JP5774352B2 - A non-contact method for continuous measurement of sample growth during culture - Google Patents

Description

  The present invention relates to a method for continuously measuring the growth of a sample in culture without contact.

  Conventionally, in the field of culturing microorganisms and the like, as a method for measuring the growth of samples (microorganisms and cells) to be cultured with shaking, the optical density (OD) of the culture solution has been adopted as an index for increasing the number of cells. The method is as shown in FIG. 7, in which a sample solution cultured in a measuring cell of a certain size (10 mm square) is collected and placed, and light (600 nm light) is irradiated from the outside in a dark place to control the amount of transmitted light. A measuring method using a spectrophotometer to measure is performed.

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  However, the measurement method using the spectrophotometer described above is not only complicated, because shaking culture must be stopped, a part of the culture sample must be taken out and transferred to a dedicated measurement cell. There is a problem that the growth state of the sample cannot be measured in real time.

  In addition to a decrease in the number of samples in culture each time it is measured, there is a problem that contamination due to miscellaneous bacteria may occur due to work.

The present invention is stretched and spread to a thickness having a thin constant fluctuation on the inner surface of the culture vessel by shaking the centrifugal pressure or the sample liquid swirl shaking in culture reciprocal shaking operation in culture vessel path which transmits light Adjust the length to be short so that light can be transmitted, and irradiate the sample liquid with infrared light that can measure the amount of transmitted light or reflected light from the outside of the container under visible light in the room . The amount of transmitted light and / or reflected light due to the increase in optical density due to growth is measured and collated with the optical density model data, the difference in the size and shape of the culture vessel, and the amount and viscosity of the culture sample solution. The initial reflection mainly consists of the reflected light from the surface of the culture vessel and the scattered light from the culture sample liquid surface and bubbles based on the shaking method, the amplitude, the slowness of the shaking speed, the magnitude of the shaking angle, and the temperature. Initially measure the light intensity in advance To reflect calculated by conversion formula determined in, and when measuring the transmitted light quantity changes in optical density than the measured value of both the variation in the amount of reflected light, the measurement value of the transmission light amount to more initial measurement of the scattered light due to the air bubbles Thus, the measurement value based on the transmitted light amount is used to correct the measurement value based on the reflected light amount so that the growth of the sample in culture can be continuously measured, thereby solving such a problem.

  The present invention measures the optical density (OD) corresponding to the growth of a culture sample in real time during culture shaking, so that the work involved in the process of sampling a sample, setting to a spectrophotometer, and measuring is conventionally performed. This reduces the burden of the culture, eliminates the decrease in the culture sample, adversely affects the suspension of the culture (depletion of dissolved oxygen necessary for the culture, etc.), and further reduces the risk of contamination.

The combined use of both the reflected light amount and the transmitted light amount measuring method brings about an effect that it is possible to accurately measure from the initial measurement to the optical density (OD) in the high density region.
It is expected to contribute to the selection of highly proliferating strains from many specimens and the selection of antibiotic resistant bacteria.

  By using infrared light as the irradiation light for measurement, the optical density can be measured during normal shaking culture under visible light indoors.

  By raising the position of the light irradiation position and the reflected light amount measuring sensor and measuring at a position where the thickness of the culture sample solution is thin, it is possible to measure even higher optical density.

  By automating the culture solution to a constant concentration by using the foot-back control of the measured value, there is an effect that continuous measurement over a long time can be made possible.

Explanatory drawing which shows the example of the implementation apparatus of the method of 1st Example Explanatory drawing which shows the example of the implementation apparatus of the method of 2nd Example Explanatory drawing which shows the example of the implementation apparatus of the method of 3rd Example Explanatory drawing which shows the example of the implementation apparatus of the method of 4th Example Explanatory drawing which shows the example of the implementation apparatus of the method of 5th Example Explanatory drawing which shows the example of the implementation apparatus of the method of 6th Example Illustration of measurement with a conventional spectrophotometer

  The present invention irradiates a sample liquid during shaking culture with light from the outside, and changes in the amount of transmitted light and / or reflected light due to an increase in the optical density accompanying the growth of the sample liquid are measured in the size of the culture vessel. Model data of optical density required based on difference in shape and shape, degree of viscosity of culture sample solution, degree of viscosity, shaking method, amplitude amplitude, slow shaking speed, shaking angle magnitude, temperature level Thus, the growth of the culture sample was continuously measured in a non-contact manner while shaking.

Embodiments will be described below with reference to the drawings.
FIG. 1 shows an example of an apparatus for carrying out the first embodiment of the present invention. The apparatus has a main body 1 having an operation unit and a calculation unit inside for performing control and input / output; LED which is connected and is equipped with a shaking table 2 on which a small Erlenmeyer flask A containing a culture sample solution is placed and swirled and irradiates infrared light (900 nm) with the Erlenmeyer flask A placed therebetween The light measurement unit 3 includes a light irradiation unit 3a and a light receiving sensor unit 3b. The calculation unit of the main body 1 stores optical density model data (concentration-transmitted light amount curve) for calculating the optical density (OD) change due to the growth of the culture sample by checking the numerical value of the difference between the irradiated light amount and the transmitted light amount. I am letting.

  In order to measure the growth of the sample due to the culture, for example, the sample solution a of the E. coli culture is put in the Erlenmeyer flask A as a sample and placed on the shaking table 2 and the concentration change accompanying the growth of E. coli while swirling. Therefore, the numerical value of the difference between the irradiated light and the transmitted light, that is, the optical density (OD) is collated with the optical density model data and calculated to measure the progress of proliferation. The growth of the culture sample can be continuously measured in real time without contact from the outside without stopping during the shaking culture.

  As shown in Table 1, the Erlenmeyer flask A has an optical path length that prevents light from passing through when the concentration of the culture solution increases from the size of its width. The sample liquid a is thinly spread on the inner wall surface of the flask A to a thickness having a certain fluctuation, so that the substantial optical path length is shortened and light can be transmitted. Therefore, it was possible to perform measurement while shaking culture using the Erlenmeyer flask A having a size much larger than the measurement cell of the conventional spectrophotometer.

  The sample solution a of E. coli culture contained in the Erlenmeyer flask A is swirled and spread on the inner wall surface of the Erlenmeyer flask A to a thickness with a certain fluctuation, so that the irradiation light is distributed near the inner center. Because the light passes through, the optical path length changes depending on the slow rotation speed, the amount of liquid, the degree of viscosity, and light scattering occurs due to the reflection and shaking of the irradiated light when it enters and exits. Even if the amount of transmitted light is measured, the value does not reflect the concentration. However, if the size of the Erlenmeyer flask A (= liquid amount), the magnitude of the amplitude, and the shaking speed are constant, it is stable. Focusing on the fact that the optical path length and the light scattering state fall within a certain range, and in this state the Lambert-Beer law that the logarithm of the reciprocal of the amount of transmitted light is proportional to the concentration of the substance

As a result, the amount of transmitted light (photosensor voltage) was measured while shaking the E. coli culture solutions of various concentrations (optical density), and it was confirmed that the fluctuation was within a certain range. Calculate the representative value of the transmitted light amount in the same state from the measured value of tens of thousands of times, obtain optical density model data (concentration-transmitted light curve) that decreases the transmitted light amount according to the concentration accompanying the growth of the sample, Optical density model data under various conditions of size, shape, liquid amount, viscosity, rotation speed, and temperature were obtained, stored in the calculation unit, and compared with the measured value for calculation.

  Table 2 shows the correlation between the cell concentration and the sensor voltage according to the method of the present application. A dilution series of the culture broth was prepared using Escherichia coli JM109 strain, and the measurement voltage under various shaking conditions was examined. As a result, it was confirmed that there is a continuous relationship in the range of optical density (OD) of 0.1 to 2.0. From this measurement result, the decrease rate of the sensor voltage is changed to the optical density (OD). Model data (conversion formula) to be converted was obtained.

  Table 3 shows growth monitored using Escherichia coli JM109 strain. From this, a time course of optical density (OD) corresponding to the value measured by the spectrophotometer could be obtained.

  Table 4 shows the optical density (OD) measured in Escherichia coli, yeast, Bacillus subtilis, Agrobacterium, bifidobacteria by the method of the present application. Although some cells with different sizes require correction, the optical density (OD) could be measured in a wide range of species including anaerobic cells.

Thus, by selecting the optical density model data under appropriate shaking conditions and calculating the optical density (OD) based on the decrease rate of the transmitted light amount from the initial transmitted light amount at the start of culture, While shaking the growth, it can be continuously measured without contact from the outside of the container.
The arithmetic unit of the main body 1 stores approximate expressions of model data (concentration-transmitted light amount curve) of various optical densities obtained for each shaking condition, so that the concentration (= optical density, OD) of an unknown sample is stored. ) Can also be calculated by inputting the transmitted light quantity into an approximate expression.

  In the case of a sample having optical characteristics that are significantly different from those of Escherichia coli, an error may occur in the measurement value comparison with the spectrophotometer, but the error can be reduced to about ± 15% by a correction function. In addition, when using an LED as the light source of irradiation light, the light quantity may vary due to the characteristics of the LED, but it is automatic by referring to the rate of change of the light quantity during measurement based on the transmitted light quantity at the start of measurement. Can be corrected automatically.

  By using infrared light (900 nm) as the irradiation light for measurement, it is possible to perform measurement during normal shaking culture under visible light indoors without requiring measurement in a conventional dark place.

Table 5 shows E. coli HB101 strain diluted to OD ₆₀₀ = 1.0 and its scattering spectrum measured. OD ₉₀₀ was about 0.6 times that of OD ₆₀₀ , but the molar extinction coefficient is a constant independent of the cell concentration, so the wavelength difference can be corrected by multiplying by the ratio coefficient. .

  FIG. 2 shows an example of an apparatus that uses a test tube B as a culture container. As in the previous example, a light irradiation unit 3a and a light receiving sensor unit for irradiating light with the test tube B containing a main body 1 and a sample to be cultured in between. The measuring part 2 which consists of 3b is provided, and the shaking stand 4 which supports the test tube B in the inclined state and performs reciprocating shaking is provided.

  The optical path length of the liquid in the test tube B in the reciprocating shaking state is shorter as the inclination of the test tube is closer to the horizontal and the shaking speed is faster, and becomes longer as the shaking speed is closer to the vertical and the shaking speed is slower (see FIG. 2). ). In addition to the optical path length, various factors such as light scattering on the liquid surface and the amount of bubbles generated in the culture solution will occur, but with stable shaking, the difference in test tube size, The optical path length and light scattering fluctuate within a certain range due to the amount of liquid, the viscosity of the liquid, the optical density and refractive index of the liquid, the magnitude of the inclination of the test tube, the magnitude of the amplitude, and the slowness of the shaking speed. Will be decided.

  In the same way as the previous example, under the condition that the test tube size, liquid volume, shaking angle, amplitude, and shaking speed are constant, the optical path length and light scattering state are within a certain range. When the amount of transmitted light (light-receiving sensor voltage) was measured while shaking the E. coli culture, it was confirmed that the fluctuation was actually within a certain range, and even in this case, it was proved that the Lambert-Beer law was established. . Furthermore, a representative value of transmitted light was obtained from tens of thousands of actual measurement values under various conditions, an optical density model data group in which the amount of transmitted light decreased according to the concentration was obtained, and stored in the arithmetic unit of the main body 1 is there.

  Table 6 shows the results obtained by measuring Escherichia coli while culturing it by the method of this example, and comparing it with the optical density (OD) value measured with a spectrophotometer after being sampled many times in the middle. It was confirmed that both measured values almost coincided with time.

  Table 7 shows a series of dilutions of the E. coli culture solution, and compares the values measured by the method of Example and the spectrophotometer. It was confirmed that the measured value obtained by the method of the example coincided with the measured value when diluted with the spectrophotometer.

  Table 8 shows the results of measuring the optical density (OD) in Escherichia coli, yeast, and Bacillus subtilis by the method of the second embodiment, and confirms that the optical density (OD) can be measured in a wide range of biological species. did.

  Since the arithmetic unit of the main body 1 stores approximate expressions of model data (concentration to transmitted light amount curve) of various optical densities, the density (= optical density, OD) of an unknown sample also approximates the transmitted light amount. It is the same as in the previous example that it can be calculated by inputting to. However, in shaking culture in test tube B, it was confirmed that it took 30 to 60 minutes to stabilize the initial transmitted light amount under shaking conditions with a large stirring effect. The measurement of density (OD) is started.

  FIG. 3 shows an example of an apparatus that uses a large Erlenmeyer flask C as a culture container. The optical sensor 3c for measuring the amount of reflected light is provided on the light irradiating part 3a side of the measuring part 3 according to the first embodiment. Same as the configuration. In this embodiment, the optical density (OD) is measured by adding the correction of the transmitted light amount around the change in the reflected light amount so that a long optical path length and a high density optical density (OD) can be measured. is there. As shown in Table 9, it can be seen that the amount of transmitted light decreases and the amount of reflected light increases as the optical density (OD) increases as the culture time elapses.

  Similar to the first embodiment, the representative values of the reflected light amount are obtained from tens of thousands of actually measured values while shaking for various concentrations of Escherichia coli culture solution, and the optical density at which the reflected light amount increases according to the concentration accompanying the growth of the sample. Model data (conversion formula of concentration-light curve), and the calculation unit of optical density conversion formula group under various conditions with different amounts of liquid, viscosity, rotation speed, and temperature of Escherichia coli culture I remembered it. Then, the optical density (OD) can be calculated from the change in the amount of reflected light through the corresponding conversion formula.

  Table 10 shows the results obtained by measuring Escherichia coli while culturing it by the method of this example, and comparing it with the optical density (OD) value measured with a spectrophotometer after sampling many times in the middle. It was confirmed that both measured values almost coincided with time.

  Table 11 shows a series of dilutions of the E. coli culture solution, and compares the values measured by the method of Example and the spectrophotometer. In general, a spectrophotometer requires dilution of a culture solution when measuring a high concentration optical density (OD), but the measurement value according to this example is the same as the measurement value when the spectrophotometer is diluted to reach a high concentration range. Matched.

  Shake the growth of the culture sample by selecting the optical density model data under the corresponding shaking conditions and calculating the optical density (OD) based on the rate of increase in the reflected light amount from the initial reflected light amount at the start of culture. It can be continuously measured without contact.

  Note that when the optical density (OD) is measured by the amount of reflected light, the reflected light from the surface of the culture vessel and the scattered light from the sample liquid surface and bubbles are mainly used at the initial stage of the culture. Although the sensitivity and accuracy of the optical density are slightly inferior to those of the transmitted light measurement method, the ratio of scattered light from the cells increases due to sample growth, and the culture concentration (optical density) increases significantly. However, it is advantageous for measurement in a large culture vessel for carrying out a large amount of permeation culture.

  In order to improve that the reflected light measurement method is inferior to the transmitted light measurement method in the initial state, the transmission method should be combined only in the initial measurement in the measurement method mainly using the reflection method. You can also. In this way, the low sensitivity and accuracy at low density, which is a disadvantage of the reflection method, can be compensated, and the calculation formula from the ratio of the initial reflected light and the amount of reflected light during cell proliferation can be corrected.

  FIG. 4 shows an embodiment in which the light irradiation unit 3a and the optical sensor unit 3c for measurement irradiation light are adjusted to a high position, that is, a portion where the thickness of the culture solution becomes thin during swirling shaking. By measuring the position where the thickness is thin, the density decreases compared to the measurement at the thick position, and measurement up to an optical density (OD) higher than usual becomes possible. According to the verification, when OD = 6 to 7 which can be measured at a low position, the optical density up to a maximum OD = 20 can be measured at a high position.

  In addition, many bubbles are generated during culture using baffle flasks or shake flasks (not shown) that improve shaking performance by combining measurement value averaging and minimum value acquisition during light intensity measurement. The optical density (OD) can be measured even under the shaking conditions.

  FIG. 5 shows an example of an implementation apparatus of a method in which a display device 5, a printing device 6, and a recording device 7 are connected to the main body 1, and the progress / results of measurement are displayed in real time, printed, and further recorded. Is. Note that these devices can be connected individually, but can also be used with other devices through a personal computer.

  FIG. 6 shows an embodiment of a method for automating the maintenance of a constant culture concentration by connecting a medium supply pump 8 and a medium solution discharge pump 9 operating on the basis of measured value feedback to the main body. is there. In this way, the burden of work during measurement can be reduced, and measurement over a long period of time can be performed.

  The present invention reduces the work burden, does not reduce the culture sample, and externally increases the optical density (OD) corresponding to the growth of the culture sample while reducing the adverse effects of the suspension of culture and the risk of contamination as much as possible. Since it can be measured in real time during culture shaking without contact, it can be widely used to improve production efficiency by applying to the management of production conditions as well as the acquisition of experimental data.

1 is a main body 2 is a shaking table 3 is a measuring unit 3a is a light irradiation unit 3b is a light receiving sensor unit 3c is a light sensor unit 4 is a reciprocating shaking table 5 is a display device 6 is a printing device 7 is a recording device 8 is for supplying a medium 9 is a pump for discharging the medium solution A is a small Erlenmeyer flask B is a test tube C is a large Erlenmeyer flask a is a sample solution

Claims (5)

Optical light path length which transmits the light to extend spread thinly thickness having a certain fluctuation in the inner surface of the culture vessel by shaking the centrifugal pressure or the sample liquid swirl shaking in culture reciprocal shaking operation in culture vessel The sample solution is propagated by irradiating it with infrared light that enables measurement of the transmitted light amount or reflected light amount from the outside of the container under visible light in the room from the outside of the container. Changes in the amount of transmitted light and / or reflected light due to an increase in optical density are measured and collated with optical density model data. Differences in the size and shape of the culture vessel, the volume and viscosity of the culture sample solution, and shaking. Based on the method, the amplitude, the slowness of the shaking speed, the magnitude of the shaking angle, and the temperature, the initial reflected light amount mainly composed of the reflected light from the culture vessel surface and the scattered light from the culture sample liquid surface and bubbles is preliminarily determined. Measured to reflect the initial value It was then calculated by conversion formula determined, and used when measuring the amount of transmitted light changes in optical density than the measured value of both the variation in the amount of reflected light, the measurement of the transmitted light amount to more initial measurement of the scattered light due to the air bubbles The measurement value based on the amount of transmitted light is used to correct the measurement value based on the amount of reflected light so that the growth of the sample in culture can be continuously measured. Method of continuous measurement with.   2. The growth of the sample during culture according to claim 1, wherein the change in the initial light amount value is corrected by reflecting the change in the transmitted light amount or the reflected light amount due to bubbles generated in the initial stage of shaking culture of the sample liquid in the initial light amount value. A method of continuous measurement by contact. In measuring the change in reflected light quantity, the growth of the sample in the culture of claim 1, the position of the measuring sensor and the irradiation position of the light amount of reflected light the thickness of the culture sample solution is adjusted to as much as possible thin portion A non-contact continuous measurement method. The non-contact growth of the sample during culture according to any one of claims 1 to 3 , wherein averaging of the measurement value and acquisition of the minimum value are combined at the time of light quantity measurement under shaking conditions containing a lot of bubbles in the baffle flask and the shake flask. Method of continuous measurement with. The growth of the sample during culture according to any one of claims 1 to 4 , wherein the supply of the medium solution or the discharge of the medium solution is automated by feedback of the measurement process or the measured value of the result to maintain the culture solution at a constant concentration. A non-contact continuous measurement method.