W. H. Nelson

UV Resonance Raman Studies of Bacteria

Abstract Rapid and reliable methods for the detection and identification of microorganisms are very important. Fortunately, many effective means for bacterial identification have been developed. On the other hand, only a few of these are rapid, accurate, and cost effective. In most instances, if general examinations of bacteria must be performed, presently used techniques are very slow and may not be helpful unless extremely tedious procedures are followed. Traditional methods, which are based upon visual microscopic examination, biochemical reactions, and physiological functions of bacteria, are inherently slow, time consuming, and tedious. Even today, important decisions related to the presence of pathogens have to be made before the results of microbiological tests are available. This situation is changing rapidly, however.

UV Resonance Raman Spectra of Bacillus Spores

UV resonance Raman spectra of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis endospores have been excited at 222.7,230.7,242.5, and 251.1 nm, and spectra have been compared with those of vegetative cells. The resonance Raman spectra of aqueous solutions of dipicolinic acid and calcium dipicolinate have been measured at the same wavelengths. Spectra of endospores and their corresponding germinated spores show only modest differences when excited at 222, 231, and 251 nm. However, very substantial differences appear when excitation occurs at 242 nm. Difference spectra obtained at 242 nm by subtracting spectra of germinated spores of Bacillus cereus from spectra of their corresponding endospores are attributed almost entirely to dipicolinate. Vegetative cells and endospores show large spectral dissimilarities at all exciting wavelengths. These spectral differences, which vary strongly with exciting wavelength, appear to be the result of large differences in the amounts and composition of proteins and nucleic acids, especially ribosomal RNA. The very substantial resonance enhancement of Raman spectra has been obtained from aqueous solutions of pure dipicolinic acid and of sodium and calcium dipicolinate salts, as well as spores at the various exciting wavelengths. The strong enhancement of dipicolinate spectra in spores, however, was noted only with 242-nm excitation. Consequently, only with 242-nm light was it possible to selectively and sensitively excite and study calcium dipicolinate in spores. Resonance enhancement of the dipicolinate spectra with 242-nm excitation appears due primarily to resonance interactions with n-π* electronic transitions associated with the pyridine ring and/or the carboxylate group.

UV resonance Raman spectra of bacteria, bacterial spores, protoplasts and calcium dipicolinate

Abstract Resonance Raman spectra have been obtained with 222.65, 230.72, 242.39 and 250.96 nm excitation for Bacillus subtilis, Enterobacter cloacae, Pseudomonas fluorescens and Staphylococcus epidermids . Endospores of Bacillus cereus and protoplasts of Bacillus megaterium have been studied also. With 251 nm excitation, bacterial nucleic acid spectra are obtained selectively. Nucleic acid also strongly excited by 242 nm light while at that wavelength aromatic amino acid spectra just begin to be detected. Aromatic amino acid spectra are observed exclusively at 231 nm and appear along with some new strong nucleic acid peaks with 222 nm excitation. Calcium dipicolinate has been excited selectively in Bacillus spores at 242 nm. Large characteristic spectral differences can be explained as due to the selective excitation of various UV-absorbing cell components. Large intensity differences seen in the tryptophan-associated 1556 cm −1 peak excited at 222 and 231 nm appear to be strongly correlated with Gram type. Results suggest that UV-absorbing bacterial taxonomic markers can be selectively excited to give rise to characteristics resonance Raman bacterial spectral fingerprints which have the potential to be used as the basis for methods of rapid identification.

The Steady-State and Decay Characteristics of Primary Fluorescence From Live Bacteria

The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coli, 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 fluorophores 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.

An Ultraviolet (242 nm Excitation) Resonance Raman Study of Live Bacteria and Bacterial Components

Ultraviolet-excited (242 nm) resonance Raman spectra have been obtained for the first time for five types of bacteria: Escherichia coli, Pseudomonas fluorescens, Staphylococcus epidermidis, Bacillus subtilis, and Enterobacter cloacae. Detailed, highly reproducible spectra show substantial differences in both the intensities and the energies of peaks, which suggests that such spectra provide unique “fingerprints” reflecting the unique combinations of chemotaxonomic markers present in each type of organism. Many of the spectral features excited by 242-nm radiation probably arise from cellular RNA, DNA, and the amino acids tyrosine and tryptophan. Background fluorescence has been shown to be negligible.

Steady-state and decay characteristics of protein tryptophan fluorescence from bacteria

The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coli, and Bacillus subtilis have been observed. Each organism exhibits a strong maximum in its emission spectrum at 330–340 nm when excited at 290 nm. Iodide quenching and denaturization experiments with 8 M urea provide strong evidence for the assignment of the 330–340-nm fluorescence to protein tryptophan. Most importantly, the decay of this bacterial protein-tryptophan fluorescence has been described by two exponential functions in all cases. The observation that characteristic protein-tryptophan fluorescence lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Direct application will most likely be found in combination with the measurement of other luminescence parameters.

Ultraviolet micro‐Raman spectrograph for the detection of small numbers of bacterial cells

The construction of a practical UV micro‐Raman spectrograph capable of selective excitation of bacterial cells and other microscopic samples has been described. A reflective objective is used to focus cw laser light on a sample and at the same time collect the scattered light at 180°. With the aid of a quartz lens the image produced is focused on the slits of a spectrograph equipped with a single 2400 grooves/mm grating optimized for 250 nm. Spectra were detected by means of a blue‐intensified diode array detector. Resonance Raman spectra of Bacillus subtilis and Flavobacterium capsulatum excited by the 257.2 nm output of a cw laser were recorded in the 900–1800 cm−1 region. Bacterial cells were immobilized on a quartz plate by means of polylysine and were counted visually. Cooling was required to retard sample degradation. Sample sizes ranged from 1 to 50 cells with excitation times varying from 15 to 180 s. Excellent spectra have been obtained from 20 cells in 15 s using a spectrograph having only 3% th...

Ultraviolet Resonance Raman Spectra of Escherichia Coli with 222.5–251.0 nm Pulsed Laser Excitation:

Resonance Raman spectra of the gram-negative organism, Escherichia coli, have been obtained with 222.5-, 230.6-, and 251.0-nm excitation, and the results have been compared with those reported earlier for 242.4-nm excitation. Major changes in bacterial spectra have been observed with changes in exciting wavelength. The origins of the major peaks in each spectrum have been explained primarily in terms of contributions of nucleic acid bases and aromatic amino acids. As an aid in making assignments, spectra of aromatic amino acids, nucleosides, and mixtures of the two have been obtained at each wavelength used to excite bacterial spectra. Background fluorescence has been observed to be negligible below 251 nm. Selective excitation of bacterial nucleic acid and protein components has been done with ease. Results suggest that an extension of the exciting wavelength range to 190–220 nm will allow the selective excitation of additional cell components.

Effect of Cultural Conditions on Deep UV Resonance Raman Spectra of Bacteria

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−1, on the one hand, as compared to a prominent protein tyrosine + tryptophan peak at 1616 cm−1, 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−1) peak to the intensity of the A + G peak (1485 cm−1) 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 222.65-nm 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−1 tryptophan and 1616-cm−1 tryptophan + tyrosine bands have been found to be strongly correlated with bacterial Gram type and nearly independent of cultural media or stage of growth.

Differentiation of algae clones on the basis of resonance Raman spectra excited by visible light

Fourteen algae clones belonging to four different classes, including clones of Pseudo-nitzschia (Bacillariophyceae), some of which are capable of producing the toxin domoic acid, have been studied by means of resonance Raman spectra excited at 457.9 and 488 nm. Spectra taken at both excitation wavelengths are of high quality and are sufficiently distinct to differentiate clones at the algal class level. All spectra contain major features near 1000, 1153, and 1523 cm(-)(1), which are strongly resonance enhanced due to carotenoid pigments. Weaker features between 920-980 and 1170-1230 cm(-)(1), also due to carotenoid pigments, are more characteristic of the algae clones and more directly reflect different carotenoid composition. Similarities and differences among spectra have been analyzed by the method of principal component analysis (PCA). A distinct clustering of spectral data according to algal class has been shown by PCA score plots. All Pseudo-nitzschia clones can be separated from other classes of algae on the basis of spectra, but it is not possible to distinguish toxic Pseudo-nitzschia from nontoxic clones on the basis of these spectra, which reflect only differences in carotenoid composition.