Effect of selective growth media on the differentiation of Salmonella enterica serovars by Fourier-Transform Mid-Infrared Spectroscopy

Salmonella enterica serovars are prevalent foodborne pathogens responsible for high numbers of salmonellosis each year. Complex Fourier-transform infrared (FTIR) spectra offer unique biochemical fingerprints of bacteria with bands due to major cellular components. Growth media effects on discrimination of Salmonella serovars by FTIR spectroscopy were investigated and a novel sample preparation technique was developed. S. enterica strains from six serovars were grown on xylose lysine desoxycholate (XLD), Miller-Mallinson (MM), and plate count (PCA) agar as a control (37 degrees C, 24 h). Isolated colonies were suspended in 50% acetonitrile and centrifuged; the remaining pellet was placed on an AMTIR (attenuated total reflectance) crystal and dried under vacuum. Classification models (Soft Independent Modeling of Class Analogy, SIMCA), generated from derivatized infrared spectra (1300-900 cm-1 or 1200-900 cm-1), successfully discriminated among Salmonella strains with major discrimination from 1000-970 cm-1 associated to stretching modes of O-specific polysaccharide chains of lipopolysaccharides. Sample treatment with acetonitrile enhanced safe handling of the bacteria, removed interfering signals and improved the discriminating ability of SIMCA. All media were able to discriminate the S. enterica strains studied, varying in discriminating peaks and class distances in SIMCA classification. This methodology, with the production of large libraries of pathogenic bacteria, could be applied for the rapid monitoring of bacterial contamination in food with minimal sample manipulation.     

Mechanical compression affecting the thermal-induced conformational stability and denaturation temperature of human fibrinogen

Thermal-induced conformational stability and changes in denaturation temperature of human fibrinogen (FBG) after different mechanical compressions were investigated by a simultaneous Fourier transform infrared microspectroscopy equipped with thermal analyzer (thermal FTIR microscopic system). The confocal Raman microspectroscopy was also applied to determine the thermal reversibility of solid FBG. FBG powder was pressed on one KBr pellet (1 KBr method) or sealed within two KBr pellets (2 KBr method) by different mechanical compressions. The result indicates that there was no marked difference in the thermal behavior for the solid FBG samples prepared by 1 KBr method in the heating process even under different mechanical compression pressures, in which the thermal-induced denaturation temperatures from native to denatured state were maintained constant at 66-67 degrees C. However, the denaturation temperature for the solid FBG samples prepared by 2 KBr method was shifted from 55 to 62 degrees C with the increase of mechanical compression pressure. A good linear correlation was also found between the denaturation temperature and mechanical compression pressure for FBG samples prepared by 2 KBr method. The solid FBG sample, whether prepared by 1 KBr or 2 KBr method, was also found to show the thermal-irreversible property.     

Screening of poly-β-hydroxybutyrate-producing microorganisms using Fourier transform infrared spectroscopy

An alternative method for the analysis of poly--hydroxybutyrate (PHB) in microbial biomass based on FTIR spectroscopy was developed in which a semi-transparent, thin pellet of biomass was made with KBr and scanned by IR spectroscopy, from 400 cm^–1to 2000 cm^–1. The IR spectra of the target biomass showed a distinct absorbance band of the carboxylic ester group at 1724.03 cm^–1. The technique can be used to screen large numbers of microorganisms for PHB production.     

Pressure dependence of human fibrinogen correlated to the conformational alpha-helix to beta-sheet transition: an Fourier transform infrared study microspectroscopic study

We used Fourier transform infrared (FTIR) microspectroscopy to investigate pressure-induced conformational changes in secondary structure of fibrinogen (FBG). Solid state FBG was compressed on a KBr pellet (1KBr method) or between two KBr pellets (2KBr method). The peak positions of the original and second-derivative ir spectra of compressed FBG samples prepared by the 1KBr method were similar to FBG sample without pressure. When FBG was prepared by the 2KBr method and pressure was increased up to 400 kg/cm(2), peaks at 1625 (intermolecular beta-sheet) and 1611 (beta-sheet aggregates structure and/or the side-chain absorption of the tyrosine residues) cm(-1) were enhanced. The peaks near 1661 (beta-sheet) and 1652 (alpha-helix) cm(-1) also exhibited a marked change with pressure. A linear correlation was found between the peak intensity ratio of 1611/1652 cm(-1) (r = 0.9879) or 1625/1652 cm(-1) (r = 0.9752) and applied pressure. The curve-fitted compositional changes in secondary structure of FBG also indicate that the composition of the alpha-helix structure (1657-1659 cm(-1)) was gradually reduced with the increase in compression pressure, but the composition of the beta-sheet structure (1681, 1629, and 1609 cm(-1)) gradually increased. This indicates that pressure-induced conformational changes in FBG include not only transformations from alpha-helix to beta-sheet structure, but also unfolding and denaturation of FBG and the formation of aggregates.     

An alternative infrared spectroscopy assay for the quantification of polysaccharides in bacterial samples

The ability of bacteria to produce extracellular polysaccharides has been regarded as an indication of biofilm-forming capacity. Therefore, the determination of the sugar content in bacterial samples becomes a significant parameter. The colorimetric methods currently used are rather sensitive to the nature of the sugars and therefore require knowledge of the sugar types present in the samples. Unfortunately, the types of sugars present in bacteria are generally unknown and often composed of a complex mixture. In this article, we propose an alternative method based on Fourier transform infrared (FTIR) spectroscopy for the estimation of the total sugar content in bacterial samples. The method is based on a systematic treatment of FTIR spectra obtained from dried bacteria samples. It is assumed that the total sugar amount can be estimated from the area of characteristic bands between 970 and 1182 cm(-1). In parallel, the amide II band (1560-1530 cm(-1)) associated with proteins, or the C-H stretching region (2820-3020 cm(-1)) associated with the biomass, can be used for normalization purposes. Therefore, the ratio of the band area in the sugar window over that of the amide II or C-H stretching can be used to report the sugar content in bacterial samples. This method has been validated on model bacterial mixtures containing sugars, proteins, and DNA. Results with real bacterial samples are also provided and show conclusively that increased sugar contents in biofilms can be identified. The proposed FTIR approach requires minimal sample preparation and a single acquisition, is rapid, and may be applied to any kind of bacterial growth.     

Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy

Crystalline structures of cellulose (named as Cell 1), NaOH-treated cellulose (Cell 2), and subsequent CO2-treated cellulose (Cell 2-C) were analyzed by wide-angle X-ray diffraction and FTIR spectroscopy. Transformation from cellulose I to cellulose II was observed by X-ray diffraction for Cell 2 treated with 15-20 wt% NaOH. Subsequent treatment with CO2 also transformed the Cell 2-C treated with 5-10 wt% NaOH. Many of the FTIR bands including 2901, 1431, 1282, 1236, 1202, 1165, 1032, and 897 cm(-1) were shifted to higher wave number (by 2-13 cm(-1)). However, the bands at 3352, 1373, and 983 cm(-1) were shifted to lower wave number (by 3-95 cm(-1)). In contrast to the bands at 1337, 1114, and 1058 cm(-1), the absorbances measured at 1263, 993, 897, and 668 cm(-1) were increased. The FTIR spectra of hydrogen-bonded OH stretching vibrations at around 3352 cm(-1) were resolved into three bands for cellulose I and four bands for cellulose II, assuming that all the vibration modes follow Gaussian distribution. The bands of 1 (3518 cm(-1)), 2 (3349 cm(-1)), and 3 (3195 cm(-1)) were related to the sum of valence vibration of an H-bonded OH group and an intramolecular hydrogen bond of 2-OH ...O-6, intramolecular hydrogen bond of 3-OH...O-5 and the intermolecular hydrogen bond of 6-O...HO-3', respectively. Compared with the bands of cellulose I, a new band of 4 (3115 cm(-1)) related to intermolecular hydrogen bond of 2-OH...O-2' and/or intermolecular hydrogen bond of 6-OH...O-2' in cellulose II appeared. The crystallinity index (CI) was obtained by X-ray diffraction [CI(XD)] and FTIR spectroscopy [CI(IR)]. Including absorbance ratios such as A1431,1419/A897,894 and A1263/A1202,1200, the CI(IR) was evaluated by the absorbance ratios using all the characteristic absorbances of cellulose. The CI(XD) was calculated by the method of Jayme and Knolle. In addition, X-ray diffraction curves, with and without amorphous halo correction, were resolved into portions of cellulose I and cellulose II lattice. From the ratio of the peak area, that is, peak area of cellulose I (or cellulose II)/total peak area, CI(XD) were divided into CI(XD-CI) for cellulose I and CI(XD-CII) for cellulose II. The correlation between CI(XD-CI) (or CI(XD-CII)) and CI(IR) was evaluated, and the bands at 2901 (2802), 1373 (1376), 897 (894), 1263, 668 cm(-1) were good for the internal standard (or denominator) of CI(IR), which increased the correlation coefficient. Both fraction of the absorbances showing peak shift were assigned as the alternate components of CI(IR). The crystallite size was decreased to constant value for Cell 2 treated at >or= 15 wt% NaOH. The crystallite size of Cell 2-C (cellulose II) was smaller than that of Cell 2 (cellulose I) treated at 5-10 wt% NaOH. But the crystallite size of Cell 2-C (cellulose II) was larger than that of Cell 2 (cellulose II) treated at 15-20 wt% NaOH.     

Synchrotron Based FTIR Spectromicroscopy of Biopolymer Blends Undergoing Phase Separation

The results of a FTIR microspectroscopy analysis performed on the mixed gelling system of a protein (gelatin) and a polysaccharide (maltodextrin) are presented. A series of spectra were acquired consecutively by raster scanning the surface over a region of interest. The analysis has been carried out on three mixed-biopolymer samples prepared following different temperature paths, to study the effects of kinetics and thermodynamics on the texture of the final product. The chemical mapping has revealed to be very sensitive to composition, physical structure and hydration of the biopolymers. The results are critically discussed in reference to the band assignments and to protocols of sample preparation on the microtexture of the thin film.     

Reflectance circular dichroism of solid-state chiral coordination compounds

Solid-state diffuse reflectance circular dichroism (DRCD) spectra of some chiral lambda- and delta-M[(A--A)(n)](m+) coordination compounds [M(III) = Cr, Co; A--A = ethylendiamine (en), O,O'-2(R)3(R)dimethylethylene dithiophosphate ion, (R,R)bdtp(-), sepulchrate (Sep); n = 1, 3; m = 0, 3] were measured and compared with their solution, nujol mull, and KBr pellet spectra in the spectral region of 300-800 nm. The validity of the DRCD method to obtain reliable CD spectra which reflect a correct absolute configuration at the metal coordination center as well as subtle changes in the stereochemistry of the compound in the solid vs. solution states was proved. DRCD is the only spectroscopic method to examine randomly oriented chiral compounds without the effect of pressure, solvent, or diluting media such as KBr or nujol (which lends the sample irrecoverable and may interact with the sample), and directly relate to the structure that is revealed by X-ray diffractometry.     

Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniques

The acyl carbonyl group of [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin (5MeTA-chymotrypsin) has been investigated by using both resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopies. The spectrum of the acyl-enzyme carbonyl group has been obtained as a function of pH over the range 3.0-10.0 in the RR experiments and over the range 3.4-7.6 (p2H) in the FTIR experiments. The carbonyl spectral profiles obtained by using FTIR spectroscopy are substantially different from the carbonyl profiles obtained by using RR spectroscopy. The FTIR spectra were obtained by subtracting the spectrum of the free enzyme from that of the acyl-enzyme. Use of the active-site inhibitor phenylmethanesulfonyl fluoride demonstrates that part of the intensity observed in the FTIR spectra of 5MeTA-chymotrypsin is due to a subtraction artifact giving rise to enzyme-associated bands, probably from peptide groups perturbed by substrate binding. The enzyme bands can be removed by subtracting the FTIR spectrum of 13C=O acyl-enzyme from that of 12C=O acyl-enzyme. Additionally, this procedure reveals that one of the acyl-enzyme carbonyl bands observed at 1727 cm-1 using RR spectroscopy is absent in the FTIR acyl-enzyme spectrum. However, a feature near 1720 cm-1 can be induced in the FTIR spectrum by actinic light in the near-UV region. Thus, it is proposed that the 1727 cm-1 RR carbonyl band results from a population of acyl-enzymes which is generated by exposure to the laser beam during RR data collection. When both the RR and FTIR data are adjusted to remove artifacts, they provide essentially identical carbonyl stretching profiles.     

Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin.

Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.     

IR spectroscopic studies of major cellular components. III. Hydration of protein,nucleic acid,and phospholipid films

The IR absorption spectra of protein, DNA, RNA, and phospholipid films as a function of the water content are reported. We find that the hydration of protein films affects the peak intensity of amide I and amide II bands and the shape of the amide III band. For nucleic acids, the symmetric (nu(S) PO(2) (-)) and antisymmetric (nu(AS) PO(2) (-)) stretching vibrations of the phosphate linkage are the most affected by hydration, because both intensity changes and frequency shifts are observed. The spectra of phospholipid films are also sensitive to hydration, and they exhibit changes in the peak intensities and frequencies of both nu(S) PO(2) (-) and nu(AS) PO(2) (-) vibrations. We interpret the spectral differences between water saturated and dried films both in terms of structural changes and the change in the local dielectric in the vicinity of the polar and solvent exposed groups. In addition, we observe that the most significant change in the absorption intensity, frequency, and shape of the water sensitive vibrations occurs at high hydration levels. The principal component analysis of hydration results and the kinetics of water removal from sample films are also discussed. In addition, protein spectra acquired using film and KBr pellet sampling techniques are compared.     

Thermally induced permutations of antigenicity and conformation of arachin (peanut globular protein)*.

The reactivity of arachin (principal storage protein of the peanut) with anti-arachin decreased after arachin was heated at certain temperatures. Infrared and circular dichroic spectra of heated and unheated samples of arachin were examined to determine whether conformational modes changed concomitantly with decreased antigenicity. Infrared absorbances of each sample at amide I, II and V bands were essentially identical; however, absorbances at amide IV and VI bands were altered in relative intensities proportionally to the temperatures of treatment. Conformational modes, determined by circular dichroism, gradually increased in content of unordered structure from x-helical and pleated sheet modes relative to the temperature of treatment. These observations are discussed in regard to relationships of the loss of antigenicity to conformatonal changes in heated arachin.     

A1 reduction in intact cyanobacterial photosystem I particles studied by time-resolved step-scan Fourier transform infrared difference spectroscopy and isotope labeling

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy, with 5 mus time resolution, has been used to produce P700(+)A(1)(-)/P700A(1) FTIR difference spectra in intact photosystem I particles from Synechococcus sp. 7002 and Synechocystis sp. 6803 at 77 K. Corresponding spectra were also obtained for fully deuterated photosystem I particles from Synechococcus sp. 7002 as well as fully (15)N- and (13)C-labeled photosystem I particles from Synechocystis sp. 6803. Static P700(+)/P700 FTIR difference spectra at 77 K were also obtained for all of the unlabeled and labeled photosystem I particles. From the time-resolved and static FTIR difference spectra, A(1)(-)/A(1) FTIR difference spectra were constructed. The A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled trimeric photosystem I particles from both cyanobacterial strains are very similar. There are some mode frequency differences in spectra obtained for monomeric and trimeric PS I particles. However, the spectra can be interpreted in an identical manner, with the proposed band assignments being compatible with all of the data obtained for labeled and unlabeled photosystem I particles. In A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled photosystem I particles, negative bands are observed at 1559 and 1549-1546 cm(-)(1). These bands are assigned to amide II protein vibrations, as they downshift approximately 86 cm(-)(1) upon deuteration and approximately 13 cm(-)(1) upon (15)N labeling. Difference band features at 1674-1677(+) and 1666(-) cm(-)(1) display isotope-induced shifts that are consistent with these bands being due to amide I protein vibrations. The observed amide modes suggest alteration of the protein backbone (possibly in the vicinity of A(1)) upon A(1) reduction. A difference band at 1754(+)/1748(-) cm(-)(1) is observed in unlabeled spectra from both strains. The frequency of this difference band, as well as the observed isotope-induced shifts, indicate that this difference band is due to a 13(3) ester carbonyl group of chlorophyll a species, most likely the A(0) chlorophyll a molecule that is in close proximity to A(1). Thus A(1) reduction perturbs A(0), probably via a long-range electrostatic interaction. A negative band is observed at 1693 cm(-)(1). The isotope shifts associated with this band are consistent with this band being due to the 13(1) keto carbonyl group of chlorophyll a, again, most likely the 13(1) keto carbonyl group of the A(0) chlorophyll a that is close to A(1). Semiquinone anion bands are resolved at approximately 1495(+) and approximately 1414(+) cm(-)(1) in the A(1)(-)/A(1) FTIR difference spectra for photosystem I particles from both cyanobacterial strains. The isotope-induced shifts of these bands could suggest that the 1495(+) and 1414(+) cm(-)(1) bands are due to C-O and C-C modes of A(1)(-), respectively.     

Characterisation of Authentic Lignin Biorefinery Samples by Fourier Transform Infrared Spectroscopy and Determination of the Chemical Formula for Lignin

Efficient methods for lignin characterisation are increasingly important as the field of lignin valorisation is growing with the increasing use of lignocellulosic feedstocks, such as wheat straw and corn stover, in biorefineries. In this study, we characterised a set of authentic lignin biorefinery samples in situ with no prior purification and minimal sample preparation. Lignin chemical formulas and lignin Fourier transform infrared (FTIR) spectra were extracted from mixed spectra by filtering out signals from residual carbohydrates and minerals. From estimations of C, H and O and adjustment for cellulose and hemicelluloses contents, the average chemical formula of lignin was found to be C₉H_10.2O_3.4 with slight variations depending on the biomass feedstock and processing conditions (between C₉H_9.5O_2.8 and C₉H_11.1O_3.6). Extracted FTIR lignin spectra showed many of the same characteristic peaks as organosolv and kraft lignin used as benchmark samples. Some variations in the lignin spectra of biorefinery lignin residue samples were found depending on biomass feedstock (wheat straw, corn stover or poplar) and on pretreatment severity, especially in the absorbance of bands at 1267 and 1032 cm^?1 relative to the strong band at ~1120 cm^?1. The suggested method of FTIR spectral analysis with adjustment for cellulose and hemicellulose is proposed to provide a fast and efficient way of analysing lignin in genuine lignin samples resulting from biorefineries.     

Infrared spectroscopy of retinoids

The Fourier transform infrared spectra of 15 purified retinoids were compared. Retinoids with conjugated C = O groups revealed the presence of a band between 1730 and 1630 cm-1 X A characteristic of retinoids is the presence of a band between 1650 and 1620 cm-1 due to C = C stretching where three to four conjugated C = O and C = C bonds were present or between 1610 and 1555 cm-1 where more conjugated unsaturations were present. The presence of cis double bonds was confirmed by a band at 1380 cm-1 while unsubstituted trans double bonds gave absorbances at 990 to 955 cm-1. Epoxy rings, which are present in some retinoids, resulted in bands at 1250, 880, and 790 cm-1 while a furan structure was confirmed by bands at 1175, 1083, and 1065 cm-1.     

Contamination of humic acid by silica gel and sodium bicarbonate

Summary The extent of contamination of soil humic fractions by silica gel, sodium bicarbonate and sodium carbonate was investigated by infrared spectroscopy. Humic fractions were extracted with 0.1 N NaOH from a surface sample of a Davidson soil (Rhodic Paleudult, Reddish-brown Lateritic soil). After careful purification, the humic preparations were characterized by scanning with the Beckman IR-18A spectrophotometer using the micro KBr pellet technique. The spectra of the various humic fractions corresponded to the respective spectra reported in the literature. No indications were obtained for the presence of silica gel, sodium bicarbonate or sodium carbonate. Strong absorption at 1000 cm^-1, as exhibited by non-purified humic acid, could be caused by Si-O vibrations. However, this band disappeared after washing humic acid with HCl-HF.Contribution of the University of Georgia, College of Agric. Expt. Sta., College Sta., Athens, Georgia, USA.Contribution of the University of Georgia, College of Agric. Expt. Sta., College Sta., Athens, Georgia, USA.     

In situ synthesis of magnetite nanoparticles in carrageenan gels

Magnetite nanoparticles have been successfully synthesized in the presence of carrageenan polysaccharides using an in situ coprecipitation method. Iron coordination to the sulfate groups of the polysaccharide was confirmed by FTIR. The polysaccharide type (kappa, iota, or lambda) and concentration have been varied and their effects on particle morphology and chemical stability of the resultant nanocomposite investigated. The presence of carrageenan induces the formation of smaller particles, compared to those formed in the absence of polymer, and their average size depends on the nature and concentration of the polysaccharide used. The chemical stability of magnetite nanoparticles toward oxidation was also seen to depend on biopolymer type with magnetite formed in iota-carrageenan showing the highest chemical stability. A general tendency toward lower stability is observed as the polysaccharide concentration is increased. It is suggested that magnetite chemical stability in the carrageenan composites is determined by a fine balance between particle size and gel strength, the latter determining oxygen diffusion rates through the medium.     

库克Noni果汁多糖含量及分子量测定

建立测定库克Noni果汁中多糖含量及分子量的方法。通过醇沉法分离Noni果汁多糖,进一步分离纯化得到均一多糖组分;用精制Noni果汁多糖测得该多糖对葡萄糖的换算因子,对多糖含量进行定量测定;并通过SE-HPLC(Size Exclusion-High Performance Liquid Chromatography)法测定了该多糖的相对分子量。结果表明,多糖含量测定方法简便可行,供试液在4 h内显色稳定,重现性较好,平均回收率为99.3%,RSD为1.93%(n=3);测得该多糖的相对分子量为1140 KDa。     

Development and validation of an NMR-based identity assay for bacterial polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.