Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. Th... more Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. The amount of heme necessary for half-maximal growth was 2 to 10 ng/ml (3.8 to 15 pmol/ml) among the Bacteroides species and strains tested. The growth rate, metabolic products from glucose fermentation, and cell yields were affected by the concentration of heme in the medium and by the length of time the culture was incubated. When heme was growth limiting (4 ng/ml), growth rates decreased by 50%, cultures started producing lactic and fumaric acids, and the cell yields declined. The cell yield for B. fragilis (ATCC 25285) at 24 h in medium containing 6.5 microgram of heme per ml was 69 g (dry weight) of cells per mol of glucose compared to 16 g (dry weight) of cells per mol of glucose with 4 ng of heme per ml. B. fragilis was unable to grow in defined medium when a porphyrin precursor, delta-aminolevulenic acid or porphobilinogen, was added in place of heme.
An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilis Morone chr... more An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilis Morone chrysops), using cells collected from the peritoneal cavity of this fish. The findings indicated that: (1) 10 days following a single intraperitoneal injection (1 ml) of Freund's incomplete adjuvant (FIA) was an appropriate time for collecting suitable working concentrations (5·3 4·0 10 7 cells ml 1 ) of peritoneal phagocytes (83·7 1·5% macrophages) from these hybrids held at 23 C; (2) these cells phagocytosed latex beads (polystyrene microspheres 3·12 m in diameter) after 30 min of in vitro incubation at room temperature (25 1 C). The phagocytic ability and phagocytic capacity in a washed adherent layer exposure system were 67·2 2·76% and 4·14 0·35 beads phagocyte 1 , respectively.
Updated information and services can be found at: These include: REFERENCES http://aac.asm.org/co... more Updated information and services can be found at: These include: REFERENCES http://aac.asm.org/content/45/11/3262#ref-list-1 at: This article cites 33 articles, 15 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on August 22, 2013 by guest http://aac.asm.org/ Downloaded from ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 0066-4804/01/$04.00ϩ0
Recently, a number of analytical methods have been successfully developed which use nucleic acid ... more Recently, a number of analytical methods have been successfully developed which use nucleic acid sequencing to identify biological warfare agents. However, the effectiveness of these methods, towards the safety and protection of US Armed Forces and their allies are limited by the period required to enumerate the nucleic acid through polymerase chain reactions or culture growth to produce sufficient quantities for analysis. To overcome this limitation, we have been investigating the ability of surface-enhanced Raman spectroscopy to detect nucleic acids with sufficient sensitivity and selectivity to eliminate the need for enumeration. The design of a small volume electrolytic sample cell will be presented along with analysis of the nucleic acid bases and preliminary analysis of model bacteria.
The ability of Raman spectroscopy to detect anthrax-causing spores as they pass through a mail so... more The ability of Raman spectroscopy to detect anthrax-causing spores as they pass through a mail sorting system was investigated. A pump was connected to an existing vacuum manifold on a commercial sorter, and a filter designed to capture 0.5-3 µm particles was placed in-line. A standard business letter containing 0.23 g of Bacillus cereus spores, a Bacillus anthracis surrogate, was placed in a stack of 20 letters and passed through the system. Raman spectra of the filter positively identified the captured material as bacterial spores by the dominant calcium dipicolinate Raman spectral bands associated with the spore core. A limit of detection, using 400 mW of 785 nm laser excitation for a 1-s acquisition, is estimated at 4.5 mg. The ability of a Raman spectroscopy based system to detect and prevent the distribution of a letter containing gram levels of anthrax spores is discussed.
The Chemical Weapons Convention prohibits the development, production, stockpiling, and use of wa... more The Chemical Weapons Convention prohibits the development, production, stockpiling, and use of warfare agents (chemical and biological), and requires their destruction. Yet their use persists and has been included in the terrorist's arsenal. Currently, a number of analytical methods are being developed to perform rapid measurements of trace agents to ensure treaty compliance, as well as safe environments for military personal and the public at large. We have been investigating the ability of surface-enhanced Raman spectroscopy to detect bacterial nucleic acid-base pairs with sufficient sensitivity and selectivity to eliminate the need for enumeration used in polymerase chain reactions and culture growth, required by other measurement techniques. The design of a small volume, fiber optic coupled, electrolytic sample cell is presented along with analysis of DNA and RNA separated from non-toxic bacteria.
Characterization of bacteria is currently an important research area in the medical, military, fo... more Characterization of bacteria is currently an important research area in the medical, military, food, and agricultural sciences. In recent years, FT-IR has found an application as a microbiological detection method and as a general research tool. When coupled with a liquid chromatographic system, a new facet of research has evolved. By utilizing the separation ability of typical liquid chromatography systems, matrix elimination is possible, therefore allowing for clean spectra of cellular components. Information about the compositional makeup of various bacteria enhances the overall understanding of biology at the cellular level, provides a quantification of the chemistry of cellular processes, and can be used as a general identification tool. Both whole cells and lysed Escherichia coli cells were investigated in the present study. The cellular components consisting of proteins, glycoproteins, phospholipids, fatty amides and acids, and genomic materials were separated, isolated, and identified by FT-IR.
Since September 11, 2001, the threat of terrorist attack and biological warfare within U.S. borde... more Since September 11, 2001, the threat of terrorist attack and biological warfare within U.S. borders has become a sobering reality. In an effort to aid military personnel and the public at large, we have been investigating the utility of surface-enhanced Raman spectroscopy (SERS) to provide rapid identification of chemical agents directly, and biological agents through their chemical signatures. This approach is based on the ability of Raman spectroscopy to identify molecular structure through the abundant vibration information provided in spectra and the ability of SERS to detect extremely low concentrations (e.g. part-per-billion) through the enhancement of Raman scattering by six orders of magnitude or more. Toward the goal of developing a portable analyzer, we have been studying the ability of two SER media to obtain continuous (i.e., reversible) and quantitative (i.e., reproducible) measurements. Here we compare measurements of nucleic acid bases, adenosine monophosphate, and ribonucleic acid extracted from Escherichia coli, Bacillus subtilis and Staphylococcus aureus obtained by electrolytic SERS and metal-doped sol-gel SERS. The capabilities of these SER media are summarized in terms of rapid detection of B. anthracis and dipicolinic acid.
Since the distribution of anthrax causing spores through the U.S. Postal System in the autumn of ... more Since the distribution of anthrax causing spores through the U.S. Postal System in the autumn of 2001, numerous methods have been developed to detect spores with the goal of minimizing casualties. During and following an attack it is also important to detect spores on surfaces, to assess extent of an attack, to quantify risk of infection by contact, as well as to evaluate post-attack clean-up. To perform useful measurements, analyzers and/or methods must be capable of detecting as few as 10 spores/cm2, in under 5-minutes, with little or no sample preparation or false-positive responses, using a portable device. In an effort to develop such a device, we have been investigating the ability of surfaceenhanced Raman spectroscopy (SERS) to detect dipicolinic acid (DPA) as a chemical signature of bacilli spores. In 2003 we employed SERS to measure DPA extracted from a 10,000 spores per μL sample using hot dodecylamine. Although the entire measurement was performed in 2 minutes, the need to heat the dodecylamine limits field portability of the method. Here we describe the use of a room temperature digesting agent in combination with SERS to detect 220 spores collected from a surface in a 1 μL sample within 3 minutes.
The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pse... more The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coil, and Bacillus subtilis have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their widespread occurrence in bacteria and their relatively high quantum yields. Candidate flnorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.
Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively... more Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively for Gram-negative Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes, as well as Gram-positive Bacillus subtilis and Staphylococcus epidermidis. Spectra have been obtained from cultures in the lag, log, and stationary growth phases excited in turn by 228.9, 244.0, and 248.2 nm light. Although Raman spectral peak positions (cm(-1)) excited by a given wavelength are very similar for all five bacterial species, the organisms are characterized by significantly different spectral intensity values. Intensity changes are associated with growth phase changes in all of the species as well. A comparison of measured with estimated average intensities has been made for spectra of log-phase E. coli. It is possible to compare measured intensities with intensities estimated for log-phase E. coli on the basis of the knowledge of its known average cellular molecular composition. A significant degree of hypochromism is observed in E. coli nucleic acid spectra. In contrast, strong average hyperchromism characterizes all aromatic amino acid peaks belonging to the same E. coli cells. Results suggest that knowledge of spectral intensity values will enhance significantly the capability to identify bacteria by means of their UV resonance Raman spectra.
Bacteria grown on trypticase soy agar (TSA), trypticase soy broth (TSB), and Davis minimal media,... more Bacteria grown on trypticase soy agar (TSA), trypticase soy broth (TSB), and Davis minimal media, and harvested at times ranging from 4.5 to 48 h have been excited at 242.54 and 222.65 nm for the purpose of generating resonance Raman spectra. When excitation with 242.54-nm light occurs, simple spectra of tyrosine and tryptophan and various nucleic acids are observed. Large changes in the relative intensities of major nucleic acid peaks at 1485 and 1575 cm -~, on the one hand, as compared to a prominent protein tyrosine + tryptophan peak at 1616 cm -~, on the other, have been attributed to very large variations in the RNA content of bacterial cells from culture to culture. The spectral changes are observed whenever differences in growth rates or variations in cultural media result in substantial changes in the amount of ribosomal RNA. In spite of very large cultural effects on peak intensities it has been possible to obtain bacterial G+C/A+T ratios from these spectra. Specifically, the ratio of the intensity of the C (1530 cm -~) peak to the intensity of the A+G peak (1485 cm -~) when plotted against the known molar percent G+C of the corresponding bacterial DNA produces a straight line. Plots have been shown to be very nearly growth-time and media independent for fourteen different types of bacteria, which range in DNA G+C content from 32 to 66%. Spectra obtained with 222.65nm light, in contrast with spectra obtained with 242.54-nm excitation, have been found to be nearly growth-rate and media independent. The excitation wavelength, 222.65 nm, appears to be the best yet found for use in rapid Raman identification of bacteria. All strong peaks which have been assigned have been attributed to protein modes. Relative intensities of 1556-cm ~ tryptophan and 1616-cm -~ tryptophan + tyrosine bands have been found to be strongly correlated with bacterial Gram type and nearly independent of cultural media or stage of growth. Index Heading: Raman spectroscopy.
Time-resolved fluorescence spectra have been obtained for Escherichia coli, Pseudomonas fluoresce... more Time-resolved fluorescence spectra have been obtained for Escherichia coli, Pseudomonas fluorescens, Staphylococcus epidermidis, and Enterobacter cloacae. Pseudomonas fluorescens has been shown to have distinctly different time-resolved spectra. Fluorescence excitation spectra for the three other organisms showing similar time-resolved spectra are very nearly alike, while spectra of Pseudomonas fluorescens are markedly different. This suggests that the changes in the average fluorescence lifetime with emission wavelength are due to the separate contributions from fluorophores of distinctly different lifetimes which have emission maxima at different wavelengths.
It has been determined that domoic acid (DA) can be quantitated from homogenized shellfish tissue... more It has been determined that domoic acid (DA) can be quantitated from homogenized shellfish tissue by means of resonance Raman spectra excited by 251 nm light. Detection limits have been found to be substantially below the 20 micrograms DA per grain tissue deemed by regulators to be unfit for human consumption. Clam tissue obtained from a supermarket has been prepared for analysis by direct homogenization for 2 minutes in a Waring blender. The homogenized samples were placed in a flow system and subjected to a 5-10 mw 251 nm excitation. Back-scattering collected for 20-30 seconds provided sufficient information for analysis. The method is extremely simple to use since the DA produces a single intense peak at 1652 cm-1. Because relatively-weak protein, nucleic acid and lipid spectra are excited from the tissue, background interference is surprisingly low. Bacteria can be detected using the same approach, but sensitivities are much lower primarily due to spectral interference from tissue nucleic acids.
The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pse... more The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coil, and Bacillus subtilis have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their widespread occurrence in bacteria and their relatively high quantum yields. Candidate flnorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.
Resonance Raman spectra of the gram-negative organism, Escherichia coil, have been obtained with ... more Resonance Raman spectra of the gram-negative organism, Escherichia coil, have been obtained with 222.5-, 230.6-, and 251.0-nm excitation,
Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. Th... more Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. The amount of heme necessary for half-maximal growth was 2 to 10 ng/ml (3.8 to 15 pmol/ml) among the Bacteroides species and strains tested. The growth rate, metabolic products from glucose fermentation, and cell yields were affected by the concentration of heme in the medium and by the length of time the culture was incubated. When heme was growth limiting (4 ng/ml), growth rates decreased by 50%, cultures started producing lactic and fumaric acids, and the cell yields declined. The cell yield for B. fragilis (ATCC 25285) at 24 h in medium containing 6.5 microgram of heme per ml was 69 g (dry weight) of cells per mol of glucose compared to 16 g (dry weight) of cells per mol of glucose with 4 ng of heme per ml. B. fragilis was unable to grow in defined medium when a porphyrin precursor, delta-aminolevulenic acid or porphobilinogen, was added in place of heme.
An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilis Morone chr... more An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilis Morone chrysops), using cells collected from the peritoneal cavity of this fish. The findings indicated that: (1) 10 days following a single intraperitoneal injection (1 ml) of Freund's incomplete adjuvant (FIA) was an appropriate time for collecting suitable working concentrations (5·3 4·0 10 7 cells ml 1 ) of peritoneal phagocytes (83·7 1·5% macrophages) from these hybrids held at 23 C; (2) these cells phagocytosed latex beads (polystyrene microspheres 3·12 m in diameter) after 30 min of in vitro incubation at room temperature (25 1 C). The phagocytic ability and phagocytic capacity in a washed adherent layer exposure system were 67·2 2·76% and 4·14 0·35 beads phagocyte 1 , respectively.
Updated information and services can be found at: These include: REFERENCES http://aac.asm.org/co... more Updated information and services can be found at: These include: REFERENCES http://aac.asm.org/content/45/11/3262#ref-list-1 at: This article cites 33 articles, 15 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on August 22, 2013 by guest http://aac.asm.org/ Downloaded from ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 0066-4804/01/$04.00ϩ0
Recently, a number of analytical methods have been successfully developed which use nucleic acid ... more Recently, a number of analytical methods have been successfully developed which use nucleic acid sequencing to identify biological warfare agents. However, the effectiveness of these methods, towards the safety and protection of US Armed Forces and their allies are limited by the period required to enumerate the nucleic acid through polymerase chain reactions or culture growth to produce sufficient quantities for analysis. To overcome this limitation, we have been investigating the ability of surface-enhanced Raman spectroscopy to detect nucleic acids with sufficient sensitivity and selectivity to eliminate the need for enumeration. The design of a small volume electrolytic sample cell will be presented along with analysis of the nucleic acid bases and preliminary analysis of model bacteria.
The ability of Raman spectroscopy to detect anthrax-causing spores as they pass through a mail so... more The ability of Raman spectroscopy to detect anthrax-causing spores as they pass through a mail sorting system was investigated. A pump was connected to an existing vacuum manifold on a commercial sorter, and a filter designed to capture 0.5-3 µm particles was placed in-line. A standard business letter containing 0.23 g of Bacillus cereus spores, a Bacillus anthracis surrogate, was placed in a stack of 20 letters and passed through the system. Raman spectra of the filter positively identified the captured material as bacterial spores by the dominant calcium dipicolinate Raman spectral bands associated with the spore core. A limit of detection, using 400 mW of 785 nm laser excitation for a 1-s acquisition, is estimated at 4.5 mg. The ability of a Raman spectroscopy based system to detect and prevent the distribution of a letter containing gram levels of anthrax spores is discussed.
The Chemical Weapons Convention prohibits the development, production, stockpiling, and use of wa... more The Chemical Weapons Convention prohibits the development, production, stockpiling, and use of warfare agents (chemical and biological), and requires their destruction. Yet their use persists and has been included in the terrorist's arsenal. Currently, a number of analytical methods are being developed to perform rapid measurements of trace agents to ensure treaty compliance, as well as safe environments for military personal and the public at large. We have been investigating the ability of surface-enhanced Raman spectroscopy to detect bacterial nucleic acid-base pairs with sufficient sensitivity and selectivity to eliminate the need for enumeration used in polymerase chain reactions and culture growth, required by other measurement techniques. The design of a small volume, fiber optic coupled, electrolytic sample cell is presented along with analysis of DNA and RNA separated from non-toxic bacteria.
Characterization of bacteria is currently an important research area in the medical, military, fo... more Characterization of bacteria is currently an important research area in the medical, military, food, and agricultural sciences. In recent years, FT-IR has found an application as a microbiological detection method and as a general research tool. When coupled with a liquid chromatographic system, a new facet of research has evolved. By utilizing the separation ability of typical liquid chromatography systems, matrix elimination is possible, therefore allowing for clean spectra of cellular components. Information about the compositional makeup of various bacteria enhances the overall understanding of biology at the cellular level, provides a quantification of the chemistry of cellular processes, and can be used as a general identification tool. Both whole cells and lysed Escherichia coli cells were investigated in the present study. The cellular components consisting of proteins, glycoproteins, phospholipids, fatty amides and acids, and genomic materials were separated, isolated, and identified by FT-IR.
Since September 11, 2001, the threat of terrorist attack and biological warfare within U.S. borde... more Since September 11, 2001, the threat of terrorist attack and biological warfare within U.S. borders has become a sobering reality. In an effort to aid military personnel and the public at large, we have been investigating the utility of surface-enhanced Raman spectroscopy (SERS) to provide rapid identification of chemical agents directly, and biological agents through their chemical signatures. This approach is based on the ability of Raman spectroscopy to identify molecular structure through the abundant vibration information provided in spectra and the ability of SERS to detect extremely low concentrations (e.g. part-per-billion) through the enhancement of Raman scattering by six orders of magnitude or more. Toward the goal of developing a portable analyzer, we have been studying the ability of two SER media to obtain continuous (i.e., reversible) and quantitative (i.e., reproducible) measurements. Here we compare measurements of nucleic acid bases, adenosine monophosphate, and ribonucleic acid extracted from Escherichia coli, Bacillus subtilis and Staphylococcus aureus obtained by electrolytic SERS and metal-doped sol-gel SERS. The capabilities of these SER media are summarized in terms of rapid detection of B. anthracis and dipicolinic acid.
Since the distribution of anthrax causing spores through the U.S. Postal System in the autumn of ... more Since the distribution of anthrax causing spores through the U.S. Postal System in the autumn of 2001, numerous methods have been developed to detect spores with the goal of minimizing casualties. During and following an attack it is also important to detect spores on surfaces, to assess extent of an attack, to quantify risk of infection by contact, as well as to evaluate post-attack clean-up. To perform useful measurements, analyzers and/or methods must be capable of detecting as few as 10 spores/cm2, in under 5-minutes, with little or no sample preparation or false-positive responses, using a portable device. In an effort to develop such a device, we have been investigating the ability of surfaceenhanced Raman spectroscopy (SERS) to detect dipicolinic acid (DPA) as a chemical signature of bacilli spores. In 2003 we employed SERS to measure DPA extracted from a 10,000 spores per μL sample using hot dodecylamine. Although the entire measurement was performed in 2 minutes, the need to heat the dodecylamine limits field portability of the method. Here we describe the use of a room temperature digesting agent in combination with SERS to detect 220 spores collected from a surface in a 1 μL sample within 3 minutes.
The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pse... more The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coil, and Bacillus subtilis have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their widespread occurrence in bacteria and their relatively high quantum yields. Candidate flnorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.
Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively... more Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively for Gram-negative Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes, as well as Gram-positive Bacillus subtilis and Staphylococcus epidermidis. Spectra have been obtained from cultures in the lag, log, and stationary growth phases excited in turn by 228.9, 244.0, and 248.2 nm light. Although Raman spectral peak positions (cm(-1)) excited by a given wavelength are very similar for all five bacterial species, the organisms are characterized by significantly different spectral intensity values. Intensity changes are associated with growth phase changes in all of the species as well. A comparison of measured with estimated average intensities has been made for spectra of log-phase E. coli. It is possible to compare measured intensities with intensities estimated for log-phase E. coli on the basis of the knowledge of its known average cellular molecular composition. A significant degree of hypochromism is observed in E. coli nucleic acid spectra. In contrast, strong average hyperchromism characterizes all aromatic amino acid peaks belonging to the same E. coli cells. Results suggest that knowledge of spectral intensity values will enhance significantly the capability to identify bacteria by means of their UV resonance Raman spectra.
Bacteria grown on trypticase soy agar (TSA), trypticase soy broth (TSB), and Davis minimal media,... more Bacteria grown on trypticase soy agar (TSA), trypticase soy broth (TSB), and Davis minimal media, and harvested at times ranging from 4.5 to 48 h have been excited at 242.54 and 222.65 nm for the purpose of generating resonance Raman spectra. When excitation with 242.54-nm light occurs, simple spectra of tyrosine and tryptophan and various nucleic acids are observed. Large changes in the relative intensities of major nucleic acid peaks at 1485 and 1575 cm -~, on the one hand, as compared to a prominent protein tyrosine + tryptophan peak at 1616 cm -~, on the other, have been attributed to very large variations in the RNA content of bacterial cells from culture to culture. The spectral changes are observed whenever differences in growth rates or variations in cultural media result in substantial changes in the amount of ribosomal RNA. In spite of very large cultural effects on peak intensities it has been possible to obtain bacterial G+C/A+T ratios from these spectra. Specifically, the ratio of the intensity of the C (1530 cm -~) peak to the intensity of the A+G peak (1485 cm -~) when plotted against the known molar percent G+C of the corresponding bacterial DNA produces a straight line. Plots have been shown to be very nearly growth-time and media independent for fourteen different types of bacteria, which range in DNA G+C content from 32 to 66%. Spectra obtained with 222.65nm light, in contrast with spectra obtained with 242.54-nm excitation, have been found to be nearly growth-rate and media independent. The excitation wavelength, 222.65 nm, appears to be the best yet found for use in rapid Raman identification of bacteria. All strong peaks which have been assigned have been attributed to protein modes. Relative intensities of 1556-cm ~ tryptophan and 1616-cm -~ tryptophan + tyrosine bands have been found to be strongly correlated with bacterial Gram type and nearly independent of cultural media or stage of growth. Index Heading: Raman spectroscopy.
Time-resolved fluorescence spectra have been obtained for Escherichia coli, Pseudomonas fluoresce... more Time-resolved fluorescence spectra have been obtained for Escherichia coli, Pseudomonas fluorescens, Staphylococcus epidermidis, and Enterobacter cloacae. Pseudomonas fluorescens has been shown to have distinctly different time-resolved spectra. Fluorescence excitation spectra for the three other organisms showing similar time-resolved spectra are very nearly alike, while spectra of Pseudomonas fluorescens are markedly different. This suggests that the changes in the average fluorescence lifetime with emission wavelength are due to the separate contributions from fluorophores of distinctly different lifetimes which have emission maxima at different wavelengths.
It has been determined that domoic acid (DA) can be quantitated from homogenized shellfish tissue... more It has been determined that domoic acid (DA) can be quantitated from homogenized shellfish tissue by means of resonance Raman spectra excited by 251 nm light. Detection limits have been found to be substantially below the 20 micrograms DA per grain tissue deemed by regulators to be unfit for human consumption. Clam tissue obtained from a supermarket has been prepared for analysis by direct homogenization for 2 minutes in a Waring blender. The homogenized samples were placed in a flow system and subjected to a 5-10 mw 251 nm excitation. Back-scattering collected for 20-30 seconds provided sufficient information for analysis. The method is extremely simple to use since the DA produces a single intense peak at 1652 cm-1. Because relatively-weak protein, nucleic acid and lipid spectra are excited from the tissue, background interference is surprisingly low. Bacteria can be detected using the same approach, but sensitivities are much lower primarily due to spectral interference from tissue nucleic acids.
The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pse... more The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coil, and Bacillus subtilis have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their widespread occurrence in bacteria and their relatively high quantum yields. Candidate flnorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.
Resonance Raman spectra of the gram-negative organism, Escherichia coil, have been obtained with ... more Resonance Raman spectra of the gram-negative organism, Escherichia coil, have been obtained with 222.5-, 230.6-, and 251.0-nm excitation,
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