Evaluating the efficacy of a phage cocktail against Pseudomonas fluorescens group strains in raw milk: microbiological, physical, and chemical analyses.

The objective of this study was to investigate the effectiveness of a phage cocktail against Pseudomonas fluorescens group and its effect on the microbial, physical and chemical properties of raw milk during different storage conditions. A phage cocktail consisting of Pseudomonas fluorescens, Pseudomonas tolaasii, and Pseudomonas libanensis phages was prepared. As a result, reductions in fluorescent Pseudomonas counts of up to 3.44 log units for the storage at 4 °C and 2.38 log units for the storage at 25 °C were achieved. Following the phage application, it is found that there was no significant difference in the total mesophilic aerobic bacteria and Enterobacteriaceae counts. However, it was observed that the number of lactic acid bacteria was higher in phage-treated groups. The results also showed that pH values in the phage added groups were lower than the others and the highest titratable acidity was obtained only in the bacteria-inoculated group. As a future perspective, this study suggests that, while keeping the number of target microorganisms under control in the milk with the use of phages during storage, the microbiota and accordingly the quality parameters of the milk can be affected. This work contributes to the development of effective strategies for maintaining the quality and extending the shelf life of milk and dairy products.

Investigation of phage and molasses interactions for the biocontrol of E. coli O157:H7.

Resistance to antibiotics is one of the most critical health problems in the world. Therefore, finding new treatment methods to be used as alternatives to antibiotics has become a priority for researchers. Similar to phages, certain products containing antimicrobial components, such as molasses, are widely used to eliminate resistant bacteria. Molasses has a strong antimicrobial effect on bacterial cells, and this effect is thought to be due to the breakdown of the cytoplasmic cell membrane and cell proteins of the polyphenols in molasses. In the present study, phage-molasses interactions were investigated to examine the effects of concomitant use. It was found that molasses samples increased the size of phage plaques by up to 3-fold, and MIC and 1/2 × MIC concentrations of molasses increased the burst size of phages. Although no synergistic effect was found between the phage and molasses, the antimicrobial activities of the components and the effect of molasses on phage activity were demonstrated.

Authentication of liquid egg composition using ATR-FTIR and NIR spectroscopy in combination with PCA.

BACKGROUND: The starting point of this work is to propose a qualitative approach for authenticity confirmation of the composition of liquid egg products. To this end, it was aimed to detect the liquid egg authenticity and adulteration (with water) by using attenuated total reflectance Fourier-transform infrared (ATR-FTIR), near infrared (NIR) spectroscopy and chemometrics. RESULTS: Liquid (n = 50) and dry (n = 50) samples of whole egg, egg yolk, egg white, and whole egg containing extra egg white (BXEW) and water (BXW) were prepared. Principal component analysis (PCA) models were formed using the data obtained from ATR-FTIR and NIR measurements of liquid and dry samples. A better classification was achieved with PCA model of ATR-FTIR measurements formed by using dry samples (100%) instead of liquid ones (80%). The best separation was obtained between dry sample groups of BXEW and BXW (adulterated). The presence of water content in liquid samples showed a negative effect on classification of the samples, while a good classification (100%) was obtained for NIR measurements of both liquid and dry sample groups. The developed PCA models achieved classification regardless of the form of egg samples (liquid or dry). CONCLUSION: The results of the study revealed that adulterated egg samples (with water) could be qualitatively detected using ATR-FTIR and NIR spectroscopy techniques in combination with PCA. © 2019 Society of Chemical Industry.

Immunomagnetic separation and Listeriamonocytogenes detection with surface-enhanced Raman scattering

Background/aim: We aimed to develop a rapid method to enumerate Listeria monocytogenes (L. monocytogenes) utilizing magnetic nanoparticle based preconcentration and surface-enhanced Raman spectroscopy measurements. Materials and methods: Biological activities of magnetic Au-nanoparticles have been observed to have the high biocompatibility, and a sample immunosensor model has been designed to use avidin attached Au-nanoparticles for L. monocytogenes detection. Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhimurium) bacteria cultures were chosen for control studies. Antimicrobial activity studies have been done to identify bio-compatibility and bio-characterization of the Au-nanoparticles in our previous study and capturing efficiencies to bacterial surfaces have been also investigated. Results: We constructed the calibration graphs in various population density of L. monocytogenes as 2.2 × 101 to 2.2 × 106 cfu/mL and the capture efficiency was found to be 75%. After the optimization procedures, population density of L. monocytogenes and Raman signal intensity showed a good linear correlation (R2 = 0.991) between 102 to 106 cfu/mL L. monocytogenes. The presented sandwich assay provides low detection limits and limit of quantification as 12 cfu/mL and 37 cfu/mL, respectively. We also compared the experimental results with reference plate-counting methods and the practical utility of the proposed assay is demonstrated using milk samples. Conclusion: It is focused on the enumeration of L. monocytogenes in milk samples and the comparision of results of milk analysis obtained by the proposed SERS method and by plate counting method stay in food agreement. In the present study, all parameters were optimized to select SERS-based immunoassay method for L. monocytogenes bacteria to ensure LOD, selectivity, precision and repeatablity.

SERS-based rapid assay for sensitive detection of Group A Streptococcus by evaluation of the swab sampling technique.

Beta-hemolytic, Group A Streptococcus pyogenes (GAS) is a life-threating pathogen and the reason for prominent disease, pharyngitis. The conventional analysis of GAS, gold standard, takes 48 hours and the related rapid tests lack in accuracy and sensitivity. In this study, firstly, the efficiency of swab sampling, which is a must in the GAS detection, was discussed with the proposed surface-enhanced Raman spectroscopy (SERS)-based batch assay and each step was controlled by the plate-counting method. Secondly, SERS-based lateral flow immunoassay (LFIA) test strips were constructed and the variation in the SERS intensity of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was observed. Thus, a linear correlation was found with a R2 value of 0.9926 and the LOD was calculated to be 0.2 CFU mL-1 of GAS which could be counted as one cell. The combination of the gold standard with the LFIA-SERS technique enabled the fast and accurate pathogen detection. In addition, GAS was quantified with paper-based test strips up to 100 CFU ml-1 level of bacteria for the first time without any interference. Besides, this study was featured with the discussion of the whole cell and pretreated cell detection of pathogens with LFIAs. Therefore, this work enlightens the points that have never been discussed on pathogen detection with paper-based platforms.

A simple and fast method for discrimination of phage and antibiotic contaminants in raw milk by using Raman spectroscopy.

Phage and antibiotic in raw milk poses significant risks for starter culture activity in fermented products. Therefore, rapid detection of phage and antibiotic contaminations in raw milk is a crucial process in dairy science. For this purpose, a preliminary novel method for detection of phage and antibiotic was developed by using Raman spectroscopy. Streptococcus thermophilus phages and ampicillin which are quite important elements in dairy industry were used as model. The phage and antibiotic samples were added to raw milk separately, and Raman measurements were carried out. The obtained spectra were processed with a chemometric method. In this study, it has been demonstrated that the presence of phage has a titer sufficient to stop the fermentation (107 pfu/ml), and antibiotic in a concentration which inhibits the growth of starter cultures (0.5 µg/ml) in raw milk could be discriminated through Raman spectroscopy with a short analysis time (30 min).

Rapid discrimination between buffalo and cow milk and detection of adulteration of buffalo milk with cow milk using synchronous fluorescence spectroscopy in combination with multivariate methods.

This research paper describes the potential of synchronous fluorescence (SF) spectroscopy for authentication of buffalo milk, a favourable raw material in the production of some premium dairy products. Buffalo milk is subjected to fraudulent activities like many other high priced foodstuffs. The current methods widely used for the detection of adulteration of buffalo milk have various disadvantages making them unattractive for routine analysis. Thus, the aim of the present study was to assess the potential of SF spectroscopy in combination with multivariate methods for rapid discrimination between buffalo and cow milk and detection of the adulteration of buffalo milk with cow milk. SF spectra of cow and buffalo milk samples were recorded between 400-550 nm excitation range with Δλ of 10-100 nm, in steps of 10 nm. The data obtained for ∆λ = 10 nm were utilised to classify the samples using principal component analysis (PCA), and detect the adulteration level of buffalo milk with cow milk using partial least square (PLS) methods. Successful discrimination of samples and detection of adulteration of buffalo milk with limit of detection value (LOD) of 6% are achieved with the models having root mean square error of calibration (RMSEC) and the root mean square error of cross-validation (RMSECV) and root mean square error of prediction (RMSEP) values of 2, 7, and 4%, respectively. The results reveal the potential of SF spectroscopy for rapid authentication of buffalo milk.

Surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin in urine.

We present a surface-enhanced Raman probe (SERS) platform for the determination of a prohibited substance, recombinant erythropoietin (rEPO), in urine matrix, using nanoparticles as substrate. Rod-shaped gold nanoparticles (GNR) were modified with a Raman label and an antibody as SERS probe. We developed two SERS-based immunoassays for detection and quantification of rEPO in urine. In the first assay, rEPO was determined by a sandwich assay with gold surfaces and GNR. In the second assay, rEPO was extracted by using core shell-structured magnetic iron oxide gold nanoparticles, and again sandwich assay was performed by using GNR. We also demonstrated the ability of the proposed method to discriminate rEPO and urinary erythropoietin (uEPO). A good linear correlation was obtained between logarithms of rEPO concentrations in urine and Raman intensities within the range of 10-1-103 pg mL-1 rEPO concentrations. Detection limits which are smaller than 0.1 pg mL-1 levels were achieved owing to the high extractive performance of the nanoextraction techniques. Graphical Abstract Schematic represantation of surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin.

Spectroscopic fingerprint of tea varieties by surface enhanced Raman spectroscopy.

The fingerprinting method is generally performed to determine specific molecules or the behavior of specific molecular bonds in the desired sample content. A novel, robust and simple method based on surface enhanced Raman spectroscopy (SERS) was developed to obtain the full spectrum of tea varieties for detection of the purity of the samples based on the type of processing and cultivation. For this purpose, the fingerprint of seven different varieties of tea samples (herbal tea (rose hip, chamomile, linden, green and sage tea), black tea and earl grey tea) combined with silver colloids was obtained by SERS in the range of 200-2000 cm(-1) with an analysis time of 20 s. Each of the thirty-nine tea samples tested showed its own specific SERS spectra. Principal Component Analysis (PCA) was also applied to separate of each tea variety and different models developed for tea samples including three different models for the herbal teas and two different models for black and earl grey tea samples. Herbal tea samples were separated using mean centering, smoothing and median centering pre-processing steps while baselining and derivatisation pre-processing steps were applied to SERS data of black and earl grey tea. The novel spectroscopic fingerprinting technique combined with PCA is an accurate, rapid and simple methodology for the assessment of tea types based on the type of processing and cultivation differences. This method is proposed as an alternative tool in order to determine the characteristics of tea varieties.

Paper membrane-based SERS platform for the determination of glucose in blood samples.

In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications.

SERS-based direct and sandwich assay methods for mir-21 detection.

In this study, two different assay methods were developed using a surface enhanced Raman scattering (SERS) label for sensitive miR-21 detection. In the first method (direct assay), the miR-21 probe was attached to SERS-labelled, rod-shaped gold nanoparticles and hybridised with the target miR-21, which was previously immobilised onto the gold slide. In the second method (sandwich assay), the target miR-21 was captured by an miR-21 probe immobilised onto the gold slide and hybridised with a second miR-21 probe immobilised on the SERS-labeled, rod-shaped gold nanoparticles. SERS signals of developed assays were obtained via a SERS spectrum of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) on the rod-shaped nanoparticles. The calibration curves were plotted to measure the different concentrations of miR-21. The detection limits of the direct and sandwich assays, which last less than 40 min, were found to be 0.36 and 0.85 nM, respectively. The developed SERS-based methods offer rapid, selective, sensitive and easy detection of miR-21, especially compared to conventional PCR-based methods.

Evaluation of long- and short-term storage conditions and efficiency of a novel microencapsulated Salmonella phage cocktail for controlling Salmonella in chicken meat.

This study aims to assess the stability and activity of using a lyophilization, formulation design and to evaluate their efficiency for controlling Salmonella in chicken meat. The phage-loaded 0.3 M sucrose gelatin mixture at 4 and 25 °C displayed significantly less phage titer loss (p < 0.05) than the other excipients and liquid phage cocktail in 12 months. The results showed that there were significant reductions of Salmonella at the end of the storage in chicken meat for newly prepared phage powder (1.86 log CFU/cm2 and 2.18 log CFU/cm2), lyophilized phage powders stored at 4 °C (1.08 log CFU/cm2 and 1.26 log CFU/cm2) and stored at 25 °C (0.66 log CFU/cm2 and 1.00 log CFU/cm2) for 10 months at MOI 100 and 1000, respectively. The results demonstrated that lyophilized phages in a simple food grade formulation can be successfully stored and might be used in biocontrol of Salmonella in meat.

Effect of wax separation on macro- and micro-elements, phenolic compounds, pesticide residues, and toxic elements in propolis.

Propolis, a natural product with many biological activities, is a resinous material produced by honeybees. It contains not only valuable components but also some possible contaminants in varying amounts. Hence, this study aimed to examine how the process step of wax separation affects certain elements, pesticide residues, and phenolic compounds in propolis. Total phenolics, elements, and some pesticide residues were analyzed in the crude propolis (CP samples), wax portion (W samples), and remaining propolis fraction (PF samples) after wax separation. Total phenolics of the CP samples were determined in the range of 31.90-45.00 mg GAE g-1 sample, while those of the PF samples were in the range of 54.97-162.09 mg GAE g-1 sample. Loss/reduction values by means of wax separation for phenolics were calculated as 10.88% and 17.89%, respectively. Pb contents of all PF samples were low (0.232-1.520 mg kg-1), but it was also noteworthy that nearly 40% or even more of Cr, As, Cd, and Pb were removed by wax separation. Removal of significant amounts of carbendazim (38.09%-67.35%), metalaxyl (81.57%-72.67%), tebuconazole (65.99%-78.36%), and propargite (88.46%-83.05%) was also achieved. Wax separation enables the removal of toxic substances from crude propolis without causing huge losses in phenolic compounds.

Estimation of time since deposition of semen stain on different fabric types using ATR-FTIR spectroscopy and chemometrics.

Various body fluids such as blood, semen, vaginal secretions, and saliva are frequently encountered at crime scene. In cases of sexual assault, semen stains are one of the most reliable evidence of biological origin. In this study, our objective was to develop a method for estimating the time since deposition of semen stains on five different fabric types using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy, with a focus on a time frame of up to 8 weeks. Semen samples from six different volunteers were dripped onto five distinct fabric materials, and ATR-FTIR measurements were obtained at 17 different time points. Principal component analysis (PCA) and partial least squares (PLS) methods were employed to differentiate semen stains on various fabric samples and estimate the age of semen stains. Models constructed using PCA and PLSR achieved high R2 values and low root-mean-square error (RMSE). While the performance varies depending on fabric types, it was observed that age estimation of semen stains can be made within following intervals: 0.39-0.76 days for 0-7 day range, 2.59-3.38 days for the 1-8 week range, and 3.98-8.1 days for the 0-56 day range. This study demonstrates the effectiveness of using ATR-FTIR spectroscopy in combination with chemometrics to estimate the age of human semen stains on various fabric types based on time-dependent spectral changes.

Identification of blood at simulated crime scenes using silver nanoparticles with SERS.

The analysis of substances and samples obtained from a crime scene is very important in solving forensic cases. To determine the variables involved in a crime and to expedite the investigation process, the rapid analysis of body fluids in small quantities and within environments containing diverse components is particularly necessary. For this reason, it is of great importance to analyze biological fluids with rapid, noncontaminating, nondestructive, low-cost, and accurate techniques. In recent years, with advancements in laser technology, spectroscopic methods have been introduced as analytical techniques in forensic medicine and chemical studies. This study focuses on surface-enhanced Raman spectroscopy (SERS) to demonstrate the detection of blood samples in simulated crime scenes. To minimize the background signal from fluorescent biomolecules in blood, dilution was performed with two different components and Raman analysis was performed for four different concentrations of blood. In general, a decrease in noise in the spectra was observed as the blood was diluted. Crime scenes consisting of pure blood, blood diluted with ethanol and distilled water (1:2, 1:4, and 1:8), a blood-mineral water mixture, a blood-cherry juice mixture, and silver nanoparticle-added mixtures were simulated, and their spectra were examined. Chemometric analyses of the data were performed. Despite high noise and low peak intensities, blood-identifying signals were detected when examining different blood concentrations. It was observed that silver nanoparticles provided high enhancement of blood peaks thanks to their strong plasmonic properties.

Gold-Coated Iron Composite Nanospheres Targeted the Detection of Escherichia coli.

We report the preparation and characterization of spherical core-shell structured Fe3O4-Au magnetic nanoparticles, modified with two component self-assembled monolayers (SAMs) consisting of 3-mercaptophenylboronic acid (3-MBA) and 1-decanethiol (1-DT). The rapid and room temperature synthesis of magnetic nanoparticles was achieved using the hydroxylamine reduction of HAuCl4 on the surface of ethylenediaminetetraacetic acid (EDTA)-immobilized iron (magnetite Fe3O4) nanoparticles in the presence of an aqueous solution of hexadecyltrimetylammonium bromide (CTAB) as a dispersant. The reduction of gold on the surface of Fe3O4 nanoparticles exhibits a uniform, highly stable, and narrow particle size distribution of Fe3O4-Au nanoparticles with an average diameter of 9 ± 2 nm. The saturation magnetization value for the resulting nanoparticles was found to be 15 emu/g at 298 K. Subsequent surface modification with SAMs against glucoside moieties on the surface of bacteria provided effective magnetic separation. Comparison of the bacteria capturing efficiency, by means of different molecular recognition agents 3-MBA, 1-DT and the mixed monolayer of 3-MBA and 1-DT was presented. The best capturing efficiency of E. coli was achieved with the mixed monolayer of 3-MBA and 1-DT-modified nanoparticles. Molecular specificity and selectivity were also demonstrated by comparing the surface-enhanced Raman scattering (SERS) spectrum of E. coli-nanoparticle conjugates with bacterial growth media.

Isolation, characterization, and application of bacteriophage cocktails for the biocontrol of Pseudomonas fluorescens group strains in whole and skimmed milk.

Pseudomonas fluorescens group strains can lead to spoilage of milk as well as loss of quality in dairy products through their heat-resistant enzymes. Phages are important alternatives for combating spoilage bacteria in food industry and used successfully in many applications. The aim of this study was the isolation and characterization of phages and to assess the efficiency of a phage cocktail in whole and skimmed milk. For this purpose, phages effective against Pseudomonas fluorescens (L23.2), Pseudomonas tolaasii (P22.1), and Pseudomonas rhodesiae (A11.1) were isolated. Their host range was found to be highly specific, and the transmission electron micrographs indicates that they belonged to Tectiviridae family. Their genome sizes were found to be vary between 38.3 and 53.5 kb. The latent periods and burst sizes were determined as 15, 10, 15 min and 91, 20, 80 PFU/infected cell for L23.2, P22.1, and A11.1, respectively. All three phages were found to be sensitive to low pH and high temperature. The effect of the phage cocktail was monitored in milk with different fat contents during storage at 4 °C for 5 days. As a result, bacterial reductions up to 4.09 and 5.29 log-units were observed for the whole and skimmed milk, respectively. Thus, the efficacy of a phage cocktail against a bacterial mixture of different P. fluorescens strains was tested in milk samples with different fat contents in accordance with real-life scenarios for the first time.

Principles, Methods, and Real-Time Applications of Bacteriophage-Based Pathogen Detection.

Bacterial pathogens in water, food, and the environment are spreading diseases around the world. According to a World Health Organization (WHO) report, waterborne pathogens pose the most significant global health risks to living organisms, including humans and animals. Conventional bacterial detection approaches such as colony counting, microscopic analysis, biochemical analysis, and molecular analysis are expensive, time-consuming, less sensitive, and require a pre-enrichment step. However, the bacteriophage-based detection of pathogenic bacteria is a robust approach that utilizes bacteriophages, which are viruses that specifically target and infect bacteria, for rapid and accurate detection of targets. This review shed light on cutting-edge technologies about the novel structure of phages and the immobilization process on the surface of electrodes to detect targeted bacterial cells. Similarly, the purpose of this study was to provide a comprehensive assessment of bacteriophage-based biosensors utilized for pathogen detection, as well as their trends, outcomes, and problems. This review article summaries current phage-based pathogen detection strategies for the development of low-cost lab-on-chip (LOC) and point-of-care (POC) devices using electrochemical and optical methods such as surface-enhanced Raman spectroscopy (SERS).

Evaluation of antibacterial efficacy of Lawsonia inermis Linn (henna) on periodontal pathogens using agar well diffusion and broth microdilution methods: An in-vitro study.

Background: Although widely explored in medicine, limited evidence exists in the literature regarding the efficacy of Lawsonia inermis Linn (henna) in the dental field. Aim: This study aimed to investigate the antibacterial effect of henna on Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in vitro. Methods: The agar well diffusion and broth microdilution methods were used to evaluate the antibacterial effect of henna extracts. Dimethyl sulfoxide was used to prepare the ethanol extract of henna, and distilled water was used to prepare the water extract. For both ethanol and water extracts, 4 different concentrations were prepared as 15, 30, 60, and 120 mg/mL. Results: It was determined that the water and ethanol extracts of the henna samples did not show an inhibition zone on P.gingivalis and A.actinomycetemcomitans. As a result of the evaluations made with the broth microdilution method, it was found that the ethanol extract had a higher inhibitory effect on both bacteria, and both extracts had more inhibitory effects against A.actinomycetemcomitans. Conclusion: To understand the effect of henna on periodontal pathogens, more comprehensive in vitro studies should be performed on henna samples at different concentrations and with different bases.

Overcoming obstacles: Analysis of blood and semen stains washed with different chemicals with ATR-FTIR.

INTRODUCTION: Blood and semen stains are the most common biological stains encountered at crime scenes. The washing of biological stains is a common application that perpetrators use to spoil the crime scene. With a structured experiment approach, this study aims to investigate the effects of washing with various chemicals on the ATR-FTIR detection of blood and semen stains on cotton. MATERIALS AND METHODS: On cotton pieces, a total of 78 blood and 78 semen stains were applied, and each group of six stains was immersed or mechanically cleaned in water, 40% methanol, 5% sodium hypochlorite solution, 5% hypochlorous acid solution, 5 g/L soap dissolved pure water, and 5 g/L dishwashing detergent dissolved water. ATR-FTIR spectra gathered from all stains and analyzed with chemometric tools. RESULTS AND DISCUSSION: According to performance parameters of developed models, PLS-DA is a powerful tool for discrimination of washing chemical for both washed blood and semen stains. Results from this study show that FTIR is promising for use in detecting blood and semen stains that have become invisible to the naked eye due to washing of the findings. CONCLUSION: Our approach allows blood and semen to be detected on cotton pieces using FTIR combined with chemometrics, even though it is not visible to the naked eye. Washing chemicals also can be distinguished via FTIR spectra of stains.