Monitoring of pathogen carrying air-borne tea dust particles by light scattering

Monitoring of pathogen carrying air-borne tea dust particles by light scattering

Journal of Quantitative Spectroscopy & Radiative Transfer 112 (2011) 1784–1791 Contents lists available at ScienceDirect Journal of Quantitative Spe...

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Journal of Quantitative Spectroscopy & Radiative Transfer 112 (2011) 1784–1791

Contents lists available at ScienceDirect

Journal of Quantitative Spectroscopy & Radiative Transfer journal homepage: www.elsevier.com/locate/jqsrt

Monitoring of pathogen carrying air-borne tea dust particles by light scattering Sanchita Roy a,n, Rupjyoti Mahatta a, Nilakshi Barua b, Alak K. Buragohain b, Gazi A. Ahmed a a b

Optoelectronics and Photonics Research laboratory, Department of Physics, Tezpur University, Tezpur 784028, Assam, India Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784028, Assam, India

a r t i c l e in f o

abstract

Available online 11 January 2011

In order to investigate whether tea (Camellia sinensis (L.) Kuntze) dust particles could be a possible carrier of the pathogen contaminated Mycobacterium a biotechnical procedure was used, and to verify the possibility of monitoring this dust, a laser based light scattering setup was designed and fabricated. Experiments were carried out using the strain Mycobacterium smegmatis mc2 155 as a model organism to study the effect on tea dust particles. Light scattering investigations on both M. smegmatis contaminated and uncontaminated tea dust particle samples were carried out as a function of scattering angle at 543.5, 594.5 and 632.5 nm wavelengths. The results have shown that the behavior of tea dust samples both with and without Mycobacterium varies significantly for all the three different incident laser wavelengths. & 2011 Elsevier Ltd. All rights reserved.

Keywords: Light scattering Mueller matrix Camellia sinensis Mycobacterium

1. Introduction Light scattering is an important tool for the optical characterization of small particles suspended in air or when dispersed in a medium. Scattering of light [1–4] by biological samples is a subject of contemporary interest and extensive research in the present time [5]. Optical characterization of such particles is very important for a wide variety of applications, for example in monitoring hazardous air-borne particles. The usual technique is to measure the intensity of light scattered by a particle as a function of the angle between the incident and the scattered radiations. The study of angular scattering dependency of such particulate matter helps to investigate the nature of the scattering particle and to understand the radiative transfer through a medium containing the scatterer [5,6]. A number of different experimental setups have been made in the past to investigate the scattering behavior of small particles [7–12].

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Corresponding author. Tel.: + 91 9706016516; fax: +91 3712267006. E-mail address: [email protected] (S. Roy).

0022-4073/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jqsrt.2011.01.001

Tea is an important and very popular beverage used all over the world and is mainly brewed from the tender leaves of the plant species Camellia sinensis (L.) Kuntze. The plant is grown in many tropical regions of the world. The selected leaves and buds of tea plants are processed to produce the tea of commerce. During the processing stages in the tea industries from raw leaves to commercially packaged industrial tea, tea dust is released into the atmosphere as an effluent. There is possibility of the tea dust acting as carrier for asthmatic triggers and also carrier for pathogens. It has been found that the adverse effect of tea dust is mostly on the pulmonary function of tea workers who are exposed to this dust. Several authors have reported tea dust as a cause of occupational asthma, chronic respiratory disease, etc. [13]. Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB). The infection of humans by M. tuberculosis dates back to antiquity [14]. M. tuberculosis is a Gram positive bacterium with its size ranging from 0.2 to 0.4 mm. TB has been exacerbated due partly to TB deaths in the AIDS patients and due to the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of the M. tuberculosis [15]. The World Health Organization (WHO) declared TB as a global

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emergency in 1993. In 2008, an estimated 390,000–510,000 cases of MDRTB emerged globally (best estimate, 440,000 cases, 2010 Global Report) [16]. In view of the alarming global assessment of the disease and the ineffectiveness of the conventional first and second line of drugs used to treat TB, there is an urgent need for discovering and developing new antimycobacterial agents and new diagnosis and detection systems. A prerequisite in the control of TB is proper understanding of the aerosolic spread and survival of M. tuberculosis in air. It must be emphasized that M. tuberculosis is non-motile and can survive for 90–120 days on inanimate surfaces [17,18]. Among the pathogens that have relatively greater likelihood of being carried by tea dust particles is M. tuberculosis. Dissemination of pathogenic aerosols in the environment requires very advanced detection systems that may be able to detect and distinguish these pathogenic aerosols from the normal environment conditions, especially while considering the particle size of 1–10 mm size [19]. It is highly desirable that the advanced systems developed for such detections are capable of generating online and rapid information about possible threats caused by these life-threatening aerosols. Among the several techniques developed so far, the sensors based on elastic scattering is quite promising as the technique could provide information about size, shape and quality of particle surface [19,20]. Interesting results based on light scattering technique by biological particles were presented by Bickel and others around 1980 [21,22]. Description on Mueller scattering matrix and how to measure the 16 elements of the matrix were also presented [1,2,23]. Most research in this area was dedicated to the development of computational methods such as Mie theory [1,2,20], but it could not provide adequate information that could characterize the non-spherical particles having random orientations. The fundamental information about such particle is obtained from the scattered intensity, I (y, ~), where y is the polar angle relative to the direction of incident beam and ~ is the azimuthal angle relative to the scattering plane. This paper reports the work done in one such region, namely Assam in India, where tea is grown industrially and processed on a very large scale. The present work was aimed at investigating whether air-borne tea dust particles are a possible carrier of Mycobacterium pathogens, and secondly, whether monitoring of such non-spherical particles can be done by light scattering techniques. Some usual techniques are based on laser induced fluorescence and scattering techniques to characterize aerosol particles [20,24–26]. A number of different experimental setups have been made in the past to investigate the scattering behavior of small particles [27–30]. Several techniques and approaches were followed by different researchers to design instruments for its successful implementation and experimental observation of scattering signatures and to determine the matrix elements. There are many such reports on sophisticated and fully computerized light scattering setup used to investigate and characterize small particles including natural and artificial aerosols [9,10,29–32]. An original laboratory light scattering instrument, which could successfully measure the volume scattering of air borne non-spherical tea dust, was designed and

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fabricated and investigations were conducted. In our present work we have determined the phase function S11 [1–3], which was obtained by measuring the scattered light intensity as a function of scattering angle. 2. Experimental details 2.1. Light scattering setup The modified version of a laser based setup [33,34] was designed and fabricated in the Optoelectronics and Photonics Laboratory, Department of Physics, Tezpur University, Assam, India, to study the light scattering characteristics of small and ultra-small particles. The essential components of the setup (Fig. 1) are a laser source, controlled sample holders, a photomultiplier tube detector, data acquisition system and associated instrumentation. The system uses He–Ne laser source used alternatively for three different wavelengths of 632.8, 594.5 and 543.5 nm having an output power of 2.0, 0.35 and 0.35 mW, respectively, for studying the phase function as a function of the scattering and azimuthal angles. The diameter of beam cross-sections was approximately 1 mm and the distance between the source and the scattering center was 200 mm. The beam intensity was observed and it was found to be homogeneous (Fig. 2). The intensity was also monitored for 60 s and there were no fluctuations in the intensity observed for all the three wavelengths. The arrangement of polarizers in our experimental setup was optional and was not used specifically in our experiment as we have attempted to measure only the scattering function. The laser light was scattered by the sample of tea dust particles placed at the scattering center held by a mechanical support. The scattered light intensity signal was sensed by the photomultiplier tube (H 5784-20, Hamamatsu, Japan) which is movable along both y and ~ directions. This was further connected to a high gain and a low noise amplifier circuit. The amplified signals were interfaced to a data acquisition system (Vinytics, PCI-9812) to store the recorded data. The system could measure scattered light signals from an angle of 10–1701 in steps of 51 for y, and from 101 to 601 in steps of

Computer for data acquisition & Stepper motor control

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M Turn table Fig. 1. Schematic diagram of the scattering setup. P—Photomultiplier tube; L-Polarizer; C—Analyzer; S—Refractive index matching basin and sample holder; and M—Stepper motor.

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101 for ~ to subsequently account for recording the volume scattering. The setup was covered by a black enclosure to cutoff electromagnetic noise and the beam stops were used at tactical points to minimize intensity of stray reflections. Light scattering experiments were carried out in the single-scattering regime. In single-scattering experiments it is essential to avoid multiple scattering. This requires the sample concentration to be very low. For low concentration samples the signal-to-noise ratio is found to be small. We have applied the method followed by Volten et al. [35] to determine the highest sample concentration for which single scattering is applicable. The detector was placed at a fixed position (y =151). A number of measurements were subsequently made with increase in sample concentration. The effect of multiple scattering is assumed to be negligible as long as the scattered flux is proportional to the sample concentration. In this way we could determine the most favorable cell density to which single scattering was applied. In our case, we found the optimum concentration of tea dust sample at three different wavelengths to be approximately 1.5  108 CFU/mL (Fig. 3). The scattering volume in the view of the detector is determined by the scattering angle, geometry of the scattering volume of particles and distance to the detector. The correction factor [35,36] as a function of scattering angle which equals to sin y was derived, which when multiplied to the measured flux gave the phase function. We have followed the method used by Volten et al. [35] for our measurements. 2.2. Validity of the measurements The accuracy and reliability of our experimental setup was verified by measurements of scattering properties of polystyrene spheres (Fig. 4a) having an average size of 2.5 mm. The polystyrene sample was dispersed in distilled water contained in a cylindrical pyrex glass cuvette. In order to minimize the effect of strong reflections that may have arisen due to large difference of refractive index between the air and the cuvette containing the polystyrene sample, the method described by Volten et al. where

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Cell density (1X108 CFU/mL)) Fig. 3. Graph for the intensity of scattered light in arbitrary units for unpolarized light versus concentration of Mycobacterium smegmatis cells in 2.0 mg/mL tea dust for a fixed position of detector at 151. The unfilled white triangle, solid gray triangle and solid black triangle corresponds to the results for 543.5, 594.5 and 632.8 nm incident laser wavelengths, respectively.

the cuvette surrounded by a high refractive index medium such as glycerin was used [37].We took our measurements in differential mode in order to minimize the background noise. The scattered light intensity was primarily measured with the cuvette containing distilled water without polystyrene particles, and consequently the measurements were taken with the cuvette containing the polystyrene particles with distilled water. The data from the set of experiments with distilled water were then subtracted from the data of the set of experiments taken with the polystyrene particles suspended in distilled water. In this manner the measurements were corrected from background measurements. Similar precautions were taken when investigating the scattering behavior of Mycobacterium smegmatis contaminated and uncontaminated tea dust particles suspended in phosphate buffer solution (PBS). The use of metallic enclosures and beam stops ensured the elimination of any other remaining stray optical and electromagnetic noise. In addition, when we use a pyrex glass cuvette, a small fraction of the incident light gets reflected by the inner wall of the cuvette and follows its trace back and is scattered again. This secondary signal is actually noise and must be corrected. We used the method [37] in which the correction function for this error was given by uncor uncor F11 ðyÞ ¼ F11 ðyÞ-F11 ð1800 yÞR uncor F11

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(y) is the actual measurement without the where uncor corrections for reflections, F11 (1801  y) is the expected measurement due to the scattering from the reflected light, F11 (y) is the final measurement with the corrections for reflections and R ( = 0.017) is the reflection coefficient for pyrex glass. We have validated our experimental results by comparing it with the theoretical predictions based on Mie theory [1,2] for spherical particles. The theory was incorporated into ANSI standard C program [38] to generate theoretical intensity spectra. In order to validate our experimental results, we have estimated

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Fig. 4. (a) Scanning electron micrograph of polystyrene spheres at 10,000  resolution (scale 1 mm); (b)–(d) are phase function, S11, for polystyrene spheres at laser wavelength of 543.5, 594.5 and 632.8 nm, respectively. Graph for Mie calculation is shown by solid black lines and experimental results are shown by solid gray line with solid gray circles.

absolute error from the experimental and the theoretical values, using the formula, Absolute error= (Estimated value–Theoretical value)/Theoretical value, and found our results to have small absolute errors. Although for the scattering angles 1501, 1551 and 1601 the absolute errors are slightly high, i.e. 0.26, for the other scattering angles the absolute error lies in the range of 0.02–0.16. The measured values for S11 were found to tally well with the theoretical results within acceptable limits of deviation (Fig. 4b–d), thereby proving the setup to be efficient for performing light scattering measurements. 2.3. Sample preparation and antimycobacterial assay of C. sinensis dust The samples of C. sinensis dust particles were collected from areas around tea factories situated in Assam, India. agar well diffusion assay [39] was performed in order to investigate whether the tea dust particles inhibit the Mycobacterial growth through polyphenols that are present in the unprocessed green tea leaves [40]. In order to maintain safe laboratory procedures, the non-pathogenic species M. smegmatis, which has all the characteristic properties of the pathogenic species M. tuberculosis except for pathogenecity, was used for the investigations. The strain M. smegmatis mc2 155 was cultured in Mueller Hinton Broth 2 media at 37 1C for 18 h. The bacterial cells

were suspended in a saline solution (0.85%NaCl) and its turbidity was adjusted to 0.5 in the McFarland standard. McFarland is a turbidity standard of BaSO4, which is used to standardize the number of colony forming units (CFU) of bacteria in an inoculum. The standard of 0.5 McFarland corresponds to approximately 108 CFU/ml. The suspension was inoculated in Mueller Hinton Agar media and four wells (6 mm) were punched. 50 ml of the sterile organic tea dust of two concentrations, i.e. 20 and 50 mg/ml, dissolved in 1% (v/v) dimethyl sulphoxide (DMSO) were added to well 1 and well 2, respectively. 1% DMSO was added to well 3 and Streptomycin was used as an antibiotic control in well 4. The plates were incubated at 37 1C for 18 h. This experiment was conducted in triplicate, that is, the agar well diffusion assay was performed three times to ascertain the validity of the biological process. Growth of the bacterial cells was not affected by 1% DMSO as shown by our control experiments in well 3 (Fig. 5). An attempt was made to differentiate sterile tea (C. sinensis (L) Kuntze.) dust from M. smegmatis contaminated tea dust by investigating the absorbance at 600 nm by the sterile tea dust and tea dust inoculated with M. smegmatis culture at different concentrations. The concentration of tea dust was kept constant for all the samples while the concentration of bacteria was changed. A 16 h M. smegmatis culture was harvested by centrifugation

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at 3000 rpm, washed twice with sterile phosphate buffer saline (PBS) pH 7.4 and resuspended in PBS (pH 7.4). This was done to prevent cell lysis. The suspension was further diluted and mixed properly in PBS (pH 7.4) containing 20 mg/ml of the tea dust to achieve McFarland standards of 0.1, 0.25, 0.5, 0.75 and 1.0 corresponding to approximately 0.3  108, 0.75  108, 1.5  108, 2.25  108 and 3  108 CFU/ml, respectively. The optical density of the samples was measured at 600 nm (Cecil Aquarius Spectrophotometer, Sl. No.146-276). The plot between the McFarland Standards and THE optical density is presented in Fig. 6, which indicates that the optical density of the tea

dust increases with the increase in bacterial load on the tea dust. 2.4. Scanning electron microscopy (SEM) Scanning electron micrograph image was utilized to observe the morphology of the tea dust particles and study the morphological changes in the bacteria on being adsorbed onto tea dust. The study was done using the method of Nair et al. [41]. 100 mL of bacteria adjusted to 0.5 McFarland standard was resuspended into 4 ml of 10 mM sodium phosphate buffer, pH 7.4, in two different 15 ml centrifuge tubes. In the solution, 100 mL of organic tea dust (20 mg/ml) was added and incubated at 37 1C for 6 h. After the incubation the bacteria were washed in the same buffer and fixed overnight at 4 1C with 2.5% (v/v) glutaraldehyde (Fluka). After fixation the cells were mounted on cover slips using Poly-L-Lysine (Sigma). The cells were then rinsed with 10 mM sodium phosphate buffer at pH 7.4, and dehydrated through an ethanol series for about 15 min each. The samples were dried at room temperature and coated with 10–15 nm thickness of platinum using a JEOL 1600 Auto Fine Coater. The samples were then examined under the SEM with an accelerating voltage of 10–15 kV (JEOL 6390). 3. Data analysis and results

Fig. 5. Antibacterial assay of Mycobacterium smegmatis with tea dust.

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3.1. SEM analysis The size distribution of these particles was calculated from SEM image and the results were extrapolated for the sample of dust particles. These dust particles are non-spherical and have a broad size distribution. It has been found that the size distribution of the particles is nearly Gaussian. In order to differentiate between the Mycobacterium contaminated tea dust samples from the uncontaminated one, SEM images of both the types were taken. Fig. 7(a) and (b) shows the image of M. smegmatis cells and M. smegmatis cell undergoing cell division, respectively, and Fig. 7(c) and (d) shows the Mycobacterium contaminated tea dust sample. The control cells and the cells incubated with tea dust (20 mg/mL) had smooth and natural surface morphology. The SEM photograph shown in Fig. 7(c) and (d) verifies that the air-borne tea dust particles act as carrier of M. smegmatis, thereby revealing an important point that these particles might also act as carrier of pathogenic M. tuberculosis species.

Fig. 7. SEM image of: (a) Mycobacterium smegmatis ; (b) M. smegmatis undergoing cell division; (c) tea dust contaminated by single M. smegmatis; and (d) tea dust contaminated by multiple M. smegmatis.

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Fig. 8. Phase function S11 for Mycobacterium contaminated and uncontaminated C. sinensis dust particles for: (a) ~ = 101, (b) ~ = 201, (c) ~ = 301, (d) ~ = 401 and (e) ~ = 501 respectively. Lines 1 and 2 in the graphs (a–e) represent the Mycobacterium uncontaminated and contaminated intensity profile at 632.8 nm, respectively; lines 3 and 4 in the graphs (a–e) represent the Mycobacterium uncontaminated and contaminated intensity profile at 594.5 nm, respectively; and lines 5 and 6 in the graphs (a–e) represent the Mycobacterium uncontaminated and contaminated intensity profile at 543.5 nm laser wavelength, respectively.

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3.2. Sample analysis using Agar well diffusion method Agar being a porous material allows the diffusion of the antibiotic and DMSO containing the dissolved plant extracts. The growth of the bacteria on the agar plate is inhibited by the antibiotic, which is displayed as a clear inhibition zone on the agar plate. In Fig. 5, an inhibition zone could be seen in well 4. However, compounds without any antibacterial property allow the growth of the bacteria up to the edge of the well as observed in well 1, well 2 and well 3. Based on the investigations using this method on tea dust samples, it was observed that these particles did not exhibit any antimycobacterial activity unlike the extracts of fresh tea leaves and hence it might act as a potent carrier. 3.3. Light scattering measurements The present work aims at developing a technique to detect the presence of M. tuberculosis cells on the surface of C. sinensis dust particles based on light scattering properties of these particles. A low frequency component can be seen in the plots for M. smegmatis contaminated tea dust particles in all the graphs (Fig. 8a–e). This may be due to a narrow size distribution developing due to the uniformity in size of the M. smegmatis bacteria. The random size of the uncontaminated tea dust possibly got dominated by the regularity in size of the bacteria. We can also observe that there is higher intensity of scattering from all contaminated dust particles compared to the uncontaminated ones. This may be due to an overall increase in the size of the contaminated particles compared to the uncontaminated ones. With this increase in size, the possibility of multiple scattering also cannot be ruled out. As mentioned earlier, the investigations were carried out by mixing tea dust in phosphate buffer solution. Although the mixture was thoroughly stirred for a considerable period of time to ensure that the non-uniform tea dust particles acquired a uniformly random orientation in the medium, it is observed from Fig. 8 that there is variation in the scattering plots with change in the azimuthal angle ~, indicating that the particles are not only non-spherical, but some amount of non-uniformity in orientation of the particles is also present. As the sample is in suspension in a liquid medium, the elongated samples may have tried to acquire a vertical alignment with the heavier end pointing downwards. This non-uniform orientation may have become more pronounced with contamination of the bacteria that has an elongated shape and a larger mass compared to the dust particles. Again on the other hand, the theoretical light scattering pattern of biological particles is also very difficult to calculate because of the complexity in their morphology [37]. As such, there is an urgent need to modify existing theories to characterize complex biological structures better. The measured phase functions of both Mycobacterium contaminated and uncontaminated tea dust particles were normalized to 1 at y =101 (Fig. 8a–e).There was a net total error of 0.4% for all the readings from the data acquisition and photomultiplier unit. The comparative analysis of our experimental

work on tea dust samples with existing theories will be subsequently done in our future work.

4. Conclusion We report the design and fabrication of a portable lightscattering instrument, which is relatively light and convenient to use. The light scattering behavior of both Mycobacterium contaminated and uncontaminated tea dust particles has been studied as a function of scattering angle. The result presented here, which is in agreement with theoretical calculations for polystyrene spheres, confirms the reliability of the device. Therefore, this instrument can be used routinely for studies of various types of small particles, aerosols and hydrosols. Scanning electron micrograph (SEM) image was utilized to observe the morphology of the tea dust particles and to study any morphological changes in the bacteria itself, which may have been brought about by any possible antibacterial property of the tea dust particles. From the SEM images it could be seen that although there was considerable morphological changes suffered by the dust particles due to contamination by the bacteria, no morphological changes in the bacteria itself were observed. This proves the lack of antibacterial property of the tea dust and hence these dust particles are suitable hosts for the bacteria (Fig. 7c). In future, comparative analysis will be made using theoretical approach for the investigations on tea dust samples.

Acknowledgements Sanchita Roy wishes to thank the Department of Science and Technology, Ministry of Science and Technology, Government of India for the project grant (sanction no. SR/WOS-A/ PS-20/2007 dated 04/08/2008) under the Women Scientist-A Scheme. The authors also wish to thank Isha Ruhullah Kamrupi, Ranjan Dutta Kalita and Ratan Boruah of Tezpur University for acquiring the SEM images. References [1] Bohren CF, Huffman DR. absorption and scattering of light by small particles. New York: Wiley; 1983. [2] Mishchenko MI, Hovenier JW, Travis LD. light scattering by nonspherical particles: theory, measurements, and applications. San Diego, CA: Academic; 2000. [3] Mishchenko MI, Travis LD, Lacis AA. Scattering absorption and emission of light by small particles. Cambridge: University Press; 2002. [4] van de Hulst HC. Light scattering by small particles. New York: Dover Publications; 1981. [5] Gilev KV, Eremina E, Yurkin MA. Maltsev VP1. Comparison of the discrete dipole approximation and the discrete source method for simulation of light scattering by red blood cells. Opt Express 2010;18(6):5681–90. [6] Kaye PH. Spatial light-scattering analysis as a means of characterizing and classifying non spherical particles. Meas Sci Technol 1998;9:141. [7] Quinby-Hunt MS, Hunt AJ, Lofftus K, Shapiro D. Polarized-light scattering studies of marine chlorella. Limnol Oceanogr 1989;34(8): 1587–600. ~ O, Hovenier JW, de Haand JF, Vassen W, Van der [8] Volten H, Munoz Zande WJ, et al. WWW scattering matrix database for small mineral particles at 441.6 and 632.8 nm. J Quant Spectrosc Radiat Transf 2005;90:191–206.

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