Quantitation and Differentiation of Bioparticles ... - ACS Publications

Aug 26, 2008 - Schizosaccharomyces pombe, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium...
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J. Phys. Chem. B 2008, 112, 11785–11793

11785

Quantitation and Differentiation of Bioparticles Based on the Measurements of Light-Scattering Signals with a Common Spectrofluorometer Cheng Zhi Huang* and Shao Fen Chen† College of Chemistry and Chemical Engineering, MOE Key Laboratory for Luminescence and Real-Time Analysis, CQKL-LTRA, Southwest UniVersity, Chongqing 400715, China ReceiVed: April 3, 2008; ReVised Manuscript ReceiVed: June 28, 2008

By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, ∆λ ) 0), we obtained synchronous light scattering (SLS) signals that related to Rayleigh and Mie scatterings. It was found that the SLS signals could be applied for quantitation and differentiation of model bioparticles such as Saccharomyces cereVisiae, Schizosaccharomyces pombe, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium. In PBS buffer, these model bioparticles could form colloidal suspensions or dispersions of sizes ranging from hundreds of nanometers to tens of micrometers, giving SLS signals with the intensity being proportional to the amount of bioparticles in the range from 1.7 × 105 to 1.7 × 109 CFU/mL. A further finding is that polarized synchronous light scattering (PSLS) signals of I0°-30° against I0°-0°, which could be obtained by introducing polarizing sheets accessory of the spectrofluorometer, and the derivative synchronous light scattering (DrSLS) signals, which could be obtained directly with the extension function of the spectrofluorometer, offer differentiation information of bioparticles connected with their size, shape, refractive indexes, and inner structure. Refractive indexes of spherical bacteria were then calculated based on light scattering signals. 1. Introduction It is very important to obtain the qualitative and quantitative information of microbial systems involving in the characterization, differentiation, identification and quantification of bioparticles, particularly within the clinical and environmental fields.1 To characterize and identify various bioparticles, approaches have been made so far including various spectroscopic methods,2-6 flow cytometry, 7 hyphenated chromatography-mass spectrometry (MS), 8,9 capillary electrophoresis,10-15 or microfluidic devices.16-18 However, most of these sophisticated instrumental techniques, enhanced Raman spectroscopy for instance,6 have been limited since the high degree of molecular selectivity of these techniques does not necessarily translate into a high degree of microbial selectivity.16 Most protocols of flow cytometry, for example, necessitate bacterial growth prior to analysis to increase the sensitivity of the assay, and the instrumental equipment, of course, is expensive.7,19 As for direct optical spectroscopic methods, they generally lack selectivity because many microbes have similar molecular components and produce similar spectra. To increase selectivity, bacteria should be incubated with various reactive fluorescence dyes, chemiluminescence substrates or selective antibodies using indirect spectroscopic approaches, which, however, suffered from much long incubation time of bacteria and time-consuming pretreatment of sample.20-22 Therefore, the characterization and identification of microorganisms continues to suffer from long detection times, limited throughput and applicability. Any particles, including biological ones, act as second radiation when excited by a light beam,23 and have strong light * Address correspondence to this author. Telephone:86-23-68254659. Fax: 86-23-68866796. E-mail: [email protected]. † Present address: Chongqing Normal University, Chongqing 400047, China.

scattering emission with the intensity and distribution related to the size, shape, refractive indexes and inner structure of these particles.24-33 These measurements, however, are generally involved the applications of flow cytometry, where cells suspended in a fluid are ejected from a nozzle and made to flow, and are then sorted according to size and shape based on the angular light scattering properties or fluorescence from attached flurophores under the excitation of laser light beam. Researches of our laboratory and other groups have showed that that light scattering signals detected by synchronously scanning both the excitation and emission monochromators of a common spectrofluorometer could be effectively applied to characterize the aggregation and assembly of biological and chemical species.24-28,34-36 Pasternack et al. proposed a resonance light scattering technique (RLS) technique based on such simple measurements with a common spectrofluorometer, and found that the RLS signals, which involve in the electron resonance of the scatterer with same frequency of the excitation light beam located in the near region of molecular absorption bands, could be applied to determine the size and shape of chromophore aggregates in the same way as the conventional steady-state and dynamic light scattering measurements, and could yield information of the size and shape of biopolymers.37,38 Compared with the aggregation and assembly of biological and chemical species, microbes are much more complex, and their light scattering signals are greatly different.39 Herein, we present a method of characterizing bioparticles, allowing them to be identified and quantified based on the measurements of synchronous light scattering (SLS) signals with a common spectrofluorometer. Different from the RLS technique proposed by Pasternack et al., our presently measured SLS signals related to bioparticles are not only involve the resonance Rayleigh scattering, where electron resonance of the molecular absorption bands (probably the absorption of proteins, nucleic acids, and

10.1021/jp802895b CCC: $40.75  2008 American Chemical Society Published on Web 08/26/2008

11786 J. Phys. Chem. B, Vol. 112, No. 37, 2008 other complicate substances) generally occurs, but also the large volumes of the bioparticles which are related to Mie, and Dyndall scatterings. The results, although the assignments of the complicate light scatterings should be further investigated, could demonstrate that the extracted light scattering information about bioparticles could be comparable with existing methods such as flow cytometry, particularly the polarized synchronous light scattering (PSLS) and derivative synchronous light scattering (DrSLS) signals could offer differentiation information connected with the size, shape, refractive indexes and inner structure of these bioparticles. 2. Experimental Section Apparatus. The measurements of synchronous light scattering (SLS) and polarized synchronous light scattering (PSLS) signals were made with a Hitachi F-4500 spectrofluorometer (Tokyo, Japan). For the measurements of PSLS signals, the polarizing sheets accessory of the spectrofluorometer (Tokyo, Japan) should be employed. Derivative signals of SLS and PSLS (DrSLS/DPSLS) were directly obtained with the extension function of the spectroflourometer. Particle sizes were made on a Beckman coulter N5 submicrometer particles size analyzer (Beckman Coulter, USA), which was based on dynamic light scattering (DLS). A QL-901 vortex mixer (Haimen Qilinbei’er Instrumental Ltd., Haimen, PRC) was employed to blend the solutions. Reagents. All seeds of bioparticles, including Saccharomyces cereVisiae, Schizosaccharomyces pombe, Staphylococcus aureus, Escherichia coli DH5R, Escherichia coli BL-21, Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium were kind gifts from Professor J. P. Xie (College of Life Sciences, Southwest University, China); 0.75 and 0.11 µm polybead carboxylate microspheres were commercially purchased from PolySciences, Inc. (Warrington, PA), and latex Beads with the diameters of 100 nm, 200 nm, 300 and 500 nm, respectively, were obtained from Beckman Coulter, Inc. (Fullerton, CA). Luria-Bertani (LB) culture medium, yeast extract (YE) culture medium, and sterile 0.01 M pH 7.4 PBS (0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.002 M KH2PO4) buffer were employed. All reagents were of analytical grade without further purification. Ultrapure water (18 MΩ cm) was used throughout. Bacterial Growth Conditions and Preparation of Bacterium Suspension. S. aureus, E. coli, B. subtilis, B. thuringiensis, and B. megaterium were cultured in LB culture medium at 37 °C, while S. cereVisiae and S. pombe were cultured in YE culture medium at 28 °C. After incubation overnight, these microbes reached the exponential growth phase, and then the suspensions were prepared by dispersing in sterilized 0.01 M PBS buffer (pH 7.4) with vortex. The bacterial concentrations were checked by plating serial dilutions of the samples on LB agar and YE agar, respectively, counting the colony forming unit (CFU) after incubation overnight. The bacterial cells were directly used as the sample without heat treatment. For safety, the bacteria were heat-killed in a water bath (100 °C) for 15 min eVen if liVe bacteria were employed in the experiment process. With 5-min ultrasonication, bacterial self-aggregation could be avoided in the bacterial suspension, and then completely dispersed bacteria could be obtained. Size Determination of Cells and Polybead Carboxylate Microspheres. The sizes of cells and polybead carboxylate microspheres were determined with N5 PCS submicrometer particle analyzer at a 90° scattering angle in a stable ambient of 20.0 °C by taking the solution viscosity and the refractive index values of 1.002 centipoises and 1.333, respectively. The

Huang and Chen diameter of the microparticles given directly by computer is calculated Via the Stokes-Einstein equation. The results were confirmed by optical microscopy. SLS and PSLS Measurements. The detection suspensions were sampled in the logarithmic growth phase, and the light scattering signals were measured with a Hitachi F-4500 spectrofluorometer which was equipped with a 150 W xenon lamp, an excitation monochromator, an emission monochromator, a photomultiplier tube (PMT). For the measurements of PSLS, the accessory of two polarizing sheets coupled by the spectrofluorometer was introduced at both the incident and detection directions, respectively. All SLS and PSLS were made with the excitation and emission slit widths of 5.0 nm and the PMT voltage of 400 V. 3. Results and Discussion SLS Spectra of Bioparticles. All the left sides of Figure 1 show the SLS spectra of the eight bioparticles investigated in this contribution. These spectra are not easily discriminated with the naked eye even though there are some differences between these organisms. Parts A and B of Figure 1 show that the SLS spectral shapes of S. cereVisiae and S. pombe are identical, but the scattering ability, characterized with the SLS intensity for same concentration of the bacterial, of S. pombe is stronger than that of S. cereVisiae. Both S. cereVisiae and S. pombe are fungi, and S. cereVisiae is ellipsoidal with the size about 10 µm, while S. pombe is a bacillus with the long axis about 20 µm and the short axis about 6 µm. Thus, the different light scattering abilities should origin from their shape, sizes, and refractive indexes.37,38 Similar phenomena could be observed from B. subtilis, B. thuringiensis, B. megaterium, and S. aureus (Figure 1C-F), and it could be seen that their scattering abilities are greatly different for the same concentration of these gram-positive bacteria. The shape of S. aureus is spherical with the size about 0.8-1.0 µm, while B. subtilis, B. thuringiensis and B. megaterium are bacillus with different sizes (Table S1 in the Supporting Information). As for gram-negative bacteria, on the other hand, the SLS spectra of E. coli BL-21 are different from that of E. coliDH5R. As can be seen from Figure 1 (G and H), the former one has a comparatively flat light scattering peak located at 470 nm, while the latter one has a strong scattering peak at 520.0 nm with a shoulder peak in its red side. Furthermore, it seems that the former one can scatter light stronger than the latter one for the same concentrations of the gram-negative bacteria. To investigate why these bacteria result in similar spectra, we measured the SLS spectra of spherical latex beads with different sizes (Figure S1 in the Supporting Information). It was found that the SLS spectra of the organic particles less than 50 nm only display one characteristic peak. With size increasing, the characteristic peak gets obvious red shift and there are two SLS peaks emerged if the size is larger than 300 nm. It is obviously that the profiles of SLS spectra depend on the size of particles. We can see that these bacteria should have similar SLS spectra to that of large spherical latex beads. For this similarity of SLS spectra, we cannot completely discriminate these cells even if they have different light scattering emission efficiency, but we can introduce polarization method in the following section to distinguish. In addition, we could quantify these bacteria at the maximum SLS wavelengths (Figure 2) since it was found that the SLS intensities of these bacteria are in proportional to their concentration of bioparticles in the range of 1.7 × 105 to 1.7 × 109

Quantitation and Differentiation of Bioparticles

Figure 1. Part 1 of 2.

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Figure 1. Part 2 of 2. Synchronous light scattering spectra (left) and derivative light scattering spectra (right) of model bioparticles. (A and B) S. pombe and S. cereVisiae (×105 CFU/mL): 1, 0.2; 2, 0.67; 3, 2.0; 4, 3.3; 5, 4.7. (C) B. megaterium (×106 CFU/mL): 1, 0.5; 2, 1.7; 3, 5.0; 4, 8.3; 5, 11.7. (D) B. subtilis (×106 CFU/mL): 1, 1.5; 2, 5.0; 3, 15.0; 4, 25.0; 5, 35.0. (E) B. thuringiensi (×106CFU/mL): 1, 0.33; 2, 1.1; 3, 3.3; 4, 5.5; 5, 7.7. (F) S. aureus (×107 CFU/ m L): 1, 1.7; 2, 5.7; 3, 11.7; 4, 28.5; 5, 40.0. (G) E. coli BL-21 (×106 CFU/ m L): 1, 0.40; 2, 1.33; 3, 4.00; 4, 6.67; 5, 9.33. (H) E.coli DH5R (×106 CFU/ mL): 1, 0.7; 2, 2.3; 3, 7.0; 4, 11.6; 5, 16.3.

Quantitation and Differentiation of Bioparticles

J. Phys. Chem. B, Vol. 112, No. 37, 2008 11789 TABLE 1: Light Scattering Yields (φLSx) of the Bioparticles

Figure 2. Calibrated curves and analytical parameters of eight suspensions of bioparticles. The Linear regression equations (c, CFU/ mL) could be regressed as ∆IE. coli BL21(470 nm) ) 33.1 + 3.27 × 10-6 5c, ∆IE. coli DH5R(520 nm) ) 37.6 + 2.63 × 10 c, ∆IS. cereVisiae (350 nm) ) 23.6 + 5.22 × 10- 4c, ∆IS. pombe (350 nm) ) 1.7 + 1.22 × 10- 3c, ∆IB. megaterium (470 nm)) 9.3 + 3.43 × 10- 5c, ∆IB. subtilis (470 nm) ) 24.2 + 1.10 × 10-5c, ∆IS. aureus (470 nm) ) -39.8 +3.57 × 10- 5c, ∆IB. thuringiensis (470 nm) ) -74.2 + 1.19 × 10- 4c over the linear ranges (×105CFU/mL) of 4.0 to 4.0 × 102, 7.0 to 6.6 × 103, 0.2-20.0, 0.2-20.0, 5.0 to 5.0 × 102, 1.5 to 6.6 × 103, 1.7 to 1.7 × 104, 3.3 to 3.3 × 102 with the correlation coefficients (r) 0.9994, 0.9995, 0.9999, 0.9991, 0.9998, 0.9997, 0.9995, and 0.9987 and LOD ( × 103CFU/ mL) 3.0, 3.0, 17.0, 40.0, 70.0, 15.0, 33.0, and 50.0, respectively. The SLS measurements were made under the excitation and emission slit widths of 5.0 nm and the PMT voltage of 400 V. Bioparticles were dispersed in pH 7.4 PBS buffer.

CFU/mL and the limit of determination could reach 3 × 103 CFU/mL (3 σ). Light Scattering Yields. To understand why S. cereVisiae and S. pombe have different light scattering extents considering that both bioparticles have the same spectral shapes, we introduce the concept of the light scattering yields (φLS) according to Yguerabide with following definition,40,41

φLS )

Csca Csca εCsca ) ) Cext Cabs + Csca 2.63 × 1020

(1)

wherein Csca, Cext, and Cabs are the light scattering cross section, extinction cross section, and absorption cross section, respectively;  is the molar decadic extinction coefficient (M-1 cm-1). By employing polybead carboxylate microspheres of 110 nm in diameter as a standard, we measured the φLS values of bioparticles (Table 1). From the table, we could conclude two results, one is that the light scattering yields of two fungi (S. cereVisia and S. pombe) are stronger than other bacteria at the maximum wavelengths, but both lower than 1. The other result is that the light scattering yields of latex beads are stronger than all bioparticles at the same wavelength (470 nm), and even if higher than 1. That is to say, the concept of light scattering yields is different from the fluorescence yields whose value is never higher 1. The reason is possibly because the light scattering signals of the bioparticles involve in the real and imaginary parts of the index in a turbid medium, and the enhanced light scattering signals exist, while these of polybead carboxylate microspheres only involve in the real part of the index, and the light scattering yield is one unit according to above definition.40,41 PSLS Spectra of Bioparticles. As Figure 1 shows, SLS signals cannot disclose elaborate information of bioparticles. In order to ascertain whether the light scattering signals could be applied for further information or not for the bioparticles, we measured the polarization features of light scattering signals when excited with polarized light beam of different polarized

sample (x)a

φLSx (λmax)

φLSx (λ ) 470 nm)

100 nm latex beads 200 nm latex beads 300 nm latex beads 500 nm latex beads S. cereVisia S. pombe E. coli BL-21 E. coli DH5R B. subtills B. thuringinesis B. megaterium S. aureus

8.20 (320 nm) 15.54 (470 nm) 1.41 (344 nm) 0.85(360 nm) 0.489 (350 nm) 0.454 (350 nm) 0.370 (470 nm) 0.346 (470 nm) 0.246 (470 nm) 0.349 (470 nm) 0.270 (470 nm) 0.337 (470 nm)

18.69 15.54 1.78 1.30 0.334 0.338 0.370 0.346 0.246 0.349 0.270 0.337

a Experimental conditions: The LS yield of 110 nm polybead carboxylate microspheres as a compare standard (φLSPM(λ))1), the sample RLS yield was φLSPM(λ) ) APM(λ)/Ax(λ) × Ix(λ)/IPM(λ). The LS intensities were made with excitation and emission slit widths of 5.0 nm and a PMT voltage of 400 V. The absorbance values were made against PBS buffer solutions.

angle considering that these bacteria have different shape. All the left sides of Figure 3 show the multiangular PSLS spectra of several microbes. From these polarized spectra, we can find that the polarized fingerprints spectra have much more clear differences than those displayed by SLS spectra (the left side of Figure 1). They almost have two characteristic peaks located at about 400 and 550 nm, respectively. With the change of polarized angle, the PSLS intensity at 550 nm exhibits a faster change than that at 400 nm. To make the comparison, we also measured the PSLS spectra of spherical latex beads with different sizes (Figure S2 in the Supporting Information). It can be seen that the PSLS spectra of latex beads are different from the SLS spectra also. Compared with SLS spectra, PSLS spectra display some red shift, and the two PSLS peaks are more obvious for large particles larger than 300 nm. The peaks at about 550 nm are higher than the peaks at about 370 nm. Angular Polarization of I0°-30° Against I0°-0°. To find the nature of the PSLS displayed, we have investigated the PSLS features at different polarization angle between the excitation (R) and emission (β) polarizing sheets. As the left sides of Figure 3 show, the PSLS intensity is the maximum when R-β couple is 0 degree, but almost becomes zero when R-β couple approaches 90-degree. These PSLS features could be classified three groups in terms of the spectral skeleton. One includes S. pombe, S. cereVisiae, B. thuringiensis, and E.coli BL-21, and all of them have a wide flat peak. The second group includes B. subtilis, B. megaterium, and S. aureus, and they have two peaks which seem are splitting from those of group one. The third group includes E.coli DH5R only and has one very sharp peak with a shoulder peak in its red side. We dealt with the PSLS intensities of different R-β couples for these bacteria, and found that I0°-30° against I0°-0° could be used for distinguishing these bacteria as different groups. The results showed that good differentiation could be made between fungi and bacteria with the data of I0°-30° against I0°-0° measured at 540 nm (the lefts of Figure 4). There is still some superposition among different bacteria, such as between E. coliBL-21 and B. megaterium, E. coli DH5R, and S. aureus (right side of Figure 4). For the situation, we need combining the SLS spectra and PSLS spectra of these bioparticles for further differentiation. For example, the PSLS spectra of E. coli DH5R and S. aureus have obvious difference (shown in the left side of Figure 3). Generally, the features related to the asymmetric shape of the scatters are ignored in the SLS measurements, while that in

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Figure 3. Part 1 of 2.

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Figure 3. Part 2 of 2. Angular dependent polarized light scattering spectra (left) and angular dependent polarized derivative light scattering spectra (right) of eight suspensions of bioparticles. The polarization angles are expressed as R-β, where R refers to the incidence direction polarization angle as 0° angle, and β angles refer to the detection direction polarization angle as 0°, 30°, 45°, 60°, and 90° angles, respectively.

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Figure 4. Groups distinguished for bioparticles based on angular dependence polarized light scattering. The data were obtained at 540 nm.

TABLE 2: Characteristics of SLS Spectra and DrSLS Spectra of Bioparticles DrSLS spectra

bioparticles

SLS spectra max scattering wavelength (λmax, nm)

main positive peak (λ+, nm)

S. pombe S. cereVisiae B. megaterium B. subtills B. thuringinesis S. aureus E. coli BL-21 E. coli DH5R

350 350 470 470 470 470 470 470

314.6 301.8 297.6 300.8 312.4 302.6 300.8 295.8

main negative spike λ+-λ(λ-, nm) (nm)a 575.8 580.2 587.0 583.4 569.2 593.4 583.0 573.4

261.2 278.4 289.4 282.6 256.8 290.8 282.2 277.8

a + λ -λ-, the distance width between the main positive peak and the main negative spike.

TABLE 3: Dependence of Relative PSLS Intensities of Single S. aureus and Polybead Carboxylate Microspheres on Polarization Angle polarization angle (β-R)a IS.a IPM ratio of IS.awith IPM av ratio of IS.a with IPM

0°-0°

0°-30°

0°-45°

0°-60°

129.2 33.3 3.88

101.4 25.6 3.9

75.1 18.9 3.97

49.0 13.0 3.77

3.88

a The polarization angles are expressed as R-β, where R refers to the incidence direction polarization angle as 0° angle, and β angles refer to the detection direction polarization angle as 0°, 30°, 45°, and 60° angles, respectively. PSLS were made with excitation and emission slit widths of 5.0 nm and a PMT voltage of 400 V.

PSLS could be stressed about the size and shape of microorganisms. As Figure 4 shows, if some appropriate information about the morphological property of interest is available, then PSLS is a preferred method for obtaining qualitative and quantitative information. We have measured the angular polarization scattering intensities of single S. aureus and polybead carboxylate microspheres (see next). Derivative Synchronous Light Scattering (DrSLS) Signals. In order to further discern the information of the bioparticles, we measured the DrSLS signals with the extension function of the spectrofluorometer. All the rights in Figures 1 and 3 show the DrSLS spectra of the eight model bioparticles, and the DrSLS spectra are the result from the first order of the left SLS spectra (Figure 1) or PSLS spectra (Figure 3). From these DrSLS spectra, we found that a significant feature for the DSLS spectra is that they have positive and negative peaks. By taking the DrSLS of S. cereVisiae as an example, we can see that there

are several positive peaks ranging from 220 to 360 nm with the main peak at 301.8 nm and several negative peaks ranging from 450 to 700 nm with the main spike at 580.2 nm. The positive peaks correspond to the increase of SLS signals, whereas negative peaks correspond to the decrease of SLS signals. The inflection of the positive and negative peak is at 350 nm, and it is also the wavelength where the strongest SLS signals could be measured. In such case, we can introduce two factors in order to discern the information of the bioparticles. One is the location of the spectral peaks either positive or negative (spike), and the other is the distance width between the main positive peak and main negative spike (∆λpp). As can be seen from Table 2, all the bioparticles have different ∆λpp values, which are possible related to the features of the size, the shapes, refractive index, and the interior structure of the bioparticles. As can be seen above, the DrSLS spectrum ranging from 360 to 450 nm in fact reflects the fluctuation of SLS signals. It is obvious that the SLS signals for different kind of bioparticles are lack of elaborate structure, and it is hard to find the difference for these SLS spectra. After being treated with the derivative, however, differences between bioparticles come out. Therefore, we suppose that DrSLS spectra are useful for the differentiation of bioparticles although further discerning the information of DrSLS of the bioparticles is necessary and now is underway. Refractive Index of Spherical Bacteria. According to the Mie theory,

IPAR)

|

|

16π4a6nmed4I0 m2 - 1 2 2 cos θ r2λ04 m2 + 2

(2)

where I0 is the intensity of incident monochromatic light, IPAR is the polarization scattering intensity, a is the radius of spherical particle, λ0 is the wavelength of the incident beam, R is the angle between the detection direction r and the direction of polarization of the incident beam (θ ) 90° - R), nmed is the refractive index of the medium surrounding the particle, and m is the relative refractive index of the bulk particle material. Eq 2 indicates the dependence of the angular PSLS intensity on the particle size and relative refractive index. By taking S. aureus and polybead carboxylate microspheres as an example, we discussed herein how to measure the index of bioparticles. According to eq 2, the IPAR values of S. aureus and polystyrene latex particles are only connected with the spherical particle radius (a) and relative refractive index (m) under the same determination condition (I0, θ, λ0) in a same surrounding medium (nmed) for S. aureus and polybead carboxylate micro-

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spheres. By fitting the data of size distributions of spherical S. aureus and polybead carboxylate microspheres (PM) to Mie theory, we could obtain additional information about relative refractive index of cell,

(

IS. aureus aS. aureus ) IPM apM

)

|m

|

mS. aureus2 - 1 2

6

|

2 S. aureus + 2 mpM2 - 1 2 mPM2 + 2

|

(3)

It should be noted that it is very difficult to obtain the relative refractive index of cell using existing methods. In the present work, however, it is very easy to determine the sizes of S. aureus and polybead carboxylate microspheres by dynamic light scattering and get the result of aS. aureus (0.94 µm) and aPM (0.75 µm), respectively (Figure S1 in the Supporting Information), then we can calculate that the value of (aS. aureus/aPM)6 of 3.87. As calculated in Table 3, the average angular scattering intensity ratio (IS. aureus/IPM) is about 3.88, so we can obtain the value of mS. aureus since we use the index of polybead carboxylate microspheres of 1.58 (supplied by Polysciences, INC). In such case, the relative refractive index of a cell could be obtained with a known relative refractive index and the size of the homogeneous shape particle. It should be, however, noted that the values of (IS. aureus/IPM) and (aS. aureus/aPM)6 should be at first measured and calculated. For different bioparticles, these values should be changed. Conclusions In summary, light scattering phenomena from the interaction of photon with microorganisms or cells typically suspended in a nonabsorbing media were investigated in this contribution. It is easy to obtain the SLS signals by coupling and adjusting the monochromators of a common spectrofluorometer, and PSLS signals with the polarizing sheets accessory equipped with the spectrofluorometer. Our investigations on these light scattering signals of microorganisms or cells suspensions demonstrated that the light scattering as a function of wavelength depends on the sizes, shape, refractive index and inner structure of the biological particle. As for PSLS, the angular polarized light scattering signals to study the properties of biological particle suspensions have good absolute sensitivity, nondestructive and compatible with many other techniques, and do not require sample preparation. In addition, the fingerprint spectra can be adequately represented with an interpretation model based on light scattering theory. These experiments performed blindly point out the possibility of investigating and identification of microorganisms by PSLS spectrometry, and it still needs further research and analysis. Acknowledgment. This work has received support from the National Natural Science Foundation of China (NSFC, No. 30570465, No.20425517). Supporting Information Available: Figures showing the SLS spectra of latex beads, angular dependent polarized light scattering spectra of different sizes latex beads, the size of S. aureus measured by microscopy and a table giving information about the dynamic light scattering methods, contents, descrip-

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