA from bacteriophage 46: local Raman tensors of base and backbone vibrations,” Biophys. J. 72(6), 2748–2762 (1997).
[CrossRef] [PubMed]

Vepachedu, V. R.

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

Whiteley, A. S.

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

Xie, C.

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

C. Xie, D. Chen, and Y. Q. Li, “Raman sorting and identification of single living micro-organisms with optical tweezers,” Opt. Lett. 30(14), 1800–1802 (2005).
[CrossRef] [PubMed]

Xie, C. G.

Anal. Chem. (5)

W. E. Huang, R. I. Griffiths, I. P. Thompson, M. J. Bailey, and A. S. Whiteley, “Raman microscopic analysis of single microbial cells,” Anal. Chem. 76(15), 4452–4458 (2004).
[CrossRef] [PubMed]

C. Xie, J. Mace, M. A. Dinno, Y. Q. Li, W. Tang, R. J. Newton, and P. J. Gemperline, “Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy,” Anal. Chem. 77(14), 4390–4397 (2005).
[CrossRef] [PubMed]

D. Chen, L. Shelenkova, Y. Q. Li, C. R. Kempf, and A. Sabelnikov, “Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis,” Anal. Chem. 81(9), 3227–3238 (2009).
[CrossRef] [PubMed]

D. Chen, S. S. Huang, and Y. Q. Li, “Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy,” Anal. Chem. 78(19), 6936–6941 (2006).
[CrossRef] [PubMed]

L. Peng, D. Chen, P. Setlow, and Y. Q. Li, “Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics,” Anal. Chem. 81(10), 4035–4042 (2009).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (1)

J. M. Benevides, M. Tsuboi, J. K. H. Bamford, and G. J. Thomas., “Polarized Raman spectroscopy of double-stranded RNA from bacteriophage 46: local Raman tensors of base and backbone vibrations,” Biophys. J. 72(6), 2748–2762 (1997).
[CrossRef] [PubMed]

Biopoly. (1)

V. J. C. Lin and J. L. Koenig, "Raman studies of bovine serum albumin," Biopoly. 15(1), 203–218 (1976).
[CrossRef]

Curr. Opin. Microbiol. (1)

P. Setlow, “Spore germination,” Curr. Opin. Microbiol. 6(6), 550–556 (2003).
[CrossRef] [PubMed]

Int. J. Food Microbiol. (1)

R. G. K. Leuschner and P. J. Lillford, “Thermal properties of bacterial spores and biopolymers,” Int. J. Food Microbiol. 80(2), 131–143 (2003).
[CrossRef]

Int. J. Food Sci. Technol. (1)

S. Ablett, A. H. Darke, P. J. Lillford, and D. R. Martin, “Glass formation and dormancy in bacterial spores,” Int. J. Food Sci. Technol. 34(1), 59–69 (1999).
[CrossRef]

J. Appl. Bacteriol. (1)

A. M. Cook and M. R. W. Brown, “Relationship between heat activation and percentage colony formation for Bacillus stearothermophilus spores: effects of storage and pH of the recovery medium,” J. Appl. Bacteriol. 28(3), 361–364 (1965).
[CrossRef] [PubMed]

J. Appl. Microbiol. (2)

P. Setlow, “Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals,” J. Appl. Microbiol. 101(3), 514–525 (2006).
[CrossRef] [PubMed]

S. Ghosh and P. Setlow, “The preparation, germination properties and stability of superdormant spores of Bacillus cereus,” J. Appl. Microbiol. (to be published).
[PubMed]

J. Bacteriol. (4)

M. Paidhungat, B. Setlow, A. Driks, and P. Setlow, “Characterization of spores of Bacillus subtilis which lack dipicolinic acid,” J. Bacteriol. 182(19), 5505–5512 (2000).
[CrossRef] [PubMed]

S. S. Huang, D. Chen, P. L. Pelczar, V. R. Vepachedu, P. Setlow, and Y. Q. Li, “Levels of Ca2+-dipicolinic acid in individual Bacillus spores determined using microfluidic Raman tweezers,” J. Bacteriol. 189(13), 4681–4687 (2007).
[CrossRef] [PubMed]

F. R. Evans and H. R. Curran, “The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria,” J. Bacteriol. 46(6), 513–523 (1943).
[PubMed]

W. H. Coleman, D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow, “How moist heat kills spores of Bacillus subtilis,” J. Bacteriol. 189(23), 8458–8466 (2007).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

A. Mahadevan-Jansen and R. Richards-Kortum, “Raman spectroscopy for the detection of cancers and precancers,” J. Biomed. Opt. 1(1), 31–70 (1996).
[CrossRef]

J. Dairy Sci. (1)

J. H. Martin and P. W. Blackwood, “Effects of sub-lethal heat-shock, β-alanine, and L-alanine on germination and subsequent destruction of Bacillus spores by pasteurization,” J. Dairy Sci. 55, 577–580 (1971).
[CrossRef]

J. Food Sci. (1)

V. Sapru and T. P. Labuza, “Glassy state in bacterial spores predicted by polymer glass-transition theory,” J. Food Sci. 58(2), 445–448 (1993).
[CrossRef]

J. Gen. Microbiol. (1)

G. W. Gould and A. D. Hitchins, “Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bonds,” J. Gen. Microbiol. 33, 413–423 (1962).

J. Phys. Condens. Matter (1)

H. Liem, J. Cabanillas-Gonzalez, P. Etchegoin, and D. D. C. Bradley, “Glass transition temperatures of polymer thin films monitored by Raman scattering,” J. Phys. Condens. Matter 16(6), 721–728 (2004).
[CrossRef]

Opt. Commun. (1)

C. Creely, G. Singh, and D. Petrov, “Dual wavelength optical tweezers for confocal Raman spectroscopy,” Opt. Commun. 245(1-6), 465–470 (2005).
[CrossRef]

Opt. Express (2)

Opt. Lett. (2)

Other (6)

S. Ghosh, P. Zhang, Y.-q. Li, and P. Setlow, "Superdormant spores of Bacillus species have elevated wet heat resistance and temperature requirements for heat activation," (unpublished).

J. R. Ferraro, K. Nakamoto, and C. W. Brown, Introductory Raman spectroscopy, 2nd Edition. (Academic, 2000), pp. 267–293.

W. L. Nicholson and P. Setlow, "Sporulation, germination and outgrowth," in Molecular Biological Methods for Bacillus, C. R. Harwood and S. M. Cutting, eds., (John Wiley, 1990), pp. 391–450.

T. Kitagawa and S. Hirota, "Raman spectroscopy of proteins," in Handbook of Vibrational Spectroscopy, volume 5, J. M. Chalmers and P. R. Griffiths, eds., (John Wiley, 2002), pp. 3426–3446.

S. J. Foster, and K. Johnstone, "The trigger mechanism of bacterial spore germination," in Regulation of Prokaryotic Development, I. Smith, R. A. Slepecky, and P. Setlow, eds., (American Society for Microbiology, 1989), pp. 89–108.

A. Keynan, and Z. Evenchik, "Activation" in The Bacterial Spore, G. W. Gould and A. Hurst, eds., (Academic, 1969), pp. 359–396.

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Figures (6)

Fig. 1
Fig. 1

Optical layout of polarized laser tweezers Raman spectroscopy

Fig. 2
Fig. 2

Non-polarized Raman spectra of B. cereus and B. subtilis spores. Curve a is for untreated spores at 25 °C; curve b for cooled heat-activated spores measured at 25 °C; curve c for spores during heat-activation measured at the elevated temperature (65 °C for B. cereus, and 70 °C for B. subtilis); curve d is the subtraction between curves a and b, magnified by a factor 5 for display, and curve e is the subtraction between curves a and c. The insets show the magnified spectra in the range of the protein amide I bands. The spectra were averaged over 50 spores and the vertical baselines were shifted for display.

Fig. 3
Fig. 3

Score plots of untreated spores (red), cooled heat-activated spores (green), and heat-activated spores held at the heat activation temperature (blue) based on non-polarized Raman spectra. The individual spores in training sets are represented with open symbols and the spores in testing sets with filled symbols, respectively. (a) B. cereus; (b) B. subtilis; (c) B . cereus with untreated and cooled heat-activated spores; (d) B. subtilis with untreated and cooled heat-activated spores . BC – B. cereus; BS – B.subtilis.

Fig. 4
Fig. 4

Averaged Raman spectra for (a) untreated and (b) cooled heat-activated B. cereus spores and the loading spectra of PC1 and PC2.

Fig. 5
Fig. 5

Polarized Raman spectra of B. subtilis spores at 25 °C (a, a’); heat-activated spores held at either 25 °C (b, b’) or at 70 °C (c, c’). The insets show the averaged spectra in the range of the protein amide I band. Curves d and d’ are the subtractions of curve b (or b’) from curve a (or a’), magnified by a factor 5 for display, and curves e and e’ are the subtractions of curve c (or c’) from curve a (or a’). Left: parallel components (I//); Right: vertical components (I). All spectra were averaged over 30 spores, and the vertical baselines were shifted for display.

Fig. 6
Fig. 6

(a) Score plots of B. subtilis spores either untreated (red) at 25 °C, heat-activated and held at 25 °C (green), and heat activated and held at 70 °C (blue) based on vertical components of polarized Raman spectra. The individual spores in training sets are represented with open symbols and the spores in testing sets with filled symbols, respectively. (b) Score plots of untreated spores of B. subtilis (red) and heat-activated spores held at 25 °C (green).

Tables (3)

Tables Icon

Table 1 Ratios of the intensities of characteristic Raman bands of B. cereus spores

Tables Icon

Table 2 Ratios of intensities of characteristic Raman bands of B. subtlis spores

Tables Icon

Table 3 Depolarization ratios of characteristic Raman bands of B. subtilis spores

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