We present a method to show that average mass extinction coefficient of microbes evaluated via Lorenz-Mie theory can be used to discriminate between viable and dead microbes. Reflectance of viable and dead self-cultured fungal spores and mycelia were measured by the Fourier transform infrared spectroscopy. Complex refractive indices and mass extinction coefficient of viable and dead fungal spores and mycelia were obtained in terms of Kramers-Kronig (KK) relation and Lorenz-Mie theory respectively. Smoke box experimental system was built to validate the effectiveness of the method. The results show that viable and dead fungal spores and mycelia via average mass extinction coefficients can be distinguished. The method can be used to discriminate the bioactivity of microbes and has potential applications in identification, detection, and optical characteristics of viable and dead microbial materials.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Bioaerosols are a subcategory of particles released from terrestrial and marine ecosystems into the atmosphere. They are composed of both living and non-living components, such as organisms, dispersal units, and various fragments or excretions . The dispersal of plant, animal, and human pathogens and allergens has major implications for agriculture and public health [2–4]. Allergenic and toxic bioaerosols need not to be viable, as also dead cells or cell fragments may provoke the same adverse health effects. Examples for biological toxins found in air particulate matter are cell wall components of bacteria or secondary metabolites produced by bacteria or fungi .
The presence of bacterial pathogens in airborne particulate matter has been of considerable concern from the public health standpoint . A lot of analysis about discrimination of viable and dead microbial materials have been carried out in the previous studies. Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage was carried out by using Ethidium monoazide (EMA), sodium deoxycholate and real-time polymerase chain reaction (PCR) . A study on discriminating viable vibrio vulnificus cells from dead cells in real-time PCR was performed . Propidium monoazide (PMA)-assisted quantitative PCR was presented as a new and rapid quantitative method to distinguish viable from non-viable potential bacterial pathogens [5, 8, 9]. PMA was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR (qPCR) . EMA/PMA in combination with PCR or qPCR has been applied to identify viable food-borne pathogens in a simple matrix [9, 11–14]. Calcofluor White M2R and Sytox Green stains used together may facilitate studies to identify viable microsporidia . To sum up, bioactive substances of microbes were almost used for discrimination of viable and dead microbial materials in the methods above-mentioned.
In our study, a method for the discrimination of viable and dead microbes based on average mass extinction coefficients of microbes was presented. The procedure of this study was, therefore, the following: (i) to prepare the microbial samples and measure the reflectance of fungal spores and mycelia by using Fourier transform infrared spectroscopy, (ii) to compute the complex refractive index and evaluate mass extinction coefficient of viable and dead fungal spores and mycelia in terms of KK relationship and Lorenz-Mie theory respectively, then discriminate them, (iii) to validate the effectiveness of method above mentioned for the discrimination of viable and dead microbes via average mass extinction coefficients obtained from smoke box experimental system.
2. Preparation of material samples
Four type of microbial materials were used to discriminate the viable and dead microbes. These were viable and dead entomogenous fungal BB0310 spores and entomogenous fungal HJ0104 mycelia. Through the process of strain activation, shaking flask culture, large tank fermentation, centrifugation, pure water cleaning, vacuum freezing and drying, grinding by superfine Chinese medicine grinder , the samples of viable entomogenous fungal BB0310 spores and entomogenous fungal HJ0104 mycelia are prepared.
The method of wet-heat inactivation was used to inactivate the entomogenous fungal BB0310 spores and entomogenous fungal HJ0104 mycelia. The principle of wet-heat method was that the protein and nucleic acid of fungal spores and mycelia were deformed, which eventually led to death of fungal spores and mycelia. The procedure of wet-heat method are as followings in details: i) fungal spores and mycelia were generally sterilized by using a steam pressure of 103.4kPa at a temperature of 121 for 20 30 minutes. ii) After sterilization, fungal spores and mycelia were incubated for a few hours at the appropriate temperature and then sterilize once again to kill fungal spores and mycelia that have just germinated. Wet-heat inactivated fungal spores and mycelia containing some moisture, which may lead to contamination of other bacteria, need to be dried . The dead entomogenous fungal BB0310 spores and entomogenous fungal HJ0104 mycelia were obtained through wet-heat inactivation. All the four type of microbial materials achieved, which are viable fungal BB0310 spores, viable fungal HJ0104 mycelia, dead fungal BB0310 spores, and dead fungal HJ0104 mycelia, were put into a dry container with silica gel and stored at room temperature.
3.1 Measurement of reflection spectra
In order to achieve the reflection spectra of microbes under study, a certain amount of microbial materials samples are weighed and smashed by grinding bowl. Then, powder tablet press, model 769YP-15A, was used as a tool to produce the samples tablets of microbes. The reflection spectra were obtained from 2.5 to 15 on a Nicolet FT-IR spectrometer coupled with a Continuum microscope. The gold-plating reflective mirror is used as the background, incident angle was 18 and three sampling points from each sample tablet are selected to determine the reflection spectra. Taking the average value of the three points as the specular reflection spectrum. The spot size in Microscopic FTIR spectrometer is 100 100 , which is smaller than that in Fourier spectrometer. Within the spot, the optically clean, inclusion-free, smooth, and crack-free areas were used for the measurement so that the reflection spectra measured were nearly closer to the ideal specular reflection spectra . According the measurement method of reflection spectra above mentioned, the specular reflectance spectra of four microbes are shown in Fig. 1.
3.2 Measurement of transmittance
In order to further validate the reliability of results above-mentioned, Large-scale smoke box experimental system for extinction performance measurement of microbes was built, which was made up of a smoke box, spraying system, test system, concentration sampling system, and exhaust system as shown in Fig. 2.
A smoke box, spraying system, and exhaust system were self-made. The volume of the smoke box was 4 2.4 3m, painted by black matter lacquer inside . Two equal height optical windows were opened along the length of the smoke box. The spraying system, which consisted of a high-pressure nitrogen cylinder, ducts and a nozzle placed in the smoke box, was used to spray microbial particles into the smoke box. The test system were composed of a receiver and a source. The receiver is a medium wave cooled FLIR SC7300 infrared camera and model LS1250-100, a high-precision and high-temperature blackbody radiation source, was used to provide an infrared radiation source with constant temperature. Model LB-120F, as an intelligent particle samples, was used to constitute the concentration sampling system. Connected with the large pump outside, the exhaust system was installed on the top of the smoke box for rapid exhausting the remaining microbial particles after each experiment. In the experiments, the fungal spores or mycelia samples were sprayed through the nozzle using nitrogen gas at 10 atm. The transmittance of viable and dead fungal spores or mycelia in the smoke box experimental system can be derived in terms of measurements of the smoke materials before and after the radiation intensity of the infrared thermal imager, which revealed the extinction effects of smoke materials on the target radiation source. The transmittances of microbial materials in the smoke box experimental system were plotted in Fig. 3.
4. Results and discussion
4.1 Computation of complex refractive index
The real part n and imaginary part k of the complex refractive index m can be computed from the reflectivity and reflective phase shift , and given by [20,21]
Complex refractive indices of fungal spores and mycelia can be achieved from spectral reflectance (0 ). However, in the course of measurement of reflection spectra, some factors, such as the effective range of electromagnetic waves, spectral response of test instrument and angles of measurement, should be taken into consideration. Nearly normal incident spectral reflectance of the microbes can only be measured in ( = 2.5, = 15) in actual spectral measurement. Except for the measurable wave band, the reflectance in other bands can be achieved via the empirical equation or constant extrapolation. The upper limit of the integral in computing equation of reflective phase shift , 100 , was used because the reflectivity above 100 had little effect on the complex refractive index in the range of . In general, at lower frequencies above was obtained by constant extrapolation and was replaced by the spectral reflectance at . Exponential extrapolation method is adopted to obtain the reflectance spectra under [17, 18]. The exponential extrapolation formula is given by . In order to extrapolate the reflection spectra under , an iterative method was used to compute the coefficients A and P so that the complex refractive indices of microbes meet the requirement of n 1 and k 0 .
For the normal incident electromagnetic waves, the reflective phase shift can be expressed as a function of spectral reflectance and wavelength in terms of KK relation [20,21].20, 21].
The real part and imaginary part of complex refractive index are drawn as Fig. 4, which shows that real part and imaginary part of viable fungal spores or mycelia are always bigger than that of dead fungal spores or mycelia. Real part of complex refractive index is used to denote the dispersion of the medium to electromagnetic waves while imaginary part of complex refractive index represents the absorption of the medium to electromagnetic waves. As shown in Fig. 4, real part of microbes at low wavelength is lower than that at high wavelength, so the dispersion of microbes to electromagnetic waves at low wavelength is less than that at high wavelength. On the contrary, the absorption of the microbes to electromagnetic waves at low wavelength is more than that at high wavelength. Extrapolation coefficients, average of reflectance and complex refractive index in 3 5 are listed in Table 1. The average of real part and imaginary part of viable fungal spores or mycelia are greater than those of the corresponding dead fungal spores or mycelia respectively, as shown in Table 1. So we can initially conclude that the extinction performance of viable spores or mycelia is better than that of the corresponding dead spores or mycelia.
4.2 Evaluation of mass extinction coefficient
The mass extinction coefficient of particles is an intrinsic physical parameter to represent extinction property of particles. So can be defined as a function of the extinction cross section , mass density , effective radius of microbes. Extinction cross section can be achieved from the complex refractive index of microbes. The mass density of four type of viable and dead fungal spores and mycelia have the same values, which was 1.12 . The effective radius of fungal spores and mycelia were 1.1 , 1.5 respectively. The mass extinction coefficient of four type of microbes can be evaluated based on Lorenz-Mie theory [22–26] and drawn in Fig. 5.
The average mass extinction coefficients of four microbes were 1.531 , 0.858 , 1.269 , and 0.561 , respectively, and shown in Table 2. It can be found from Table 2 that there are obvious differences of between viable and dead spores or mycelia of the same microbe, which are 43.96% and 55.79% for BB0310 and HJ0104 respectively. Drying procedure in the process of wet-heat inactivation will make water content of dead microbes less than that of viable microbes, which will greatly reduce the absorption of infrared band. Thus mass extinction coefficients of dead microbes will be less than those of viable microbes. So a conclusion can be drawn that viable fungal spores or mycelia have better extinction performance than dead fungal spores or mycelia in 35 , and it is consistent with the initial result derived from the complex refractive index of microbial materials.
4.3 Validation of results
The average transmittances of four type of microbes were 12.057%, 34.575%, 13.703%, and 47.272% respectively. Mass density c of viable and dead microbial spores and mycelia were 0.611 , 0.605 , 0.608 , and 0.602 respectively, which were detected by using the concentration sampling system. The optical path length is 4m. Average mass extinction coefficient were obtained based on Beer-Lambert law and shown in Fig. 6.
The average mass extinction coefficients of four type of fungal spores and mycelia were 0.866 , 0.440 , 0.823 , and 0.313 respectively. Concentration, optical path length, transmittance, average mass extinction coefficient of four type of microbes in 35 are shown in Table 3. As shown in Table 3, it is obvious that differences of between viable and dead spores or mycelia of the same microbe are 49.19% and 61.97% for fungal BB0310 spores and fungal HJ0104 mycelia respectively. This result further verified the conclusion that extinction performance of viable fungal spores or mycelia are stronger than those of dead fungal spores or mycelia for the same microbes in 35 , and it is consistent with the result derived from the mass extinction coefficients of microbes evaluated by Lorenz-Mie theory. So, average mass extinction coefficients of microbes in the light of Lorenz-Mie theory can be used as a parameter to discriminate the viable and dead fungal spores or mycelia. We can collect a lot of average mass extinction coefficients of viable and dead fungal spores or mycelia and put into a microbial extinction characteristics data set at ordinary times. If fungal spores or mycelia need to be discriminate their bioactivity, average mass extinction coefficient of them can be achieved based on the method above-mentioned, bioactivity of fungal spores or mycelia can be easily obtained by comparing the average mass extinction coefficient computed and those come from microbial extinction characteristics data set. If computed value is nearly close to that of viable microbes, the fungal spores or mycelia should be discriminated as viable, and vice versa.
We have presented a novel method to discriminate the viable and dead fungal spores or mycelia. The method is based on Fourier transform infrared spectroscopy, and each microbial materials have specific reflection spectra, complex refractive index of each microbe can be achieved by KK relation, then average mass extinction coefficient of microbe can be evaluated based on Lorenz-Mie theory. The major findings obtained from this study include three points. Firstly, although this method has been applied to successfully discriminate the viable and dead fungal spores or mycelia, this method can also be applied in any other microbes. Secondly, the results also show that viable microbes have great potential as the mid-infrared extinction materials because of its strong extinction properties, thus certain microbes can be screened by the method above mentioned as new optical functional materials in the field of mid-infrared extinction. Finally, it is also obvious that bioactivity of microbes play a vital role in extinction performance of microbes in 3-5 micrometers. Specific storage method for microbial extinction materials are needed to keep microbes active so as to maintain their extinction performance. Overall, it is a practical and reliable method to discriminate the viable and dead microbes by the average mass extinction coefficient evaluated by Lorenz-Mie theory and has potential applications in identification, detection, and optical characteristics of viable and dead microbial materials.
National Natural Science Foundation of China (NSFC) (60908033, 61271353); Natural Science Foundation of Anhui Province (1408085MKL47).
References and links
1. J. Frohlich-Nowoisky, C. J. Kampf, B. Weber, J. A. Huffman, C. Pohlker, M. O. Andreae, N. Lang-Yona, S. M. Burrows, S. S. Gunthe, W. Elbert, H. Su, P. Hoor, E. Thines, T. Hoffmann, V. R. Despres, and U. Poschl, “Bioaerosols in the earth system: climate, health, and ecosystem interactions,” Atmos. Res. 182, 346–376 (2016). [CrossRef]
2. J. Fröhlich-Nowoisky, D. A. Pickersgill, V. R. Després, and U. Pöschl, “High diversity of fungi in air particulate matter,” Proc. Natl. Acad. Sci. U.S.A. 106(31), 12814–12819 (2009). [CrossRef] [PubMed]
3. V. R. Despres, J. A. Huffman, S. M. Burrows, C. Hoose, A. S. Safatov, G. Buryak, J. Frohlich-Nowoisky, W. Elbert, M. O. Andreae, U. Poschl, and R. Jaenicke, “Primary biological aerosol particles in the atmosphere: a review,” Tellus B Chem. Phys. Meterol. 64(1), 1–58 (2012). [CrossRef]
4. E. L. Brodie, T. Z. DeSantis, J. P. M. Parker, I. X. Zubietta, Y. M. Piceno, and G. L. Andersen, “Urban aerosols harbor diverse and dynamic bacterial populations,” Proc. Natl. Acad. Sci. U.S.A. 104(1), 299–304 (2007). [CrossRef] [PubMed]
5. R. Kaushik and R. Balasubramanian, “Discrimination of viable from non-viable gram-negative bacterial pathogens in airborne particles using propidium monoazide-assisted qPCR,” Sci. Total Environ. 449, 237–243 (2013). [CrossRef] [PubMed]
6. J. L. Lee and R. E. Levin, “Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR,” J. Microbiol. Methods 79(2), 184–188 (2009). [CrossRef] [PubMed]
8. A. Nocker, P. Sossa-Fernandez, M. D. Burr, and A. K. Camper, “Use of propidium monoazide for live/dead distinction in microbial ecology,” Appl. Environ. Microbiol. 73(16), 5111–5117 (2007). [CrossRef] [PubMed]
9. Y. Pan and F. Breidt Jr., “Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells,” Appl. Environ. Microbiol. 73(24), 8028–8031 (2007). [CrossRef] [PubMed]
10. S. Bae and S. Wuertz, “Discrimination of viable and dead fecal bacteroidales bacteria by quantitative PCR with propidium monoazide,” Appl. Environ. Microbiol. 75(9), 2940–2944 (2009). [CrossRef] [PubMed]
11. G. Flekna, P. Stefanic, M. Wagner, F. J. M. Smulders, S. S. Mozina, and I. Hein, “Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR,” Res. Microbiol. 158(5), 405–412 (2007). [CrossRef] [PubMed]
12. A. Nocker and A. K. Camper, “Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide,” Appl. Environ. Microbiol. 72(3), 1997–2004 (2006). [CrossRef] [PubMed]
13. A. Nocker, C. Y. Cheung, and A. K. Camper, “Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells,” J. Microbiol. Methods 67(2), 310–320 (2006). [CrossRef] [PubMed]
14. K. Rudi, B. Moen, S. M. Drømtorp, and A. L. Holck, “Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples,” Appl. Environ. Microbiol. 71(2), 1018–1024 (2005). [CrossRef] [PubMed]
15. L. C. Green, P. J. LeBlanc, and E. S. Didier, “Discrimination between viable and dead Encephalitozoon cuniculi (microsporidian) spores by dual staining with sytox green and calcofluor white M2R,” J. Clin. Microbiol. 38(10), 3811–3814 (2000). [PubMed]
16. L. Li, Y. H. Hu, Y. L. Gu, W. Chen, Y. Z. Zhao, and S. J. Chen, “Measurement and analysis on complex refraction indices of pear pollen in infrared band,” Guangpuxue Yu Guangpu Fenxi 35(1), 89–92 (2015). [PubMed]
17. Y. L. Gu, C. Wang, L. Yang, Z. W. Ou, Y. H. Hu, L. Li, Y. Z. Zhao, W. Chen, and P. Wang, “Infrared extinction before and after aspergillus niger spores inactivation,” Infrared Laser Eng. 44(1), 36–41 (2015).
18. Z. W. Dou, X. X. Li, and J. J. Zhao, “Complex refractive indices of expanded graphite deduced from its reflection spectra in infrared band,” Acta Armamentarii 32(4), 498–502 (2011).
19. M. I. Mishchenko, L. D. Travis, and A. A. Lacis, Scattering, Absorption, and Emission of Light by Small Particles(University of Cambridge, 2002).
20. O. Meitav, O. Shaul, and D. Abookasis, “Determination of the complex refractive index segments of turbid sample with multispectral spatially modulated structured light and models approximation,” J. Biomed. Opt. 22(9), 1–10 (2017). [CrossRef] [PubMed]
21. M. Tasumi, Introduction to Experimental Infrared Spectroscopy: Fundamentals and Practical Methods(John Wiley & Sons,2015).
23. S. Vandewiele, F. Strubbe, C. Schreuer, K. Neyts, and F. Beunis, “Low coherence digital holography microscopy based on the Lorenz-Mie scattering model,” Opt. Express 25(21), 25853–25866 (2017). [CrossRef] [PubMed]
25. M. I. Mishchenko, Z. M. Dlugach, and N. T. Zakharova, “Direct demonstration of the concept of unrestricted effective-medium approximation,” Opt. Lett. 39(13), 3935–3938 (2014). [CrossRef] [PubMed]
26. M. Yang, K. F. Ren, M. Gou, and X. Sheng, “Computation of radiation pressure force on arbitrary shaped homogenous particles by multilevel fast multipole algorithm,” Opt. Lett. 38(11), 1784–1786 (2013). [CrossRef] [PubMed]
27. L. Li, Y. Hu, Y. Gu, X. Zhao, S. Xu, L. Yu, Z. M. Zheng, and P. Wang, “Infrared extinction performance of randomly oriented microbial-clustered agglomerate materials,” Appl. Spectrosc. 71(11), 2555–2562 (2017). [CrossRef] [PubMed]