Article pubs.acs.org/est
Raman Microspectroscopy-Based Identification of Individual Fungal Spores as Potential Indicators of Indoor Contamination and Moisture-Related Building Damage Sutapa Ghosal,*,† Janet M. Macher,† and Kadra Ahmed‡ †
Environmental Health Laboratory Branch, California Department of Public Health, Richmond, California 94804, United States School of Public Health, University of California, Berkeley, California 94702, United States
‡
S Supporting Information *
ABSTRACT: We present an application of Raman microspectroscopy (RMS) for the rapid characterization and identification of individual spores from several species of microfungi. The RMS-based methodology requires minimal sample preparation and small sample volumes for analyses. Hence, it is suitable for preserving sample integrity while providing micrometer-scale spatial resolution required for the characterization of individual cells. We present the acquisition of unique Raman spectral signatures from intact fungal spores dispersed on commercially available aluminum foil substrate. The RMS-based method has been used to compile a reference library of Raman spectra from several species of microfungi typically associated with damp indoor environments. The acquired reference spectral library has subsequently been used to identify individual microfungal spores through direct comparison of the spore Raman spectra with the reference spectral signatures in the library. Moreover, the distinct peak structures of Raman spectra provide detailed insight into the overall chemical composition of spores. We anticipate potential application of this methodology in the fields of public health, forensic sciences, and environmental microbiology.
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and asthma exacerbation.12 This was further confirmed by a subsequent review.15 Given the diverse ecological and social significance of fungi, a rapid and accurate method for the identification of fungi is of immediate relevance to many aspects of human health and comfort as well as plant, animal, and microbial ecology. Traditional methods of fungal identification rely on the cultivation of sampled organisms along with careful examination of their macro- and microscopic morphological characteristics. These methods are typically labor and time intensive, requiring extensive sample preparation as well as well-trained experienced personnel for the analysis. The information acquired from the morphology of single spores is often limited, e.g., the genera Aspergillus and Penicillium cannot be readily distinguished based on morphological features alone, and the use of definitive stains or labels to facilitate the identification process involves sample processing steps that further delay the identification and may also render the sample material unavailable for other types of analyses. Furthermore, not all fungal species are culturable and the sampled organism may not be in a viable state.13 These limitations have prompted interest
INTRODUCTION Fungi constitute a ubiquitous indoor and outdoor presence and play significant roles in the natural environment as well as our daily lives. Release of fungal spores into the air results in their widespread distribution throughout the environment and is the primary mechanism for fungal dispersal. In fact, recent studies suggest that fungal spores along with other particles of biological origin represent significant proportions of the aerosol particle mass worldwide.1−6 For instance, Elbert et al. used air sample data and budget calculations to estimate the global, average, emission rate of basidiospores (∼17 Tg/yr) and total fungal spores (∼50 Tg/yr).2 Furthermore, according to Bauer et al. fungal spores can be regarded as primary components of PM10, total organic carbon (OC), and, most importantly, coarse OC even in urban areas.3 Fungi are indispensable in nature for biodegradation and are beneficial to humans in terms of food production, medicine, and so forth.7−9 However, a number of fungi are major pathogens/allergens for humans, animals, and plants and have significant adverse health effects as well as economic impacts.7,10−13 For instance, fungi are potentially important contributors to indoor air pollution and have been consistently implicated in building-related illnesses and symptoms.10−14 In 2009 the World Health Organization concluded that sufficient epidemiological evidence was available to show that occupants of damp or moldy buildings are at increased risk of respiratory symptoms, respiratory infections, © 2012 American Chemical Society
Received: Revised: Accepted: Published: 6088
October 25, 2011 April 24, 2012 April 25, 2012 April 25, 2012 dx.doi.org/10.1021/es203782j | Environ. Sci. Technol. 2012, 46, 6088−6095
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Figure 1. Substrate selection. Comparison of (A) background signals of different substrate materials and (B) single basidiomycete spore Raman spectra acquired on the different substrates using identical acquisition conditions. (a.u. = arbitrary units).
identification without the need for culturing or additional sample preparation steps. The various steps involved in the method development process were (1) identification of an optimal substrate for sampling and spectral acquisition, (2) development of a library of Raman spectral signatures of relevant organisms, and (3) identification of spores through the comparison of their acquired spectra with spectral signatures in the library. As a quality control step during the method development process, we examined spores from a macrofungus (basidiomycete) acquired locally in Berkeley, California (USA) for comparison with the previously published Raman spectra of commonly available macrofungi spores.28
in the application of alternative analytical methods for fungal identification with access to distinct compositional information which can be used as chemotaxonomical markers.16−18 In this paper we present the application of Raman microspectroscopy (RMS) for the rapid identification of individual, airborne fungal spores based on their unique spectroscopic fingerprints. This method enables the direct analysis of intact spores sampled in indoor and outdoor environments without any additional sample preparation steps. Furthermore, the method is based on an inexpensive and easy to use sampling format that is compatible with multiple configurations as well as complementary analytical approaches such as electron microscopybased morphological characterization. The RMS-based methodology offers a novel approach toward rapid, noninvasive characterization and identification of environmentally relevant biological particles with access to detailed chemical information at the micrometer scale. In recent years the application of vibrational spectroscopy techniques, specifically Raman spectroscopy, in biological sciences has attracted considerable interest and effort.19 This has led to significant advances in the field of spectroscopy-based biodetection, which can be applied to investigations of microbial exposures in indoor and outdoor environments. A Raman spectroscopy approach to microbial identification offers several advantages in terms of being a relatively rapid, nondestructive technique requiring minimal sample preparation and with access to molecular-level compositional information. In addition, Raman spectroscopy is characterized by minimal interference from water, thus making it suitable for analyzing biological samples. Raman microspectroscopy combines confocal microscopy with Raman spectroscopy, and is therefore suitable for analyzing small sample volumes (even as small as 1 μm3), such as individual cells (spores).19−24 There are several reports on Raman spectroscopic analyses of macro-fungi and microcolonies of Candida species which address a variety of objectives including taxonomic, medical, and pharmaceutical applications.17,25−29 However, to the best of our knowledge RMS-based identification of individual airborne microfungal spores for environmental analyses has not been reported previously. To demonstrate the feasibility of RMS-based rapid identification of individual microfungal spores in environmental samples we examined several species of microfungi which have been identified as potential indicators of indoor contamination. The goal was to develop a robust RMS methodology for spore
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EXPERIMENTAL SECTION
RMS-based analyses of fungal spores were performed using two commercially available micro-Raman setups equipped with near-infrared 785-nm diode lasers: (1) Senterra Dispersive Raman microscope (Bruker Optics Inc., Billerica, MA), and (2) Renishaw inVia Raman microscope (Renishaw Plc., Old Town, Wotton-under-Edge, Gloucestershire, U.K.). A number of different substrate materials were surveyed to identify the optimal substrate for sampling and spectral analyses of spores. These included commercially available household aluminum foil, KBr disks (Real Crystal IR Cards, Garfield, NJ), Tedlar film (Dupont, Wilmington, DE), mica, and quartz slides (SPI Supplies, West Chester, PA). The fungi samples examined in this study included one locally acquired macrofungus (basidiomycete) and several species of microfungi, i.e., Aspergillus versicolor, Cladosporium herbarum, Epicoccum nigrum, Eurotium herbariorum, Penicillium brevicompactum, Penicillium coprophilum, and Rhizopus stolonifer. Prior to analyses the spores were dispersed onto the substrate and analyzed directly without any additional sample preparation steps. A different sampling format was also investigated to demonstrate the compatibility of the RMS-based identification method with other sampling formats. In this case, a cartridge slit sampler (Air-O-Cell sampling cassette; Zefon International, Ocala, FL) was used to sample air at a rate of 15 L min−1 from a 0.9-m3 chamber containing an aerosol of A. versicolor spores.30 A more detailed description of the experimental procedures is provided in the Supporting Information. 6089
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Article
RESULTS AND DISCUSSION
Selection of Substrate for Sampling and Spectral Analyses. Several different substrate materials were screened in order to identify the optimal substrate for this study. The various criteria considered in the substrate selection process are as follows. The first and foremost consideration was that the substrate should display minimal spectral features in the region relevant for fungal spore signature, which may otherwise interfere with spore-specific peaks and thus compromise the identification process. Second, it was essential that the substrate be sufficiently robust to withstand the variety of environmental conditions encountered during field sampling. Finally, cost and availability of the substrate material were also taken into consideration. Figure 1A shows Raman spectra of the various blank substrates. The spectral region (200−1800 cm−1), shown in Figure 1A, is the most relevant in terms of spore signatures. As is evident from the figure, quartz, aluminum foil, and KBr disks have minimal spectral features in this region, in contrast to mica and Tedlar film, which display several distinct peaks in the region. Figure 1B shows Raman spectra of single spores from a local basidiomycete acquired on the various substrates using identical acquisition conditions. The best resolved spore spectrum was obtained on the aluminum foil substrate. As a substrate aluminum foil offers several advantages including its ready availability and inexpensiveness, as well as being a robust material suitable for environmental sampling. In addition, aluminum foil is compatible with multiple sampling configurations and alternate analytical techniques such as scanning electron microscopy (SEM) based morphological characterizations. Given these advantages, commercially available aluminum foil was identified as the substrate of choice for these experiments. Circular disks of the foil were mounted on SEM stubs or glass slides using carbon tape or double-sided tape, respectively, prior to sampling and analyses. Aluminum has been used previously as a substrate though in a different and less versatile format, i.e., specialized microscope slides with aluminum coating.31,32 RMS Analyses of Basidiomycete Spores. Several species of commonly occurring macrofungus (basidiomycete) spores have previously been characterized by Raman microspectroscopy and their spectral signatures are available in the literature for comparison.28,29 Therefore, during the method development process we examined the spores of a local macrofungus, primarily as a quality control step, to validate and optimize the RMS-based methodology for single spore analyses, through direct comparison of the acquired spectra with spectra available in the literature. We also performed RMS analyses of bulk spore print of the basidiomycete for comparison with single spore spectra. Figure 2A shows the Raman spectra of bulk spore print and that of a single spore. Both spectra were acquired using an integration time of 60 s and 100 mW of laser power, and are essentially identical in terms of the primary spectral features, i.e., peak position and shape. The single spore spectrum has better signal-to-noise ratio compared to bulk spore print spectrum, a likely consequence of the inherent difficulty in achieving optimal focus on the uneven bulk powder surface. These results demonstrate our ability to acquire comparable good quality spectra from a single spore as bulk spore print using the RMS methodology and without any evidence of laserinduced sample degradation at the single spore level. Figure 2B shows a single basidiomycete spore imaged before and after spectral analysis and there are no obvious signs of
Figure 2. Basidiomycete spore spectra. (A) Comparison of Raman spectra of bulk spores versus a single spore. (B) Optical images of single spores acquired before and after Raman spectral analysis. Scale bar 10 μm.
laser-induced degradation. One of the effects of laser damage in organic materials is graphitization, which results in the appearance of amorphous carbon-related Raman bands (∼1324 and ∼1589 cm−1) in the spectrum of the damaged material.33 Because carbon has a large Raman cross-section, the associated bands tend to overwhelm all other spectral contributions rendering the spectrum unusable for spore identification. Hence, care has to be taken to minimize laserinduced damage of the sample during analysis. This is typically achieved by selecting an appropriate laser power to ensure adequate signal-to-noise ratio while minimizing laser damage during analysis. In our study the optimal laser power for spectral acquisition was found to vary depending on the spore type and had to be determined empirically. We also investigated different sample preparation methods which included dry versus wet sampling (see Supporting Information). Again in both cases the spectra were identical in terms of their primary features. In addition to serving as a unique spectral fingerprint to aid spore identification, Raman spectra also provide valuable insights into the chemical composition of spores. For instance, De Gussem et al. compared the spore spectrum of a macrofungus (Lactarius species) with the spectra of reference materials known to be present in macro-fungi.28 They were thus able to assign bands in the spore spectrum to specific biomolecules and found that the dominant spectral contributions were from lipids. They concluded that their observations were consistent with the spore’s primary biological function as an energy reserve for enabling dispersal and survival through reproduction. The same group also used Raman spectroscopy to do chemotaxonomical analyses of spores belonging to several different genera of macro-fungi.29 The local basidiomycete spore spectrum examined in this study bears close resemblance to the Raman spectra acquired by De Gussem et al. for Laccaria species in the Hydnangiaceae family or Lacatarius species in the Russulaceae family.29 RMS Analyses of Microfungi Spores Relevant to Indoor Contamination. RMS-based characterization of the basidiomycete spore described above confirmed the feasibility of making these measurements at the single spore level, especially since our results are in excellent agreement with the previously reported Raman studies of macrofungi.28,29 Therefore, we applied the same RMS-based methodology toward analyzing individual microfungi spores. Microfungi species 6090
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the band assignments. Unlike the basidiomycete spore spectrum discussed previously, the spectra of microfungi spores were not readily assignable to any specific biomolecule. However, in most cases primary bands were observed in the 1000−1700 cm−1 spectral region, which is typically associated with fats and fatty acids.38 For instance, the broad band near ∼1450 cm−1 could be attributed to methyl and methylene deformation modes of fatty acids, the peak at ∼1600 cm−1 is typically assigned to conjugated υ(CC) vibrations, a characteristic feature of phenylalanine and ergosterol, both of which are known components of fungal spores.19,28,39 Hence, the microfungi spore spectra represented the overall biochemical composition of the spore and not one specific cellular component. It is evident from Figure 4 that microfungi spores belonging to different genera possess distinct Raman spectral signatures and are easily distinguishable based on their spectra. In addition to visual comparisons, library spectral signatures belonging to the different species were examined using an automated correlation-based search algorithm in order to compare and determine the similarity between the various library spectra. Within this algorithm the quality of the spectral match is determined based on the value of the correlation coefficient (r) wherein the highest value of r is determined to be a match and r value of 1 corresponds to a perfect match. Members of the microfungal spectral library were readily distinguishable from one another based on this search algorithm (see Supporting Information, Table S2). However, Raman spectra of spores belonging to the two Penicillium species, i.e., P. coprophilum and P. brevicompactum, were very similar in terms of their primary spectral features. Previous studies involving macrofungi spores have reported similar observations whereby spectral signatures of spores belonging to different species within the same genus have shown great similarities.17,29 The observed spectral similarities can potentially be attributed to the close taxonomical relationships among species within the same genus. Fungal taxonomy has traditionally been based on morphological characteristics such as the size and shape of spores or fruiting bodies. However, these morphological variations may or may not relate to a distinct variation in the spore’s chemical composition as probed by the RMS-based method. In fact, fungal taxonomy is in a state of continual flux especially since the advent of molecular tools, such as DNA sequencing, continue to offer new insights thereby challenging the traditional morphology-based groupings in fungal taxonomy.40,41 Furthermore, in biology chemical composition of a structure is often intimately related to its function. The primary function of fungal spores is energy storage for the germination of new individuals. Therefore, the observed similarity between spectral signatures of spores within the same genus and their overall resemblance to spectral features of lipids (as energy reserve) is consistent with the spore’s biological function. For instance, in a study involving wild growing mushrooms and toadstools Mohaček-Grošev et al. reported great similarities in the spectra of spores belonging to the same genus, but significant differences between the spectra belonging to the various parts, i.e., cap, stalk, and spores of the mushroom. Despite the above-mentioned similarities, closer inspection of the spore spectra for the two Penicillium species reveal a few subtle differences, particularly in the 1490−1540 cm−1 region, which corresponds to intense Raman bands for carotenes.42,43 This slight spectral variation is likely representative of the
selected for this study are identified as potential indicators of indoor contamination and moisture-related building damage.34−37 Microfungi spores were observed to be more susceptible to laser-induced degradation and thus necessitated the use of the lowest possible laser power (∼1 mW) for their analyses. In a few cases, the use of lowest available laser power was insufficient to completely eliminate the onset of laser-induced graphitization. This is suggested by the appearance of a broad background in the spectral region typically associated with amorphous carbon (soot) peaks (Figure 3). It is also possible
Figure 3. Comparison of Raman spectra of amorphous carbon (soot) and a microfungus spore showing the correspondence between localized increase in the spore spectrum background and position of the amorphous carbon bands.
that appearance of the broad band is the result of sample fluorescence or perhaps some combination of the two effects. However, in a number of the cases spore-related spectral features were clearly distinguishable above this broad background and thus offered a unique spectral signature necessary for the spore identification process. Figure 4 shows the preprocessed, average Raman spectra of single spores of the microfungi species examined in this study. The observed bands positions are given in Table 1 along with
Figure 4. Acquired reference Raman spectra of single spores belonging to different microfungi species. 6091
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Table 1. Positions in Wavenumbers (cm−1) of the Raman Bands Observed in the Spore Spectra P. brevicompactum
A. versicolor
E. herbarhmm
C. herharum
E. nigrum
R. stolonifer
257 294 364 406
400 426 435 44J 510
436
506
503
564
571
525 609
Band assignment19,28,38 chitin trehalose saccharides saccharides saccharides saccharides, ergosterol saccharides, ergosterol saccharides proteins saccharides saccharides
625 739
733
758 788 817 845 860 871 908
9̂4
941
926
967 1003 1052 1065 1116 1145 1163 1185 1199
1204 1216
1225 1265
1269
1286 1318
1281
1281 1312
1338 1351 1365
1349 1373 1427
1370 1424
1426 1434
1455 14S4 1493 1500 1515 1528
1527
1525 1540
1550 1596 1606
1604
1600 1619
1643
treliaiose polysaccharides l-rgasiicrol lipids lipids lipids and saccharides lipids and saccharides polysaccharide, trehalose lipids, ergosterol lipids chitin saccharides, chitin lipids lipids saccliarides lipids cytosine, guanine, adenine saccharides, chitin lrcosjiirrol unstaturated fatty acids chitin saccharides, ergosterol ril’osicri’l saccharides. ergosterol lipids lipids and saccharides deoxyribose lipids lipids adenine amino acids lrgoik’iol glycme carotenoid amide amide adenine i-iiw.icrol chitin lipids
RMS-Based Identification of Individual Fungal Spores. The primary objective of this study was to determine the feasibility of using Raman microspectroscopy for the identification of individual microfungal spores. In particular, we focused on microfungi that have potentially greater presence in damp indoor environments. The sampling method used in this study is suitable for passive air sampling, which is easy to
differences in spore coloration between the two species, P. coprophilum spores are red−brown while P. brevicompactum spores are typically lighter in color. Hence, in this case a morphological difference between the two spore types is related to a variation in the spores’ chemical compositions which is then detected by the RMS-based analyses. 6092
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spectra both in terms of their band shapes and relative peak intensities. Specifically, E. herbariorum has a prominent peak at ∼1519 cm−1 which is absent in the C. herbarum spectrum. The identifications were further confirmed by the automated correlation based search algorithm mentioned previously (see Supporting Information, Figure S1). Identification of the C. herbarum spore was also validated by the spore’s distinct morphology. The samples discussed above were all prepared in the laboratory. However, we also looked at an ambient air sample collected in the field. The concentration of fungal spores in the ambient air sample was quite low; nonetheless we were able to identify individual spores of Cladosporium species based on RMS analyses. The RMS-based identification method described above is compatible with a variety of sampling formats, provided the substrate is suitable for RMS analyses and there is minimal spectral interference from the substrate itself. To demonstrate this possibility we examined a cartridge slit sampler (Air-O-Cell sampling cassette; Zefon International, Ocala, FL) previously used to collect A. versicolor spores in a chamber study.30 Figure 7 shows single spore Raman spectrum of A. versicolor on the
deploy and enables the screening of airborne organisms. Our goal is to couple passive air sampling with Raman microspectroscopy-based analysis, for the identification of individual fungal spores present in an environment of interest. This method of identification relies on unique spectral signatures associated with the spores’ chemical composition. Therefore, minimal visual inspection of the sample is necessary; instead spores having similar morphology can be differentiated and identified on the basis of their unique Raman spectra. This is evident in Figure 5, which shows optical images as well as
Figure 5. Spectral signature versus morphology. (A) Comparison of Raman spectra of two different types of spores showing their distinct spectral signatures. (B) Optical images of the two spore types showing their morphological similarity. Scale bar 10 μm.
Raman spectra of A. versicolor and P. brevicompactum spores. The two spore types are morphologically quite similar as is evident from their optical images, but have very distinctive Raman spectra. The ability to distinguish fungal spores based on their Raman spectra is further demonstrated in Figure 6, which represents a sample of mixed spore composition wherein one is able to differentiate and identify two different spore types within the sample. The two distinct spore types were identified as E. herbariorum and C. herbarum based on their Raman spectra. E. herbariorum and C. herbarum spores have distinctly different
Figure 7. Raman spectra of A. versicolor spore acquired using two different sampling formats: (a) spores deposited directly onto aluminum foil substrate, (b) spores collected onto a cartridge slit sampler through impaction.
cartridge sampler along with the reference spectrum of A. versicolor spore dispersed on aluminum foil substrate as described previously. Both spectra display good correspondence in terms of their primary spectral features. Finally, a rough estimation of the limit of detection by the RMS-based method can be expressed in terms of the smallest spore size for which we were able to acquire “good” spectra. A “good” spectrum is defined qualitatively as one where the relevant peaks are clearly distinguishable above the noise level. The spores examined in this study ranged in size from 2 to 15 μm and hence the smallest analyzed spore size was 2 μm. A spherical spore of 2 μm diameter corresponds to a volume of ∼4 μm3 and equivalent mass of 4 pg (10−12 g), assuming a density of 1000 kg/m3. Hence, the detection limit of the method was at the individual spore level or 4 pg in terms of mass. It is worth noting that we have also acquired “good” spectra from individual Escherichia coli cells, with a corresponding cell volume of approximately 1 μm3, by this methodology. We have explored the application of Raman microspectroscopy as a potential tool for the identification of individual
Figure 6. Spore identification. Comparison of Raman spectra of two unknown spores with the reference Raman spectra of E. herbariorum and C. herbarum spores. Based on their spectral signatures, spores A and B were identified as E. herbariorum and C. herbarum, respectively. Inset: Optical image of the sample showing the two spores. Scale bar 10 μm. 6093
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Notes
microfungi spores in indoor and outdoor environmental samples. As a part of the method development process we examined a variety of sampling formats and analytical conditions for the acquisition of optimal Raman spectra of individual spores. Commercially available household aluminum foil was found to be a suitable sampling substrate in terms of minimal spectral interference and robustness as well as ease of use. Several different microfungi relevant to indoor contamination were examined in this study. Distinct Raman spectra of their individual spores were acquired successfully, thereby demonstrating the feasibility of using RMS as an identification tool for microfungi spores. Spectral signatures of spores belonging to different species in the same genus were remarkably similar in terms of their primary features, however, a few subtle spectral differences could be detected that were consistent with known spore characteristics. In general microfungi spore spectra appear to reflect the overall biochemical composition of the spore with peaks representing most of the known major components such as lipids, proteins, and carbohydrates. At the same time the spectra exhibit sufficient variation in terms of peak positions and relative intensities to enable RMS-based differentiation of spores that are morphologically similar, but taxonomically different. Further work needs to be done to achieve consistent differentiation of spores at the species level using the RMSbased methodology. In terms of the development of a library of Raman spectral signatures, an important consideration is the determination of the optimal number of library members necessary to create an effective library with a reasonable investment of time and resources. The number of fungal species encountered in air or dust samples is numerous. However, culture and PCR-based investigations have shown that the number of species most frequently encountered in indoor samples is approximately 100. Although there are some regional variations (temperate zones versus the tropics) in fungal species, no significant differences have been observed worldwide or by building type.44,45 Hence, the number of specimens to be analyzed for the development of a spectral library relevant for indoor sampling is quite reasonable. Some of the other potential challenges for library development include acquisition of the necessary specimens and the time involvement for the acquisition of good quality spectra to facilitate the identification process. Overall, a successful implementation of the RMS-based methodology for efficient identification of fungal spores in indoor and outdoor air samples will be helpful in addressing a number of issues related to public health, ecology, and forensic sciences and is therefore worth the effort.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Drs. Stephen Wall, Kazukiyo Kumagai, and Mark Mendell for helpful discussions. K.A., MPH fellow, thanks LabAspire and Dr. Gertrude C. Buehring for summer internship support.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*Phone: (510) 620-2815; fax: (510) 620-2825; e-mail: Sutapa.
[email protected]; mail: Environmental Health Laboratory Branch, California Department of Public Health, 850 Marina Bay Parkway, Mailstop G365/EHLB, Richmond, California 94804, USA. 6094
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dx.doi.org/10.1021/es203782j | Environ. Sci. Technol. 2012, 46, 6088−6095
