JPH05215666A - Method and device for measuring number of bacteria - Google Patents
Method and device for measuring number of bacteriaInfo
- Publication number
- JPH05215666A JPH05215666A JP4021491A JP2149192A JPH05215666A JP H05215666 A JPH05215666 A JP H05215666A JP 4021491 A JP4021491 A JP 4021491A JP 2149192 A JP2149192 A JP 2149192A JP H05215666 A JPH05215666 A JP H05215666A
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- JP
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- Prior art keywords
- light
- bacteria
- electric signal
- liquid
- lens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims description 50
- 230000003287 optical effect Effects 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 230000001678 irradiating effect Effects 0.000 claims abstract 3
- 239000013307 optical fiber Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 230000009466 transformation Effects 0.000 abstract description 3
- 239000002245 particle Substances 0.000 description 18
- 238000005259 measurement Methods 0.000 description 10
- 238000000691 measurement method Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000002847 impedance measurement Methods 0.000 description 2
- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 description 2
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- NAWXUBYGYWOOIX-SFHVURJKSA-N (2s)-2-[[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]amino]-4-methylidenepentanedioic acid Chemical compound C1=CC2=NC(N)=NC(N)=C2C=C1CCC1=CC=C(C(=O)N[C@@H](CC(=C)C(O)=O)C(O)=O)C=C1 NAWXUBYGYWOOIX-SFHVURJKSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
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- 238000003756 stirring Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By Optical Means (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は培養液中の菌の数を計測
する菌数測定方法およびその装置に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for measuring the number of bacteria in a culture solution.
【０００２】[0002]
【従来の技術】菌数の計測は食品、醸造、薬品などの分
野において、菌の培養過程の管理を行なう上で最も重要
な事項の一つであり、従来、微生物の菌数または微生物
活性の計測には以下のような方法が用いられている。 （１）コロニー計数法 寒天培地に生じたコロニーを肉眼的に計測して微生物菌
数を測定する。 （２）濁度計測法 培養液中に浮遊する菌体による光散乱現象を光学的に測
定し、その濁度により微生物菌数を求める。 （３）ＡＴＰ測定法 ＡＴＰをルシフェリン−ルシフェラーゼなどの生物発光
を利用して、その発光量から計測する。 （４）インピーダンス測定法 微生物の物質代謝による発酵培地中の電気的インピーダ
ンスの変化を測定し、微生物活性を測定する。2. Description of the Related Art The measurement of the number of bacteria is one of the most important items for controlling the culture process of bacteria in the fields of food, brewing, medicine, etc. The following methods are used for measurement. (1) Colony counting method Colonies generated on the agar medium are visually measured to determine the number of microbial cells. (2) Turbidity measurement method The light scattering phenomenon due to the bacterial cells floating in the culture solution is optically measured, and the microbial cell count is determined from the turbidity. (3) ATP measurement method ATP is measured from the amount of luminescence using bioluminescence of luciferin-luciferase or the like. (4) Impedance measurement method Changes in electrical impedance in the fermentation medium due to substance metabolism of microorganisms are measured to measure microbial activity.
【０００３】[0003]
【発明が解決しようとする課題】しかし、上記の（１）
〜（４）の方法にはそれぞれ次のような欠点がある。 （１）コロニー計数法 操作が煩雑で培地に散布後、生育に長時間（２４〜４８
時間）を要し、その計測は必ずしも正確ではない。 （２）濁度計測法 試料液が可視領域で分光学的に吸収の少ないものである
ことが必要であるが、実際には高着色度の培地を使う例
が多く、また、セルの汚れなどの影響で長期に亘る連続
インライン測定は困難である。 （３）ＡＴＰ測定法 培地中に共存する蛍光物質やＡＴＰが背景光となった
り、微生物または細胞１個当たりに含まれるＡＴＰ量が
種類や状態により異なるために、正確な菌数を求めるこ
とが困難である。 （４）インピーダンス測定法 通気攪拌のある条件下では測定することができず、また
試料溶液中に導電性物質が存在すると測定することがで
きない。[Problems to be Solved by the Invention] However, the above (1)
Each of the methods (4) to (4) has the following drawbacks. (1) Colony counting method Since the operation is complicated, it is necessary to grow for a long time (24 to 48
It takes time) and the measurement is not always accurate. (2) Turbidity measurement method It is necessary for the sample solution to have spectroscopically low absorption in the visible region, but in reality, there are many cases in which a medium with a high degree of coloring is used, and cell contamination, etc. Due to this, continuous in-line measurement over a long period of time is difficult. (3) ATP measurement method Since a fluorescent substance or ATP coexisting in the medium becomes background light, or the amount of ATP contained in each microorganism or cell varies depending on the type and state, it is necessary to obtain an accurate number of bacteria. Have difficulty. (4) Impedance measurement method It cannot be measured under the condition of aeration and stirring, and also cannot be measured if a conductive substance is present in the sample solution.
【０００４】本発明は上述の欠点を解決するためになさ
れたものであり、その目的は簡便な構造を持つセンサー
で、インラインで使用することができ、しかも長期間安
定な菌数測定方法とその装置を提供することにある。The present invention has been made to solve the above-mentioned drawbacks, and its object is a sensor having a simple structure, which can be used in-line and is stable for a long period of time, and a method for measuring the number of bacteria. To provide a device.
【０００５】[0005]
【課題を解決するための手段】上記の課題を解決するた
めに、本発明の菌数測定方法について、第１の方法は、
菌を含む流動する液体中に光を照射し、透過した光を電
気信号に光電変換し、この電気信号の標準偏差値を電気
信号の平均値で割った結果Ａと、菌を含まない液を測定
した結果を電気信号の平均値で割ったものを対数変換し
た結果Ｂとを用いて、演算結果Ｂを演算結果Ａで割り算
した値に所定の定数を乗じた結果を自乗することにより
菌数を測定する。第２の方法は、菌を含む流動する液体
中に光を照射し、菌体によって散乱された光を所定の角
度の後方から受光して光電変換し、この電気信号の標準
偏差値を電気信号の平均値で割り算し、所定の定数を乗
じ、自乗することにより菌数を測定するものである。In order to solve the above-mentioned problems, the first method of the method for measuring the number of bacteria of the present invention is as follows:
The flowing liquid containing bacteria is irradiated with light, the transmitted light is photoelectrically converted into an electric signal, and the standard deviation value of this electric signal is divided by the average value of the electric signal to obtain the result A and the liquid containing no bacteria. By using the result B obtained by dividing the measured result by the average value of the electric signal by the logarithmic transformation and multiplying the result obtained by dividing the value obtained by dividing the operation result B by the operation result A by a predetermined constant, the number of bacteria To measure. The second method is to irradiate light into a flowing liquid containing bacteria, photoelectrically convert the light scattered by the bacteria from the rear of a predetermined angle, and convert the standard deviation value of this electric signal into an electric signal. The number of bacteria is measured by dividing by the average value of, multiplying by a predetermined constant, and squaring.
【０００６】以上の方法を実施するための本発明の装置
は、透過光または後方散乱光を光量に比例した電気信号
に変換する光電変換素子と、この光電変換素子によって
得られた電気信号を所定の倍率に増幅する増幅回路と、
増幅した電気信号（Ｖ）の変動成分のみ増幅する交流増
幅回路と、交流増幅された変動信号から実効値を出力す
るためのＲＭＳ／ＤＣ変換器と、ＲＭＳ／ＤＣ変換器の
出力を、電気信号（Ｖ）の平均化直流信号で割り算する
ための第１の割り算器と、この平均化直流信号の逆数を
対数化する対数変換器と、この対数変換器の出力を第１
の割り算器の出力に所定の定数を乗じたもので割り算す
るための第２の割り算器と、この割り算器の出力を自乗
する自乗演算器と、この自乗演算器の出力を表示するた
めの表示器からなる電気回路を有し、とくに第２の方法
を行なう装置の光センサー部の構成は、光軸上の一端に
配した発光素子と、この発光素子からの光線を平行にす
るための第１レンズまたはレンズ群と、第１レンズまた
はレンズ群を通過した光を液中で一点に集束させるため
の第２レンズまたはレンズ群と、液体と第１，第２レン
ズまたはレンズ群とを隔てるための透明な光学窓と、第
２レンズまたはレンズ群の液中における焦点を中心とす
る後方散乱光を、第２レンズまたはレンズ群を通して受
光するための光ファイバー列と、この光ファイバーの他
端面で少なくとも発光素子の発する波長の光を透過させ
る干渉フィルターと、この干渉フィルターを通過した光
を受光して電気信号に変換する光電変換素子からなる。The apparatus of the present invention for carrying out the above method is a photoelectric conversion element for converting transmitted light or backscattered light into an electric signal proportional to the amount of light, and an electric signal obtained by the photoelectric conversion element. An amplification circuit that amplifies to a magnification of
An AC amplifier circuit that amplifies only the fluctuation component of the amplified electric signal (V), an RMS / DC converter for outputting an effective value from the AC-amplified fluctuation signal, and an output of the RMS / DC converter A first divider for dividing by the averaged DC signal of (V), a logarithmic converter for logarithmizing the reciprocal of the averaged DC signal, and an output of the logarithmic converter for the first
2nd divider for dividing by the output of the divider of a given constant, a square calculator that squares the output of this divider, and a display for displaying the output of this square calculator The configuration of the optical sensor section of an apparatus having an electric circuit including a vessel and particularly performing the second method is such that a light emitting element arranged at one end on the optical axis and a light beam from the light emitting element are parallel to each other. To separate one lens or lens group, a second lens or lens group for converging light passing through the first lens or lens group into one point in the liquid, and the liquid and the first or second lens or lens group Transparent optical window, an optical fiber array for receiving backscattered light centered on the focus of the second lens or lens group in the liquid through the second lens or lens group, and at least the other end surface of the optical fiber. An interference filter which transmits light of a wavelength emitted by the optical device, a photoelectric conversion element for converting into an electric signal by receiving the light transmitted through the interference filter.
【０００７】[0007]
【作用】本発明の菌数測定方法は上記のようにしたため
に、第１の方法では、菌体の大きさと屈折率によって決
まる散乱係数に依存することなく、しかもセルを流れる
液の流量にも依存せずに菌数を測定することができ、第
２の方法では第１の方法の作用に加え、セルや光学窓の
汚れ、光源の光強度変動に依存することなく長期的に安
定な菌数測定が可能である。また、これらの方法を実施
するために、上記の回路構成とすることにより、簡単で
安価な装置とすることができ、とくに第２の方法を行な
う光学センサー部をコンパクトにして、正確な後方散乱
光成分を測定できるようにしている。Since the method for measuring the number of bacteria of the present invention is as described above, the first method does not depend on the scattering coefficient determined by the size and the refractive index of the cells, and also the flow rate of the liquid flowing through the cell. The number of bacteria can be measured independently, and in the second method, in addition to the effect of the first method, the bacteria are stable for a long period of time without depending on the contamination of the cell or the optical window and the fluctuation of the light intensity of the light source. It is possible to measure a few. Further, in order to carry out these methods, the above-mentioned circuit configuration can be used as a simple and inexpensive device, and in particular, the optical sensor section for carrying out the second method can be made compact to provide accurate backscattering. The light component can be measured.
【０００８】[0008]
【実施例】以下、本発明を実施例により説明する。本発
明の第１の方法は、上水分野における凝集プロセスに適
用された光変動測定法に基づくものである。この方法
は、本来広い範囲の粒径分布を持つような懸濁液には適
用できなかったが、比較的粒径の揃った菌体に適用する
ことによって、菌数の正確な測定が可能になることに着
目したものである。この測定方法の原理を以下に説明す
る。EXAMPLES The present invention will be described below with reference to examples. The first method of the present invention is based on the optical fluctuation measurement method applied to the coagulation process in the waterworks field. Originally, this method could not be applied to suspensions with a wide range of particle size distribution, but by applying it to cells with a relatively uniform particle size, it is possible to accurately measure the number of bacteria. It focuses on becoming. The principle of this measuring method will be described below.
【０００９】懸濁液に照射された光ビームの体積中に存
在する懸濁粒子数の確率分布はＰｏｉｓｓｏｎ分布に従
う。したがって、ある時刻の懸濁粒子数の分散と平均粒
子数が等しいことから、透過光強度の標準偏差
（Ｖrms ）は次の式で表わされる。 Ｖrms ＝Ｖ0 ｅｘｐ（−νＣ／Ａ）ｓｉｎｈ（ν1/2 Ｃ／Ａ） ＝Ｖm ｓｉｎｈ（ν1/2 Ｃ／Ａ） （１） 但し、Ｖ0 ：照射透過光量 Ｖm ：平均透過光量 ν ：光路体積中懸濁質平均個数濃度 Ｃ ：懸濁質散乱断面積 この式は、ν1/2 Ｃ／Ａ≪１の条件下で次のように近似
することができる。 Ｖrms ＝Ｖm （ν1/2 Ｃ／Ａ） （２）The probability distribution of the number of suspended particles existing in the volume of the light beam with which the suspension is irradiated follows the Poisson distribution. Therefore, since the dispersion of the number of suspended particles at a certain time is equal to the average number of particles, the standard deviation (V rms ) of transmitted light intensity is expressed by the following equation. V rms = V 0 exp (-νC / A) sinh (ν 1/2 C / A) = V m sinh (ν 1/2 C / A) (1) where V 0 : irradiation transmitted light amount V m : average Amount of transmitted light ν: Average number concentration of suspended solids in optical path volume C: Scattered material scattering cross section This formula can be approximated as follows under the condition of ν 1/2 C / A << 1 . V rms = V m (ν 1/2 C / A) (2)
【００１０】一方、平均透過光強度はＬａｍｂｅｒ−Ｂ
ｅｅｒ式より次式で表わされる。 Ｖm ＝Ｖ0 ｅｘｐ（−νＣ／Ａ） （３） ｌｎ（Ｖ0 ／Ｖm ）＝νＣ／Ａ （４） 式（１）と式（４）より、懸濁質散乱断面積Ｃと光路断
面積Ａを消去し次の式が得られる。 ν＝Ａ｛ｌｎ（Ｖ0 ／Ｖm ）／（Ｖrms ／Ｖm ）｝2 （５） 単位体積当たりの懸濁粒子個数濃度をＮとすると、式
（５）より次の式を得ることができる。 Ｎ＝｛ｌｎ（Ｖ0 ／Ｖm ）／（Ｖrms ／Ｖm ）｝2 ／（ＬＡ）（６） 但し、Ｌ：光路長On the other hand, the average transmitted light intensity is Lamber-B.
It is expressed by the following equation from the eer equation. V m = V 0 exp (−νC / A) (3) ln (V 0 / V m ) = νC / A (4) From the formulas (1) and (4), the suspended matter scattering cross section C and the optical path are obtained. The cross-sectional area A is deleted and the following equation is obtained. ν = A {ln (V 0 / V m ) / (V rms / V m )} 2 (5) Given that the number concentration of suspended particles per unit volume is N, the following formula is obtained from formula (5). You can N = {ln (V 0 / V m ) / (V rms / V m )} 2 / (LA) (6) where L: optical path length
【００１１】このように、単分散（粒径が揃っている）
粒子の懸濁液では、懸濁粒子の散乱断面積という厄介な
パラメータを消去できるので、光学系が一定であれば、
被測定粒子の粒径を問わない普遍的な測定法である。し
かし、多くの場合、懸濁粒子の粒径は数桁の広い分布を
持っており、式（１）のν1/2 Ｃと式（４）のν1/2は
それぞれ各粒径についての総和であり、単純な割り算で
は散乱断面積を消去することができない。この意味で、
一定の培養条件下における菌体の粒径がほぼ揃っている
（２〜３倍程度）という事実は、上述の測定方法を単純
な形で適用することができる極めて稀で有用な例であ
る。As described above, monodispersion (particle size is uniform)
In a suspension of particles, the annoying parameter of the scattering cross section of suspended particles can be eliminated, so if the optical system is constant,
This is a universal measurement method regardless of the particle size of the particles to be measured. However, in many cases, the particle size of the suspended particles have a wide distribution of several orders of magnitude, equation (1) of [nu 1/2 C and the formula (4) [nu 1/2 is for each particle size respectively It is the sum, and the scattering cross section cannot be eliminated by simple division. In this sense
The fact that the cell diameters of cells are almost uniform under constant culture conditions (about 2 to 3 times) is an extremely rare and useful example to which the above-mentioned measurement method can be applied in a simple form.
【００１２】本発明の第２の方法は、前述の本発明の第
１の方法を改良したものであり、以下にその測定原理を
説明する。第１の方法では、透過光強度を測定量として
いたが、第２の方法は、懸濁粒子による散乱光のある角
度成分の強度を測定量とする。ここで、照射光光軸にあ
る有限の微小領域に存在する粒子のある角度成分（ｐ）
の平均散乱光強度Ｓmeanは、単純に次の式で表わされ
る。 Ｓmean.p＝νＶ0 Ｃp （７） Ｃp ：角度ｐの立体角積分散乱断面積 一方、散乱光強度の標準偏差Ｓrms は、前述のＰｏｉｓ
ｓｏｎ分布の仮定より、次式で表わされる。 Ｓrms.p ＝ν1/2 Ｖ0 Ｃp （８） したがって、式（７）と式（８）から次の式が得られ
る。 ν＝（Ｓmean.p／Ｓrms.p ）2 （９） The second method of the present invention is an improvement of the above-mentioned first method of the present invention, and the measurement principle thereof will be described below. In the first method, the intensity of transmitted light is used as the measurement amount, but in the second method, the intensity of a certain angular component of scattered light due to suspended particles is used as the measurement amount. Here, an angle component (p) of a particle present in a finite minute region on the irradiation light optical axis
The average scattered light intensity S mean of is simply expressed by the following equation. S mean.p = νV 0 C p (7) C p : solid angle integral scattering cross section of angle p On the other hand, the standard deviation S rms of scattered light intensity is the Pois described above.
It is expressed by the following formula based on the assumption of the son distribution. S rms.p = ν 1/2 V 0 C p (8) Therefore, the following equation is obtained from the equations (7) and (8). ν = (S mean.p / S rms.p ) 2 (9)
【００１３】上述のある有限の微小領域の体積は、その
微小領域が照射光の集束点であるから、照射光集束レン
ズの有効径、波長、焦点距離によって決まるビームウェ
ストの径ＡS と散乱光受光レンズの焦点深度Ｌw の積で
ある。したがって、懸濁粒子の個数濃度は次式で表わさ
れる。 Ｎ＝（Ｓmean.p／Ｓrms.p ）2 ／（Ｌw ＡS ） （１０） 式（１０）では、角度ｐの立体角積分散乱断面積Ｃp の
他に、照射光強度も消去されていることがわかる。即
ち、光源光強度の変動や光学窓の汚れの影響で容易に変
動する照射光強度に依存しない安定な菌数測定が可能に
なることを意味している。これは、本発明の第１の方法
が、式（６）で明らかなように、照射光強度を変数とし
て含むことに比べ大きな長所である。The volume of the above-mentioned finite minute region is the focal point of the irradiation light, and therefore the beam waist diameter A S and scattered light determined by the effective diameter, wavelength and focal length of the irradiation light focusing lens. It is the product of the depth of focus L w of the light receiving lens. Therefore, the number concentration of suspended particles is expressed by the following equation. N = (S mean.p / S rms.p ) 2 / (L w A s ) (10) In the formula (10), the irradiation light intensity is also erased in addition to the solid angle integral scattering cross section C p of the angle p. You can see that it is done. That is, it means that it is possible to stably measure the number of bacteria independent of the irradiation light intensity which is easily changed due to the fluctuation of the light intensity of the light source and the influence of the contamination of the optical window. This is a great advantage as compared with the first method of the present invention, which includes the irradiation light intensity as a variable, as is clear from the equation (6).
【００１４】図１は、本発明の第１の方法を実施するた
めの回路ブロック図である。図１において、液中を透過
した光または散乱光を受光して、光電変換するためのフ
ォトダイオード（ＰＤ）の電流信号をＩ／Ｖ変換器で光
量に比例する電圧信号Ｖに変換する。この電圧信号Ｖ
は、ＡＣアンプによって変動成分のみを抽出して増幅し
た後、ＲＭＳ／ＤＣコンバータによって標準偏差を与え
る直流信号Ｖrms を得る。また、同時に電圧信号ＶはＤ
Ｃアンプで増幅された後、ローパスフィルター（ＬＰ
Ｆ）によって変動成分を平均化した直流電圧信号Ｖmean
に変換する。得られた二つの信号Ｖrms とＶmeanを第１
の割り算器（ＤＩＶ）で（Ｖrms ／Ｖmean）を計算す
る。また、Ｖmeanは同時に対数変換器（ＬＯＧ）で逆対
数変換される。これら二つの演算結果は、第２の割り算
器（ＤＩＶ）および自乗演算器（ＳＱ）で式（６）の演
算を施し、その結果を表示器（ＤＩＳＰ）で表示する。
但し、式（６）中の照射光強度Ｖ0 は、懸濁質を含まな
い液を測定したときのＶmeanが１Ｖとなるように、ＤＣ
アンプの増幅率を調整するものとする。また、ＡＣアン
プの増幅率は第１の割り算器で定数として増幅率を補正
するものとする。FIG. 1 is a circuit block diagram for implementing the first method of the present invention. In FIG. 1, light transmitted through liquid or scattered light is received, and a current signal of a photodiode (PD) for photoelectric conversion is converted into a voltage signal V proportional to the amount of light by an I / V converter. This voltage signal V
After extracting and amplifying only the fluctuation component by the AC amplifier, the RMS / DC converter obtains the DC signal V rms giving the standard deviation. At the same time, the voltage signal V is D
After being amplified by the C amplifier, a low pass filter (LP
DC voltage signal V mean obtained by averaging the fluctuation component by F)
Convert to. The two obtained signals V rms and V mean are
(V rms / V mean ) is calculated by the divider (DIV) of. Further, V mean is inversely logarithmically converted by a logarithmic converter (LOG) at the same time. These two operation results are subjected to the operation of the equation (6) by the second divider (DIV) and the square operator (SQ), and the results are displayed by the display (DISP).
However, the irradiation light intensity V 0 in the equation (6) is DC so that V mean when the liquid containing no suspended matter is measured becomes 1V.
The amplification factor of the amplifier shall be adjusted. Further, the amplification factor of the AC amplifier is corrected by the first divider as a constant.
【００１５】図２は本発明の第２の方法を実施するため
の回路ブロック図である。図２が図１と異なる点は、散
乱光が微弱であるために、光電変換素子に高感度の光電
子増倍管（ＰＭＴ）を用いた点、対数変換器と第２の割
り算器が不要となる点である。したがって、本発明の第
２の方法は、第１の方法に比べて簡単な回路構成で実現
することができる。FIG. 2 is a circuit block diagram for carrying out the second method of the present invention. The difference between FIG. 2 and FIG. 1 is that a highly sensitive photomultiplier tube (PMT) is used for the photoelectric conversion element because the scattered light is weak, and the logarithmic converter and the second divider are unnecessary. That is the point. Therefore, the second method of the present invention can be realized with a circuit configuration simpler than that of the first method.
【００１６】図３は、本発明の第１の方法を実施するた
めの光学センサーの構成を示し、図３（ａ）は外観図、
図３（ｂ）は部分断面図を表わす。但し、図３は模式的
に光学センサーの構成を表わしたものであって、図の上
では（ａ）と（ｂ）の大きさは一致していない。この光
学センサーは、菌数測定のインラインセンサーを目的と
しており、センサー部は光ファイバーを用いてコンパク
ト化してある。本発明の第１の方法の測定原理は、測定
値が試料液の流速に依存しないという特徴を持っている
ことと、一般に、培養中の培養液は攪拌されていること
から、センサー部は開放型にして、照射光を直接流動す
る培養液に照射するように浸漬するのがよい。このよう
な理由から図３（ａ）のように、センサー部１は円柱状
とし、図３（ａ），（ｂ）に示すように、底面に切り込
みを入れて測定部とし、その内面に正確に光軸を一致さ
せて向き合うように、照射用光ファイバー２と受光用光
ファイバー３の各端面を配置してある。図３（ａ）の４
は光照射窓を表わす。また、測定部の幅、即ち、図３
（ｂ）に矢印で示した光路長５は１０ｍｍとした。セン
サー部１は、センサホルダー６から袋ねじ７を弛めるこ
とにより着脱可能としたために、種々の光路長５のセン
サー部１を用意しておき、これらを有効に使用すること
ができる。光ファイバー２，３は、コア径が０．５ｍｍ
の耐熱性石英ファイバーを用いた。これは、図３（ｂ）
に示すように、光センサー部１を樹脂性として、ファイ
バー埋め込みの射出成形を可能とするためである。光セ
ンサー部１とセンサーホルダー６は、Ｏリング８を介し
て袋ねじ７で密着させ、液のファイバーカップリング部
９への侵入を防いでいる。FIG. 3 shows a structure of an optical sensor for carrying out the first method of the present invention, and FIG. 3 (a) is an external view.
FIG. 3B shows a partial sectional view. However, FIG. 3 schematically shows the configuration of the optical sensor, and the sizes of (a) and (b) do not match in the figure. This optical sensor is intended as an in-line sensor for measuring the number of bacteria, and the sensor section is made compact by using an optical fiber. The measurement principle of the first method of the present invention is characterized in that the measured value does not depend on the flow rate of the sample solution, and in general, the culture solution during culturing is agitated, so the sensor section is opened. It is preferable to make a mold and immerse it so that irradiation light is directly irradiated to the flowing culture solution. For this reason, as shown in FIG. 3 (a), the sensor unit 1 has a columnar shape. The end faces of the irradiation optical fiber 2 and the light receiving optical fiber 3 are arranged so as to face each other with their optical axes aligned with each other. 4 in FIG. 3 (a)
Represents a light irradiation window. In addition, the width of the measurement unit, that is, FIG.
The optical path length 5 indicated by the arrow in (b) was set to 10 mm. Since the sensor unit 1 can be attached and detached by loosening the cap screw 7 from the sensor holder 6, it is possible to prepare the sensor units 1 having various optical path lengths 5 and use them effectively. The optical fibers 2 and 3 have a core diameter of 0.5 mm.
The heat-resistant quartz fiber of was used. This is shown in Fig. 3 (b).
This is because the optical sensor unit 1 is made of a resin, as shown in, to enable fiber-embedded injection molding. The optical sensor unit 1 and the sensor holder 6 are closely attached with a cap screw 7 via an O-ring 8 to prevent liquid from entering the fiber coupling unit 9.
【００１７】図４は本発明の第２の方法を実施するため
の光学センサー部を示す側面図であり、一部を断面で表
わしてある。この光学センサー部は、液中の懸濁粒子か
らの散乱光のうち、ある特定の角度成分のみを受光する
ことを目的とし、かつ、前述の図３に示した光学センサ
ーと同様に、インライン測定のためにコンパクト化を図
ったものである。図４においてこの光学センサー部は、
図示を省略した光源として波長７８０ｎｍの半導体レー
ザー装置と、レーザービームを液中で平行光線にするコ
リメートレンズ（第１レンズ）１０、コリメートされた
レーザービームを液中でスポット状に集束させ、かつ、
このスポットの中に存在する菌１１からの散乱光（後方
散乱光）１２を集光する集束レンズ（第２レンズ）１３
と、このレンズで集光されコリメートされた散乱光を、
光軸の所定の同心円上で受光するための複数本の光ファ
イバー１４および光学窓１５から主として構成されてい
る。光ファイバー１４は多芯ケーブル１６により、図示
していない光電変換素子（光電子増倍管）に干渉フィル
ターを介して接合されている。１７はレーザ光用電源か
らのリード線である。受光散乱角度は、第２レンズ１３
の焦点距離および光軸を中心とする円周上に配列した光
ファイバー１４の位置の二つで厳密に決定される。この
ように、本発明の第２の方法を行なう装置では、投光系
と受光系でレンズを共有することによって、光学センサ
ー部をコンパクトにし、受光散乱角の決定を簡略にして
いる。FIG. 4 is a side view showing an optical sensor portion for carrying out the second method of the present invention, and a part thereof is shown in cross section. This optical sensor part is intended to receive only a specific angle component of the scattered light from the suspended particles in the liquid, and, like the optical sensor shown in FIG. 3, the in-line measurement is performed. It is intended to be compact. In FIG. 4, this optical sensor unit is
A semiconductor laser device having a wavelength of 780 nm as a light source (not shown), a collimator lens (first lens) 10 for collimating the laser beam into a parallel beam in the liquid, a collimated laser beam is focused into a spot in the liquid, and
Focusing lens (second lens) 13 that collects scattered light (backscattered light) 12 from bacteria 11 present in this spot
And the scattered light that is condensed and collimated by this lens,
It is mainly composed of a plurality of optical fibers 14 and an optical window 15 for receiving light on a predetermined concentric circle of the optical axis. The optical fiber 14 is joined by a multi-core cable 16 to a photoelectric conversion element (photomultiplier tube) (not shown) via an interference filter. Reference numeral 17 is a lead wire from the power source for laser light. The light receiving / scattering angle is determined by the second lens 13
It is strictly determined by the focal length and the position of the optical fiber 14 arranged on the circumference of the circle centered on the optical axis. As described above, in the apparatus for performing the second method of the present invention, the light projecting system and the light receiving system share the lens, so that the optical sensor unit is made compact and the determination of the received light scattering angle is simplified.
【００１８】[0018]
【発明の効果】以上述べてきたように、本発明の菌数測
定方法の第１の方法は、菌を含む流動する液体中に光を
照射し、透過した光を電気信号に光電変換し、この電気
信号の標準偏差値を電気信号の平均値で割った結果Ａ
と、菌を含まない液を測定した結果を電気信号の平均値
で割ったものを対数変換した結果Ｂとを用いて、演算結
果Ｂを演算結果Ａで割り算した値に所定の定数を乗じた
結果を自乗することにより菌数を測定するようにしたた
めに、菌体の大きさと屈折率によって決まる散乱係数に
依存することなく、しかもセルを流れる液の流量にも依
存せずに菌数を測定することができる。本発明の第２の
方法では、菌を含む流動する液体中に光を照射し、菌体
によって散乱された光を所定の角度の後方から受光して
光電変換し、この電気信号の標準偏差値を電気信号の平
均値で割り算し、所定の定数を乗じ、自乗することによ
り菌数を測定するようにしたため、第１の方法の動作に
加えて、セルや光学窓の汚れ、光源の光強度変動に依存
することなく長期間安定な菌数測定が可能となる。一
方、これら二つの方法に適するように、各部材を組み合
わせて構成した実施例で述べた本発明の装置は、簡単で
安価なものであり、とくに本発明の第２の方法で用いる
光学センサー部は、コンパクトに形成され、正確に後方
散乱光成分を測定することができるという利点を持って
いる。As described above, the first method for measuring the number of bacteria of the present invention is to irradiate light into a flowing liquid containing bacteria and photoelectrically convert the transmitted light into an electric signal. The result of dividing the standard deviation value of this electric signal by the average value of the electric signal A
And the result B obtained by dividing the result of measuring the liquid containing no bacterium by the average value of the electric signal by the logarithmic transformation, the value obtained by dividing the calculation result B by the calculation result A was multiplied by a predetermined constant. Since the number of bacteria is measured by squaring the result, the number of bacteria can be measured without depending on the scattering coefficient determined by the size and refractive index of the cells, and also on the flow rate of the liquid flowing through the cell. can do. In the second method of the present invention, light is irradiated into a flowing liquid containing bacteria, the light scattered by the bacteria is received from the rear of a predetermined angle and photoelectrically converted, and the standard deviation value of this electric signal is obtained. Was divided by the average value of the electric signal, multiplied by a predetermined constant, and the number of bacteria was measured by squaring. Therefore, in addition to the operation of the first method, dirt on the cell or optical window, light intensity of the light source A stable number of bacteria can be measured for a long period of time without depending on fluctuations. On the other hand, the device of the present invention described in the embodiment in which the respective members are combined so as to be suitable for these two methods is simple and inexpensive, and in particular, the optical sensor unit used in the second method of the present invention is used. Has the advantage of being compactly formed and capable of accurately measuring the backscattered light component.
【図１】本発明の第１の方法を実施するための回路ブロ
ック図FIG. 1 is a circuit block diagram for implementing the first method of the present invention.
【図２】本発明の第２の方法を実施するための回路ブロ
ック図FIG. 2 is a circuit block diagram for implementing the second method of the present invention.
【図３】本発明の第１の方法を実施するための光学セン
サーの（ａ）は外観図、同じく（ｂ）は要部断面図FIG. 3A is an external view of the optical sensor for carrying out the first method of the present invention, and FIG.
【図４】本発明の第２の方法を実施するための光学セン
サー部の一部断面を含む側面図FIG. 4 is a side view including a partial cross section of an optical sensor unit for carrying out the second method of the present invention.
１ センサー部 ２ 照射用光ファイバー ３ 受光用光ファイバー ４ 光照射窓 ５ 光路長 ６ センサーホルダー ７ 袋ねじ ８ Ｏリング ９ ファイバーカップリング部 １０ 第１レンズ １１ 菌 １２ 散乱光 １３ 第２レンズ １４ 光ファイバー １５ 光学窓 １６ 多芯ケーブル １７ リード線 1 sensor part 2 irradiation optical fiber 3 light receiving optical fiber 4 light irradiation window 5 optical path length 6 sensor holder 7 bag screw 8 O-ring 9 fiber coupling part 10 first lens 11 bacteria 12 scattered light 13 second lens 14 optical fiber 15 optical window 16 Multi-core cable 17 Lead wire
───────────────────────────────────────────────────── フロントページの続き (72)発明者 財津 靖史 神奈川県川崎市川崎区田辺新田１番１号 富士電機株式会社内 (72)発明者 丹保 憲仁 北海道札幌市北区屯田２条４丁目10−33 (72)発明者 松井佳彦 北海道札幌市東区北16条東13丁目ヒュース 北16Ａ201号 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yasushi Zaitsu, Yasushi Zaitsu, Tanabe Nitta 1-1, Kawasaki-ku, Kawasaki-shi, Kanagawa Fuji Electric Co., Ltd. −33 (72) Inventor Yoshihiko Matsui North 16th East 13th Hughes, Higashi-ku, Sapporo-shi, Hokkaido Hughes North 16A201
Claims (4)
し、透過した光を電気信号に光電変換し、この電気信号
の標準偏差値を前記電気信号の平均値で割った演算結果
Ａと、菌を含まない液体を測定した結果を前記電気信号
の平均値で割ったものを対数変換した演算結果Ｂとを用
いて、前記演算結果Ｂを前記演算結果Ａで割り算した値
に所定の定数を乗じた結果を自乗することにより菌数を
測定することを特徴とする菌数測定方法。1. A calculation result A obtained by irradiating light into a liquid containing bacteria and photoelectrically converting the transmitted light into an electric signal, and dividing a standard deviation value of the electric signal by an average value of the electric signal. And a calculation result B obtained by logarithmically converting the result obtained by measuring the liquid containing no bacteria by the average value of the electric signal, the calculation result B is divided by the calculation result A to a predetermined value. A method for measuring the number of bacteria, which comprises measuring the number of bacteria by squaring the result of multiplication by a constant.
し、菌体によって散乱された光を所定の角度の後方より
受光して電気信号に光電変換し、この電気信号の標準偏
差値を前記電気信号の平均値で割り算し、所定の定数を
乗じ、自乗することにより菌数を測定することを特徴と
する菌数測定方法。2. A liquid containing a bacterium and irradiating with light, illuminating light scattered by the bacterium from a rear of a predetermined angle and photoelectrically converting it into an electric signal, and a standard deviation value of the electric signal. Is divided by the average value of the electric signal, multiplied by a predetermined constant, and then squared to measure the number of bacteria.
る菌数を測定する装置であって、透過光を光量に比例し
た電気信号に変換する光電変換素子と、この光電変換素
子によって得られた電気信号を所定の倍率に増幅する増
幅回路と、増幅した電気信号（Ｖ）の変動成分のみ増幅
する交流増幅回路と、交流増幅された変動信号から実効
値を出力するＲＭＳ／ＤＣ変換器と、ＲＭＳ／ＤＣ変換
器の出力を、前記電気信号（Ｖ）の平均化直流信号で割
り算する第１の割り算器と、前記平均化直流信号の逆数
を対数化する対数変換器と、この対数変換器の出力を前
記第１の割り算器の出力に所定の定数を乗じた値で割り
算する第２の割り算器と、この割り算器の出力を自乗す
る自乗演算器と、この自乗演算器の出力を表示する表示
器からなる電気回路を有することを特徴とする菌数測定
装置。3. A device for measuring the number of bacteria contained in a liquid by the method according to claim 1, which comprises a photoelectric conversion element for converting transmitted light into an electric signal proportional to the amount of light, and a photoelectric conversion element obtained by this photoelectric conversion element. An amplifier circuit for amplifying the electric signal thus obtained to a predetermined magnification, an AC amplifier circuit for amplifying only the fluctuation component of the amplified electric signal (V), and an RMS / DC converter for outputting an effective value from the AC amplified fluctuation signal. A first divider for dividing the output of the RMS / DC converter by the averaged DC signal of the electric signal (V), a logarithmic converter for logarithmizing the reciprocal of the averaged DC signal, and this logarithm A second divider that divides the output of the converter by the output of the first divider multiplied by a predetermined constant, a square calculator that squares the output of this divider, and an output of this square calculator An electric circuit consisting of an indicator that displays Number of bacteria measuring apparatus characterized by having a.
ンサー部は、光軸上の一端に配した発光素子と、この発
光素子からの光線を平行にする第１レンズまたはレンズ
群と、第１レンズまたはレンズ群を通過した光を液中で
一点に集束させる第２レンズまたはレンズ群と、液体と
前記第１，第２レンズまたはレンズ群とを隔てる透明な
光学窓と、前記第２レンズまたはレンズ群の液中におけ
る焦点を中心とする後方散乱光を、前記第２レンズまた
はレンズ群を通して受光する光ファイバー列と、この光
ファイバーの他端面で少なくとも前記発光素子の発する
波長の光を透過させる干渉フィルターと、この干渉フィ
ルターを通過した光を受光して電気信号に変換する光電
変換素子からなることを特徴とする菌数測定装置。4. An optical sensor unit of an apparatus for performing the method according to claim 2, wherein a light emitting element disposed at one end on the optical axis and a first lens or a lens group for collimating light rays from the light emitting element are parallel. A second lens or lens group for converging light that has passed through the first lens or lens group into a single point in the liquid; a transparent optical window separating the liquid from the first and second lenses or lens groups; An optical fiber array that receives backscattered light having a focal point in the liquid of the lens or lens group as the center through the second lens or lens group, and transmits at least light having a wavelength emitted by the light emitting element at the other end surface of the optical fiber. A bacteria count measuring device comprising an interference filter and a photoelectric conversion element that receives light passing through the interference filter and converts the light into an electric signal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4021491A JP2868947B2 (en) | 1992-02-07 | 1992-02-07 | Bacteria count method and device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4021491A JP2868947B2 (en) | 1992-02-07 | 1992-02-07 | Bacteria count method and device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05215666A true JPH05215666A (en) | 1993-08-24 |
| JP2868947B2 JP2868947B2 (en) | 1999-03-10 |
Family
ID=12056447
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4021491A Expired - Fee Related JP2868947B2 (en) | 1992-02-07 | 1992-02-07 | Bacteria count method and device |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2868947B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001041878A (en) * | 1999-07-28 | 2001-02-16 | Meidensha Corp | Calibration cell of turbidimeter |
| JP2004109010A (en) * | 2002-09-19 | 2004-04-08 | Otsuka Denshi Co Ltd | Scattered light measuring device |
| WO2007047382A2 (en) * | 2005-10-12 | 2007-04-26 | California Institute Of Technology | Optoelectronic system for particle detection |
| WO2011152236A1 (en) * | 2010-06-04 | 2011-12-08 | Obata Toru | Gel particle measurement device |
| JP2017198529A (en) * | 2016-04-27 | 2017-11-02 | Ｋｙｂ株式会社 | Fluid state detection system |
-
1992
- 1992-02-07 JP JP4021491A patent/JP2868947B2/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001041878A (en) * | 1999-07-28 | 2001-02-16 | Meidensha Corp | Calibration cell of turbidimeter |
| JP2004109010A (en) * | 2002-09-19 | 2004-04-08 | Otsuka Denshi Co Ltd | Scattered light measuring device |
| WO2007047382A2 (en) * | 2005-10-12 | 2007-04-26 | California Institute Of Technology | Optoelectronic system for particle detection |
| WO2007047382A3 (en) * | 2005-10-12 | 2007-06-07 | California Inst Of Techn | Optoelectronic system for particle detection |
| WO2011152236A1 (en) * | 2010-06-04 | 2011-12-08 | Obata Toru | Gel particle measurement device |
| JP2011257156A (en) * | 2010-06-04 | 2011-12-22 | Kohata Toru | Gell particle measurement device |
| CN102869976A (en) * | 2010-06-04 | 2013-01-09 | 小幡彻 | Gel particle measurement device |
| KR101485233B1 (en) * | 2010-06-04 | 2015-01-22 | 토루 오바타 | Gel particle measurement device |
| US8980180B2 (en) | 2010-06-04 | 2015-03-17 | Toru Obata | Gel particle measurement device |
| EP2579020A4 (en) * | 2010-06-04 | 2017-12-20 | Toru Obata | Gel particle measurement device |
| JP2017198529A (en) * | 2016-04-27 | 2017-11-02 | Ｋｙｂ株式会社 | Fluid state detection system |
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| Publication number | Publication date |
|---|---|
| JP2868947B2 (en) | 1999-03-10 |
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