Process for measuring microbial activity state

Abstract: The invention relates to a process for measuring the state of microbial activities comprising: calling microorganisms into activity in a process or environment in which microorganisms are desired to be active; cultivating the microorganisms in a culture medium; observing a change of chemical substances varied by the then metabolism of the microorganisms; and obtaining the activity state of the microorganisms based on said change.

Description: BACKGROUND OF THE INVENTION This invention relates to a process for measuring the state of microbial activities according to the preamble of claim 1. Activities of microorganisms are extensively used in a wide variety of industrial fields such as brewing of liquors and wines, manufacturing of drugs, restoration of environmental pollution, etc. On the other hand, microorganisms do harm in various fields, such as causing many diseases or putrefying foods. In either case, it is essential to observe whether any microorganism exists or not and if it does exist, how active it is. In general, to carry out the above-mentioned observation, microorganisms are actually applied to the process (for example, the brewing process) and evaluation is made on whether the required effects are obtained or not, or microorganisms are incubated in a suitable culture medium and the results of propagation of microorganisms are determined. Document WO 94/26870 describes, for example, a method for rapid quantification of microorganism growth, wherein changes in specified indicia of colony growth in the early stages of incubation are monitored to provide the early detection and enumeration of colonies in the growth medium. Data from images collected at different times is processed to enhance the detection of subtle changes in the specified indicia of colony growth and provide an early indication of the number of colonies present in the growth medium. Said images are recorded by using a camera. G. B. Williams et al. (Annals of the N. Y. Academy of Sciences 501, 350-353, 1987) show a method for metabolic detection of cell viability employing gel microdroplets (GMDs). This method provides a rapid means for detecting single cells, using E. coli as a model organism. The detection is based on the use of a microscope. SUMMARY OF THE INVENTION This invention is accomplished with the above-mentioned matters taken into account, and it is an object of this invention to provide an alternative method for detecting and quantifying the presence of microorganisms which allows to determine in real time both the positions of microorganisms in a culture and the corresponding activity states. In order to achieve the above-mentioned object, the process for measuring the state of microbial activities according to this invention (hereinafter simply called the "measuring method") is characterized in that the concentration distribution images are obtained by the use of an optical scanning two-dimensional concentration distribution measuring apparatus. In actuality, it has become possible to directly observe the state of microbial activities without waiting the final results by grasping the change in chemical substances near the microorganism caused by metabolisms of the microorganism, when the microorganism is placed under the active conditions, in the form of two-dimensional concentration distribution images. That is, by incorporating the two-dimensional images in real time, either of the following methods can be used to determine the activity state of the microorganisms: (1) a) measuring of a concentration distribution image of at least one of said chemical substances before incubation starts, said concentration distribution image covering the complete environment, b) measuring a plurality of concentration distribution images of said chemical substances in regular time intervals after incubation has been started, said plurality of concentration distribution images covering the complete environment, c) calculating the microbial activity state by comparison between the concentration distribution images measured in step a) and step b). (2) a) measuring of a concentration distribution image of at least one of said chemical substances before incubation starts, said concentration distribution image covering the complete environment, b) starting incubation, c) locating the part of said environment which first of all parts shows a difference of concentration of said chemical substance compared to the concentration measured in step a) by measuring concentration distribution images, d) measuring a plurality of concentration distribution images of said chemical substances in regular time intervals, said plurality of concentration distribution images only covering the part of said environment found in previous step, e) calculating the microbial activity state by comparison between the concentration distribution images measured in step a) and step d). (3) bringing a single microorganism into said evironment, cultivating said microorganism in said environment in order to form a colony, locating said colony by measuring a concentration distribution image and dissolving said colony of microorganisms in a solution and bringing said solution again into a new environment, cultivating said microorganisms in said new environment in order to form a plurality of colonies, counting the number of colonies by measuring a concentration distribution image. Chemical substances varied by the microbial activities are measured using an optical scanning two-dimensional concentration distribution measuring apparatus. One of the optical scanning two-dimensional concentration distribution measuring apparatus is LAPS (Light-Addressable Potentiometric Sensor). The LAPS has recently been developed for two-dimensional measurement of the pH dissolved in the liquid or in the liquid soaked into the substance, and this kind of sensor scans the semiconductor substrate forming a sensor surface reacting to ions, etc. with suitable light and measures the pH by taking out the light current induced in the semiconductor substrate by this scanning as described in Jpn. J. Appl. Phys. Vol. 33 (1994) pp L394-L397, 1994. The distribution of concentration of the dissolved substance is measured by inserting the sensor surface of the said equipment into the object to be directly measured or bringing it into contact with the object. The data obtained is computer-processed and outputted as a two-dimensional or three-dimensional concentration distribution image. It can trace not only the concentration distribution at a specified time but also the condition of changes in real time. The images obtained in real time can easily be compared with electromagnetic wave images obtained visually or by a CCD camera. Examples of the above-mentioned chemical substances include various inorganic ions, organic ions, organic substances, substances related to enzyme reactions, substances related to antigen-antibody reaction, and substances related to inheritance, which dissolve in the liquid and are able to be measured by properly modifying the above-mentioned optical scanning two-dimensional concentration distribution measuring apparatus surface. In addition, it is possible to compare the said two-dimensional concentration distribution image with the electromagnetic wave image and determine the correlationship between the concentration distribution of the chemical substance caused by the microorganism and the actual existing position of the microorganism. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation showing one example of the optical scanning two-dimensional concentration distribution measuring apparatus used in the measuring process according to this invention. FIG. 2 is a view to illustrate the first embodiment. FIG. 3 is a view to illustrate the operation. FIG. 4 is a view to illustrate the second embodiment. FIG. 5 is a view to illustrate the third embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to the drawings, the present invention will be described in detail hereinafter. (Embodiment 1) Referring now FIG. 1 and FIG. 2, an example in which the measuring process according to this invention is applied to the food manufacturing process such as fermentation and brewing is described. In the process in which microbial capabilities are utilized, the microorganism best suited for the process must be chosen (screened), and the best suited microorganism can be determined by applying the microorganism to the actual process and observing the activity state by the method described below. In this event, the substance changed by the metabolism of microorganism is the hydrogen ion, and an optical scanning two-dimensional concentration distribution measuring apparatus which is sensitive to pH is used for measurement. That is, FIG. 1 schematically illustrates an optical scanning two-dimensional concentration distribution measuring apparatus used for implementing the measuring process according to this invention. In FIG. 1, numeral 1 is a sensor portion of the optical scanning two-dimensional concentration distribution measuring apparatus, and this sensor portion 1 comprises SiO 2 layer 3 and Si 3 N 4 layer 4 successively formed on one surface (top surface in the illustrated example) of the substrate 2 which comprises a semiconductor such as silicon, etc. by thermal oxidation, CVD, or other technique and is formed to respond to hydrogen ions. And CE and RE are a counter electrode and a reference electrode, respectively, installed above the Si 3 N 4 layer 4, which is the sensor surface, and are connected to a stabilized bias circuit 10 later discussed. OC is an ohmic electrode installed on the semiconductor substrate 2 for taking out current signals, and is connected to the stabilized bias circuit 10 via a current-voltage converter 11 and an operational amplifier circuit 12 later discussed. The elements 10, 11 and 12 are members of a potentiostat 18. Numeral 5 is a light irradiating portion for irradiating the semiconductor substrate 2 of the said sensor portion 1 and is installed on the bottom surface side (opposite to the sensor surface 4) of the semiconductor substrate 2, and comprises a laser beam source 7 for intermittently emitting the light adjusted to provide an optimum beam diameter by control signals from a computer 15 later discussed via an interface board 13 later discussed as probe light 6 and a scanning controller 8 for controlling the laser beam source 7 to scan in the two-dimensional directions (X and Y directions in the figure). Numeral 9 is a control box for controlling the said sensor portion 1 and the light irradiating portion 5 and comprises the stabilized bias circuit 10 for applying suitable bias voltage to the semiconductor substrate 2, the current-voltage converter 11 for converting current signals taken from the ohmic electrode OC formed on the semiconductor substrate 2 to voltage signals, the operational amplifier circuit 12 to which signals from this current-voltage converter 11 are inputted, and the interface board 13 for exchanging signals with this operational amplifier circuit 12 or outputting control signals to the scanning controller 8. Numeral 14 is the computer as a control and a processor for carrying out various controls and computations, which is equipped with image processing capabilities, and numeral 15 is a display for displaying the pH concentration distribution. This optical scanning two-dimensional concentration distribution measuring apparatus is not at all different from the one shown in the said Japanese Patent Application No. Hei 7-39114. The sample used in this measuring process according to this invention is formed by a generally used method and, for example, a sample of agar culture medium is formed. This method is described, for example, in the microbial inspection (Encyclopedia of Clinical Inspection Technique 7, published by Igaku Shoin). For an applicable process, as shown in FIG. 2 (A), agar 23 is added to a solution 22 adjusted to the conditions suited for microbial activities in a suitable container 21, is housed in a Petri dish 24 and, is designated as ge125. On the other hand, as shown in (B) of the same drawing, the microorganism B to be evaluated in the test tube 26 is diluted with a suitable diluent 27, and this diluted microorganism B is applied to the surface of the gel 25 housed in the said Petri dish 24 to have a sample 28. As shown in FIG. 2 (C), sample 28 is turned upside down and placed on the sensor surface 4 of the optical scanning sensor portion 1 of two-dimensional concentration distribution measuring apparatus, the environmental temperature is properly set, and incubation of microorganism B takes place. In the above optical scanning two-dimensional concentration distribution measuring apparatus, the counter electrode CE and the reference electrode RE are inserted into the gel 25 of the sample 28 and direct current voltage from the stabilized bias circuit 10 is applied across the reference electrode RE and the ohmic electrode OC so as to generate a depletion layer in the semiconductor substrate 2, and a specified bias voltage is applied to the semiconductor substrate 2. Irradiating the semiconductor substrate 2 with the probe light 6 intermittently at given intervals (for example, 10 kHz), alternate current optical current is generated in the semiconductor substrate 2. In addition, by moving the light irradiating portion 7 in X or Y direction, the semiconductor substrate 2 is irradiated with the probe light 6 as if it is scanned in the two-dimensional direction and a two-dimensional image 16 indicating pH is displayed on the screen of the display 15 of the computer 14 by the positional signal (X, Y) at the sample 28 and the alternate current optical current value observed at the position. Then, the pH value at the incubation start point is recorded (in this event, at Sample 28, pH distribution is not observed) and incubation takes place. The place where the first pH value change occurs after the start of incubation is designated as the pH measuring point, and the pH value at the measuring point is recorded at regular intervals, and the difference of pH value at the start of incubation and after incubation takes place is measured and recorded at regular intervals. The difference of the pH values tend to increase logarithmically with respect to time. FIG. 3 shows the relationship between the pH value and the time in this event, with time taken as abscissa and the pH value as ordinate (displayed in logarithmic scales), respectively. Let the time till then be T b and the logarithmic value of the increment per unit time of the pH value when the pH value increases logarithmically with respect to time be S, these values T b and S are indices 17 of the activity state (see FIG. 1). That is, when several species of microorganism B are screened with the activity conditions of microorganism B set constant, the microorganism with smaller T b and larger S provides capabilities to more efficiently carry out the intended process. The above-mentioned process is an example in which the activity conditions of microorganism B are set constant and activities of a plurality of microorganism B species are evaluated for screening, but the same technique can be used when the optimum conditions are determined to carry out the process with respect to microorganism B chosen by screening. However, in this event, T b and S should be compared, respectively, with the specie of microorganism set constant while gel sample conditions and process conditions are varied. In the above-mentioned example, there is a case in which the pH value change first occurs in a plurality of points. In this event, an optional point may be designated as a pH value change observation point and several or all points of them may be designated as pH value change observation points. Which to adopt must be determined and the observation points should be set constant in a series of screening or investigation of activity conditions. The above-mentioned embodiment is an example in which the present invention is applied to a food manufacturing process such as fermentation and brewing, but it is needless to say that it can be applied to other processes which utilize capabilities of microorganism B. (Embodiment 2) In this embodiment, as an index of the activity state of microorganism B, the ratio of increase in population of microorganism B is used. Same as in the case of Embodiment 1, incubation of microorganism B is allowed to take place and two-dimensional concentration distribution images of chemical substance discharged are successively obtained. The chemical substance measured is the hydrogen ion same as that in Embodiment 1 but may be any other chemical substances. Microorganism B increases population as incubation takes place for a specified time and forms a colony 29 in which several individuals (microorganism B) aggregate as shown in FIG. 4 (A). Because around this colony 29, the pH value differs from that at the start of incubation, this can be easily identified by observing the two-dimensional concentration distribution images. As described above, colony 29 is formed in a plurality, and therefore, one each of them is cut for each gel 25 as shown in the conceptual line in FIG. 4 (A) at regular intervals. The cut colony 29 is dissolved in the culture liquid 31 in the test tube 30 and is isolated into one individual microorganism B as shown in (B) of the same drawing, and again in the method described above, it is fixed to the sensor surface 4 of the optical scanning sensor portion 1 of two-dimensional concentration distribution measuring apparatus and incubated. Each of the isolated microorganisms B repeats multiplication and forms colonies. Because around the colony, the pH value differs from that at the start of incubation, it can be easily identified by observing the two-dimensional concentration distribution images and this time, the total number of the colonies can be determined. This total number of colonies corresponds with the number of microorganisms B in the colony cut together with the gel before and indicates the number of individuals multiplied since it is cut. Now, it is possible to know how one individual of microorganism increases the population as time passes by carrying out the initial microorganism incubation and cutting of the colony at regular intervals and determining the number of microorganisms in the colony. The increase of population becomes logarithmic after a specified time (for example, T b '), and letting the logarithmic value of the then-increment be S', the microorganism with smaller T b ' and larger S' can be regarded as the microorganism which provides capabilities to efficiently carry out the intended process, and therefore, using these values, screening and optimum activity conditions of microorganism B can be obtained. (Embodiment 3) In this embodiment, description is made on an application example in which soils contaminated by crude oil leaking from tankers, petroleum plants, etc. due to accidents or other petroleum waste are decontaminated with the microbial power. The said contamination varies in forms and requires more suitable screening of microorganism B and quick determination of the conditions in which the microorganism B is allowed to be active to the maximum. The specific application method is described as follows. In this embodiment, the measured chemical substance is also the hydrogen ion, but other various types of ion species and organic substances may be acceptable. For the measuring equipment, an optical scanning two-dimensional concentration distribution measuring apparatus which is induction-reactive to hydrogen ions is used, but it is needless to say that the optical scanning two-dimensional concentration distribution measuring apparatus with the sensor surface 4 modified with suitable induction-reacting substances in accord with chemical substances to be measured is used. In this embodiment, as shown in FIG. 5 (A), the sensor surface 4 is divided into several measuring zones 4a, 4b, 4c, 4d, ..., 4n so that several types of candidate microorganisms B can be simultaneously evaluated, and in relevant measuring zones 4a-4n, different microorganisms B 1 , B 2 , B 3 , B 4 , ..., B n (not illustrated) are incubated, and their activity state is observed. After each of microorganisms B 1 -B n is diluted with a suitable solution, it is applie d to each of measuring zones 4a-4n and thereafter, placing the soil 32 to be decontaminated on each zone as shown in (B) of the same drawing can fix relevant microorganisms B 1 -B n . Measurement after this should be carried out in the same manner as in the case of Embodiment 1. The measurement can be made in the same manner not only in screening of optimum microorganism B but also in the process of determining conditions in which the screened microorganism B can be in active to the maximum. However, in this event, same microorganism B should be applied to each of the measuring zones 4a-4n, and nutrients, etc. added to each of the measuring zones 4a-4n should be properly varied. In stead of placing the soil 32 to be decontaminated on the sensor surface 4, screening is possible by directly embedding the sensor portion 1 with each of microorganism B 1 -B n applied into the soil to be decontaminated. (Embodiment 4) All of the above-mentioned embodiments describe the case in which the chemical substance to be changed by the metabolism of microorganism B is the hydrogen ion, but in place of this, the change of, for example, various inorganic ions such as calcium ions, etc., organic acids such as lactic acid, etc., and other various chemical substances can also be observed. In this event, the sensor surface 4 of the sensor portion 1 of the optical scanning two-dimensional concentration distribution measuring apparatus should be modified with the substance which responds selectively to the substance observed. (Embodiment 5) In this embodiment, there described is the case in which the existence of harmful microorganisms is found. The sample used in this method is a generally used one, and for example, a sample of agar culture medium is formed. This method is described, for example, in the microbial inspection (Encyclopedia of Clinical Inspection Technique 7, published by Igaku Shoin). That is, as in the case of Embodiment 1, the change in the chemical substance is observed as two-dimensional concentration distribution images at regular intervals as soon as incubation begins to take place. Examples of the chemical substance to be measured include any chemical substances including hydrogen ions. If any microorganism exists, continuing incubation for a specified time allows the microorganism to increase the population and form colonies in which several individuals aggregate. Because the concentration of chemical substance differs around this colony from that at the start of incubation, the change in the chemical substance can quickly and easily be identified by two-dimensional concentration distribution images. With the above-mentioned two-dimensional concentration distribution images, it is not only possible to confirm the existence of microorganism but also to identify the portion of interest, such as at which portion on the culture medium the microorganism exists, and in addition, in which portion of the microorganism the change in concentration of chemical substance is generated, that is, to identify the correlationship between chemical information and physical information, based on the actual images (electromagnetic wave images) obtained visually or by CCD camera. In particular, the calcium ions or lactic acid ions are substances frequently found in the vital reaction, and the measuring process according to this invention can be applied not only to the process effectively utilizing the microorganism B but also to researches on vital reactions, etc. Additionaly, in the above-mentioned optical scanning two-dimensional concentration distribution measuring apparatus, probe light 6 is designed to be irradiated from the rear surface side (opposite to the sensor surface 4) of the semiconductor substrate 2, but in lieu of this, it may be designed to be irradiated from the sensor surface 4 side. As described above, the process of this invention enables easy, quick, and real-time evaluation of the activity state of the microorganism, such as whether the microorganism exists or not, and if it does exist, how active it would be. Consequently, it is possible to quickly and easily carry out screening of optimum microorganism in various processes in which microorganisms are utilized as well as to determine optimum activity conditions of the microorganism. This also enables quick determination of existence of harmful microorganism.

Claims: A process for measuring the state of microbial activities comprising the steps of: a) bringing microorganisms into an environment in which said microorganisms are desired to be active, said environment containing a composition of chemical substances, b) cultivating said microorganisms in said environment, c) observing a change in said composition of chemical substances caused by a metabolism of said microorganisms, said change being observed by two-dimensional concentration distribution images of said chemical substances, d) using said concentration distribution images to determine the activity state of the microorganisms, characterized in that said concentration distribution images are obtained by the use of an optical scanning two-dimensional concentration distribution measuring apparatus. The process as claimed in claim 1, characterized in that said concentration distribution images are concentration distribution images of the pH-value. The process as claimed in claim 1 or 2, characterized in that the determination of the activity state in step d) is achieved by the following steps: e) measuring of a concentration distribution image of at least one of said chemical substances before incubation starts, said concentration distribution image covering the complete environment, f) measuring a plurality of concentration distribution images of said chemical substances in regular time intervals after incubation has been started, said plurality of concentration distribution images covering the complete environment, g) calculating the microbial activity state by comparison between the concentration distribution images measured in step e) and step f). The process as claimed in claim 1 or 2, characterized in that the determination of the activity state in step d) is achieved by the following steps: h) measuring of a concentration distribution image of at least one of said chemical substances before incubation starts, said concentration distribution image covering the complete environment, i) starting incubation, k) locating the part of said environment which first of all parts shows a difference of concentration of said chemical substance compared to the concentration measured in step h) by measuring concentration distribution images, l) measuring a plurality of concentration distribution images of said chemical substances in regular time intervals, said plurality of concentration distribution images only covering the part of said environment found in previous step. m) calculating the microbial activity state by comparison between the concentration distribution images measured in step h) and step 1). The process as claimed in claim 1 or 2, characterized in that the determination of the activity state in step d) is achieved by the following steps: n) bringing a single microorganism into said environment, o) cultivating said microorganism in said environment in order to form a colony, p) locating said colony by measuring a concentration distribution image and dissolving said colony of microorganisms in a solution and bringing said solution again into a new environment, q) cultivating said microorganisms in said new environment in order to form a plurality of colonies, r) counting the number of colonies by measuring a concentration distribution image. The process as claimed in one of claims 3 to 5, characterized in that the comparison between the concentration distribution images is numerically done by a computer unit by subtraction of the concentration values of the corresponding concentration distribution images from each other. The process as claimed in one of the preceeding claims, characterized in that a sensor surface of the optical scanning two-dimensional concentration distribution measuring apparatus is divided into a plurality of measuring zones, each zone corresponding to a different concentration distribution image.

Inventors: AKANO HIROFUMI, FUKUSHIGE TOMOAKI, IWASAKI HIROSHI, NOMURA SATOSHI, TOMITA KATSUHIKO, AKANO, HIROFUMI, FUKUSHIGE, TOMOAKI, IWASAKI, HIROSHI, NOMURA, SATOSHI, TOMITA, KATSUHIKO
Applicants: HORIBA LTD, HORIBA, LTD.
Application year: 1996
Main classification: C12Q1
IPC codes: C12Q1