CN104614341A - Determination method for complex refractive index of entomogenous fungus beauveria bassiana or green muscardine fungus - Google Patents

Abstract

The invention discloses a determination method for complex refractive index of entomogenous fungus beauveria bassiana or green muscardine fungus. A specular reflection method is adopted for determining complex refractive index of beauveria bassiana or green muscardine fungus: pure spores and hyphae of the beauveria bassiana or green muscardine fungus obtained by fermentation are subjected to freeze drying and then the pure spores and hyphae subjected to freeze drying are pressed into tablets, and spectrographic determination is carried out on the pure spores and hyphae of the beauveria bassiana or green muscardine fungus which are pressed into tablets by using a Fourier infrared spectrometer and taking a gilded reflection mirror as a background. Compared with the prior art, the determination method disclosed by the invention has the following advantages: special treatment does not need to be carried out on samples, so that physical conditions are not damaged, the related problems of scattered radiation of irregular particles inverted by Mie scattering theory are avoided, and the morphology and particle size of particles are not limited.

Description

A kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus

Technical field

The present invention relates to a kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus.

Background technology

Muscardine and green muscardine fungus are a kind of entomogenous fungis of Fungi Imperfecti, have nutrition organs mycelia and organ of multiplication spore, are mainly used in the control of insect aspect of forest at present.About the research of these two kinds of fungies, mainly concentrate on cultural method, genetic modification, toxicological study aspect, also nobody studies optical characteristics.In the present invention, we adopt mirror-reflection method, utilize micro ft-ir spectroscopy instrument to be studied its optical characteristics.The acquisition of muscardine and muscardine spore and mycelial optical property, for the application of microorganism in electromagnetic attenuation lays the foundation, opens the new application approach of microorganism.

Optical characteristics is characterized by complex index of refraction N=n+i k, and n is real part, and be the parameter characterizing reflection, k is imaginary part, relevant with the absorption of medium.The size of K value and n value, characterizes these two kinds of entomogenous fungis to the power of electromagnetic attenuation.The method of traditional measurement complex index of refraction is based on Mie-scattering lidar and K-K (kramers-kronig) relation, and this method requires that dielectric distribution is even, and must be circle or the ellipse of rule.But the mycelia of muscardine and green muscardine fungus and spore do not meet above requirement, the spore of muscardine is almost spherical, and particle diameter is 3-4 μm, muscardine spore is similar to elliposoidal, particle diameter is 4-5 μm, and the mycelia of these two kinds of fungies is long column types, and length is at hundreds of μm.So we adopt mirror-reflection method to measure real part and the imaginary part of complex index of refraction, mirror-reflection is the light reflection of specular surface, is wherein reflected to a single exit direction from the light of a single incident direction.The advantage of the method is, they avoid the relevant issues by the scattered radiation of the irregular particle of Mie scattering theory inverting, and does not limit the shape of particle and particle diameter.In addition, the method does not need sample special processing, and physical state is not destroyed; Therefore, be suitable to the measurement of muscardine and muscardine spore and mycelial complex index of refraction.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, provide a kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus, for the application of microorganism in electromagnetic attenuation lays the foundation, open the new application approach of microorganism.

The present invention is achieved by the following technical solutions: a kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus, mirror-reflection method measures the complex index of refraction of muscardine or green muscardine fungus: by compressing tablet after the pure spore of ferment gained muscardine or green muscardine fungus and mycelia freeze drying, background is done with kBr, use Fourier's micro ft-ir spectroscopy instrument, using gold-plated mirror surface as back end, spectroscopic assay is carried out to the pure spore of the muscardine after compressing tablet or green muscardine fungus and mycelia.

As the further optimization of such scheme, adopt the test of Fourier's micro ft-ir spectroscopy instrument at the mirror-reflection spectrum of 2.5-25 mu m waveband, using gold-plated mirror surface as back end, spectrometer spot size is 100 × 100 μm, incident angle is zero, carries out spectroscopic assay to the pure spore of the muscardine after compressing tablet or green muscardine fungus and mycelia.

As the further optimization of such scheme, the complex index of refraction N=n+ik of described muscardine or green muscardine fungus, n are real part, and k is imaginary part, $n\left(\mathrm{\λ}\right)=\frac{1-R\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)},$ $k\left(\mathrm{\λ}\right)=\frac{-2\sqrt{R}\sin \mathrm{\Θ}\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)}$

Wherein, R is plane reflection rate, and Θ is reflection phase shift, and λ is wavelength.

The present invention has the following advantages compared to existing technology: a kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus of the present invention, spore due to muscardine is almost spherical, particle diameter is 3-4 μm, muscardine spore is similar to elliposoidal, particle diameter is 4-5 μm, the mycelia of these two kinds of fungies is long column types, length is at hundreds of μm, mirror-reflection method is adopted to measure real part and the imaginary part of complex index of refraction, mirror-reflection is the light reflection of specular surface, is wherein reflected to a single exit direction from the light of a single incident direction.A kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus of the present invention does not need sample special processing, physical state is not destroyed, avoid by the relevant issues of the scattered radiation of the irregular particle of Mie scattering theory inverting, and the shape of particle and particle diameter are not limited.

Accompanying drawing explanation

Fig. 1 is the state change map of the muscardine k value utilizing a kind of method measuring the complex index of refraction of entomogenous fungi muscardine of the present invention to measure.

Fig. 2 is the state change map of the muscardine n value utilizing a kind of method measuring the complex index of refraction of entomogenous fungi muscardine of the present invention to measure.

Fig. 3 is the state change map of the green muscardine fungus k value utilizing a kind of method measuring the complex index of refraction of entomogenous fungi green muscardine fungus of the present invention to measure.

Fig. 4 is the state change map of the green muscardine fungus n value utilizing a kind of method measuring the complex index of refraction of entomogenous fungi green muscardine fungus of the present invention to measure.

Embodiment

Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

Embodiment one: the k value of beauveria bassiana spore and mycelia, the mensuration of n value.

Adopt mirror-reflection method to measure the method for the complex index of refraction of entomogenous fungi muscardine, be made up of following steps:

(1) activation of bacterial strain: transferred on PDA inclined-plane by the muscardine be kept in glycerine pipe, cultivates 7 days for 26 DEG C respectively.

(2) cultivation of seed: scrape 3 ring muscardines from inclined-plane, is transferred in 50mLPDB triangular flask, cultivates 2 days, as the seed of solid culture for 26 DEG C.

(3) solid fermentation is cultivated: by liquid-solid ratio be 2:1 rice based on nutrient culture media, another add a certain amount of KNO ₃and MgSO ₄7H ₂o is as inorganic salts.With the inoculum concentration inoculation solid medium of 10%.Cultivate 7 days for 26 DEG C.

(4) collection of spore and mycelia: culture matrix is dried to constant weight at 60 DEG C.Collect spore and mycelia.

(5) preparation of sample: take pure mycelia and spore 20mg respectively, utilizes infrared sheeter to press 1min under the pressure of 10MPa, makes compressing tablet.

(6) mensuration of complex index of refraction, the pure spore of fermentation gained muscardine and mycelia drying are distinguished compressing tablet afterwards, use the test of Fourier's micro ft-ir spectroscopy instrument at the mirror-reflection spectrum of 2.5-25 mu m waveband, adopt gold-plated mirror surface as back end, spectrometer spot size is 100 × 100 μm, incident angle is zero, carries out spectroscopic assay to the pure spore of the muscardine after compressing tablet and mycelia.Preferably, each compressing tablet is chosen 3 sampled points and is carried out spectroscopic assay, gets the mean value of 3 measurement results.

The complex index of refraction N=n+ki of muscardine, n are real part, and k is imaginary part,

$n\left(\mathrm{\λ}\right)=\frac{1-R\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)},$ $k\left(\mathrm{\λ}\right)=\frac{-2\sqrt{R}\sin \mathrm{\Θ}\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)}$

Wherein, R is plane reflection rate, and Θ is reflection phase shift, and λ is wavelength.See Fig. 1, it is the state change map of the k value of the beauveria bassiana spore of the preferred embodiments of the present invention measurement.At 3-5 mu m waveband, the k value of beauveria bassiana spore is 0.3111, and the k value of mycelia is 0.2247, and at 8-14 mu m waveband, the k value of beauveria bassiana spore is 0.2137, and the k value of mycelia is 0.1027.See Fig. 2, it is the state change map of the n value of the beauveria bassiana spore of the preferred embodiments of the present invention measurement.At 3-5 μm, the n value of beauveria bassiana spore is 1.2437, and the n value of mycelia is 1.1507, and at 8-14 μm, the n value of beauveria bassiana spore is 1.3455, and the n value of mycelia is 1.2553.

At 3-5 mu m waveband, the real part n of negative index and imaginary part k during change, can meet the application conditions as electromagnetic attenuation material respectively within the scope of 1.2-1.7,0.05-0.4.Because n is the parameter characterizing reflectivity, so when n changes at 1.2-1.7, more close to 1.7, its attenuating is better.K characterizes the parameter absorbed, and when k is in 0.05-0.4 range, the value of k is more close to 0.4, and its attenuating is better.At 8-14 mu m waveband, the real part n of negative index and imaginary part k during change, can meet the application conditions as electromagnetic attenuation material respectively within the scope of 1.1-1.4,0.04-0.38.Because n is the parameter characterizing reflectivity, so when n changes at 1.1-1.4, the value of n is larger, its attenuating is better.K characterizes the parameter absorbed, and when k is in 0.04-0.38 range, the value of k is larger, and its attenuating is better.

The mycelia of the muscardine more than measured and the k value of spore and n value, 3-5 mu m waveband, the k value 0.3111 of beauveria bassiana spore is greater than the k value 0.2247 of mycelia.With regard to n value, n value 1.2437 n value 1.1507 of mycelia in the scope of 1.2-1.7 of beauveria bassiana spore, not in scope.So at 3-5 mu m waveband, the electromagnetic attenuation effect of beauveria bassiana spore is better than the attenuating of mycelia.When 8-14 mu m waveband, the k value of beauveria bassiana spore is 0.2137, and the k value of mycelia is 0.1027, all meets the scope of 0.04-0.38, but the k value 0.2137 of spore is greater than the k value 0.1027 of mycelia.The n value of beauveria bassiana spore is 1.3455, and the n value of mycelia is 1.2553, also all in the scope of 1.1-1.4, but the n value 1.3455 of the beauveria bassiana spore of spore, closer to 1.4, so the attenuating of spore is better than mycelia.It can thus be appreciated that when 3-5 μm and 8-14 μm of two wave band, the attenuating of the spore of muscardine is all better than the attenuating of mycelia.

Embodiment two: the k value of muscardine spore and mycelia, the mensuration of n value.

Adopt mirror-reflection method to measure the method for entomogenous fungi green muscardine fungus complex index of refraction, be made up of following steps:

(1) activation of bacterial strain: transferred on PDA inclined-plane by the green muscardine fungus be kept in glycerine pipe, cultivates 13 days for 26 DEG C respectively.

(2) cultivation of seed: scrape 3 ring green muscardine fungus from inclined-plane, is transferred in 50mLPDB triangular flask, cultivates 2 days, as the seed of solid culture for 26 DEG C.

(3) solid fermentation is cultivated: by liquid-solid ratio be 2:1 rice based on nutrient culture media, another add a certain amount of KNO ₃and MgSO ₄7H ₂o is as inorganic salts.With the inoculum concentration inoculation solid medium of 10%.Cultivate 13 days for 26 DEG C.

(4) collection of spore and mycelia: culture matrix is dried to constant weight at 60 DEG C.Collect spore and mycelia.

(5) preparation of sample: take pure mycelia and spore 20mg respectively, utilizes infrared sheeter to press 1min under the pressure of 10MPa, makes compressing tablet.

(6) mensuration of complex index of refraction, the pure spore of fermentation gained green muscardine fungus and mycelia drying are distinguished compressing tablet afterwards, use the test of Fourier's micro ft-ir spectroscopy instrument at the mirror-reflection spectrum of 2.5-25 mu m waveband, adopt gold-plated mirror surface as back end, spectrometer spot size is 100 × 100 μm, incident angle is zero, carries out spectroscopic assay to the pure spore of the muscardine after compressing tablet and mycelia.Preferably, each compressing tablet is chosen 3 sampled points and is carried out spectroscopic assay, gets the mean value of 3 measurement results.

The complex index of refraction N=n+ki of green muscardine fungus, n are real part, and k is imaginary part,

$n\left(\mathrm{\λ}\right)=\frac{1-R\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)},$ $k\left(\mathrm{\λ}\right)=\frac{-2\sqrt{R}\sin \mathrm{\Θ}\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)}$

Wherein, R is plane reflection rate, and Θ is reflection phase shift, and λ is wavelength.See Fig. 3, it is the state change map of the k value of the muscardine spore of the preferred embodiments of the present invention measurement.At 3-5 mu m waveband, the k value of muscardine spore is 0.3973, and the k value of mycelia is 0.3875, and at 8-14 μm, the k value of muscardine spore is 0.3265, and the k value of mycelia is 0.2626.See Fig. 3, it is the state change map of the n value of the muscardine spore of the preferred embodiments of the present invention measurement.At 3-5 mu m waveband, the n value of muscardine spore is 1.2470, and the n value of mycelia is 1.2101, and at 8-14 μm, the n value of muscardine spore is 1.3530, and the n value of mycelia is 1.3473.

At 3-5 mu m waveband, the real part n of negative index and imaginary part k during change, can meet the application conditions as electromagnetic attenuation material respectively within the scope of 1.2-1.7,0.05-0.4.Because n is the parameter characterizing reflectivity, so when n changes at 1.2-1.7, more close to 1.7, its attenuating is better.K characterizes the parameter absorbed, and when k is in 0.05-0.4 range, the value of k is more close to 0.4, and its attenuating is better.At 8-14 mu m waveband, the real part n of negative index and imaginary part k during change, can meet the application conditions as electromagnetic attenuation material respectively within the scope of 1.1-1.4,0.04-0.38.Because n is the parameter characterizing reflectivity, so when n changes at 1.1-1.4, the value of n is larger, its attenuating is better.K characterizes the parameter absorbed, and when k is in 0.04-0.38 range, the value of k is larger, and its attenuating is better.

The mycelia of the green muscardine fungus more than measured and the k value of spore and n value, 3-5 mu m waveband, the k value 0.3973 of muscardine spore is greater than the k value 0.3875 of mycelia.With regard to n value, the n value that the n value 1.2470 of muscardine spore is greater than mycelia is 1.2101.So at 3-5 mu m waveband, the electromagnetic attenuation effect of muscardine spore is better than the attenuating of mycelia.When 8-14 mu m waveband, the k value that the k value 0.3265 of muscardine spore is greater than mycelia is 0.2626, and the n value that the n value 1.3530 of muscardine spore is greater than mycelia is 1.3473.It can thus be appreciated that when 3-5 μm and 8-14 μm of two wave band, the attenuating of the spore of green muscardine fungus is all better than the attenuating of mycelia

This preferred embodiment adopts mirror-reflection method to measure the method for entomogenous fungi complex index of refraction, spot size due to micro ft-ir spectroscopy instrument is less than conventional Fourier spectrometer, utilize micro ft-ir spectroscopy instrument, by advantageously in the smooth surface degree controlling sample within the scope of single hot spot, improve mirror-reflection efficiency.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (3)

1. one kind measures the method for the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus, it is characterized in that: adopt mirror-reflection method to measure the complex index of refraction of muscardine or green muscardine fungus: by compressing tablet after the pure spore of ferment gained muscardine or green muscardine fungus and mycelia drying, use Fourier's micro ft-ir spectroscopy instrument, using gold-plated mirror surface as back end, spectroscopic assay is carried out to the pure spore of the muscardine after compressing tablet or green muscardine fungus and mycelia.

2. a kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus according to claim 1, it is characterized in that: adopt the test of Fourier's micro ft-ir spectroscopy instrument at the mirror-reflection spectrum of 2.5-25 mu m waveband, using gold-plated mirror surface as back end, spectrometer spot size is 100 × 100 μm, incident angle is zero, carries out spectroscopic assay to the pure spore of the muscardine after compressing tablet or green muscardine fungus and mycelia.

3. a kind of method measuring the complex index of refraction of entomogenous fungi muscardine or green muscardine fungus according to claim 1 and 2, it is characterized in that: the complex index of refraction N=n+i k of muscardine or green muscardine fungus, n are real part, k is imaginary part $k\left(\mathrm{\λ}\right)=\frac{-2\sqrt{R}\sin \mathrm{\Θ}\left(\mathrm{\λ}\right)}{1+R\left(\mathrm{\λ}\right)+2\sqrt{R}\cos \mathrm{\Θ}\left(\mathrm{\λ}\right)}$

Wherein, R is plane reflection rate, and Θ is reflection phase shift, and λ is wavelength.