JP3788478B1 - Microbe count measuring method and microbe count measuring apparatus - Google Patents

Abstract

The present invention proposes a method for early evaluation of the number of microorganisms based on oxygen current.
In step S100, various settings used in processing according to the flowchart are reset. In step S101, the oxygen current Inew is measured. In step SA, the slope of the oxygen current is obtained. In step SB, it is determined whether or not it is possible to determine that the inclination is stable. In step SC, a first measurement time is determined. In order to determine whether the slope is stable in step SB, the oxygen current Inew is sequentially measured, and the slope of the oxygen current is updated until it is determined to be stable. Accordingly, two destinations are designated in step SB. When it is determined that the inclination is stable, the process proceeds from step SB to step SC. When it is determined that the inclination is not, the process returns to step S101 to further measure the oxygen current Inew.
[Selection] Figure 2

Description

  The present invention relates to a method for calculating the number of microorganisms.

  Various methods for calculating the number of microorganisms have been proposed. For example, as a method adopted as an official method, there is a method in which bacteria are enriched and cultured in a medium, and colonies are counted after a predetermined time to determine the initial number of bacteria.

  However, it takes nearly 20 hours to perform enrichment culture to such an extent that colonies can be visually observed. Therefore, a method for measuring the dissolved oxygen concentration in a medium in which microorganisms are cultured has been proposed. For example, the dissolved oxygen concentration in the culture medium is measured by measuring the current flowing through the oxygen electrode (hereinafter referred to as “oxygen current”). Such a technique is disclosed in Patent Document 1, for example.

JP 2003-235599 A

  However, in the method disclosed in Patent Document 1, the time until the oxygen current reaches a predetermined threshold is measured. An object of the present invention is to propose a method that can evaluate the number of microorganisms based on oxygen current at an earlier stage.

  In the first aspect of the method for measuring microorganisms according to the present invention, an oxygen current (Inew) that flows in accordance with the amount of oxygen is sequentially measured in a medium containing microorganisms whose initial number is to be measured (S101, S112). ), A method for obtaining a first measurement time (ts) that is a time required for the oxygen current to decrease beyond the first threshold (IP) a predetermined number of times (Z2), and includes the following steps (a) to ( c). That is, (a) a step of obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA); (b) the step (a) Step (SB) for determining whether or not the time difference value obtained in step (b) is stable; (c) If the determination in step (b) is affirmative, the time difference value measured in the most recent measurement period is measured. A step of setting the first threshold value depending on a value based on the oxygen current (I1 to IZ + 1) and lower than the value (S114a, S114b);

  A second aspect of the method for measuring microorganisms according to the present invention is the first aspect of the method for measuring the number of microorganisms, wherein the first threshold (IP) was measured most recently in the step (c). It is set by subtracting a predetermined threshold value (Δ) from the oxygen current (IZ + 1).

  A third aspect of the method for measuring microorganisms according to the present invention is the second aspect of the method for measuring the number of microorganisms, wherein the first threshold (IP) was measured most recently in the step (c). It is set by multiplying the oxygen current (IZ + 1) by a coefficient (q) greater than 0 and less than 1.

  Desirably, in the third aspect of the method for measuring microorganisms, the coefficient (q) is set to 0.8.

  According to a fourth aspect of the method for measuring microorganisms according to the present invention, an oxygen current (Inew) that flows in accordance with the amount of oxygen is sequentially measured in a medium containing microorganisms whose initial number is to be measured (S101, S112). ), A method for obtaining a first measurement time (tu) in which the decrease in the oxygen current becomes gentler than a threshold value (E3), and includes the following steps (a) to (c). That is, (a) a step of obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA); (b) the step (a) Step (SB) for determining whether or not the time difference value obtained in step (b) is stable; (c) If the determination in step (b) is affirmative, the difference value (Knew) measured thereafter ) Increases in increments exceeding the threshold with respect to the difference value (Kold) in step (b), the step (SC) of determining the measurement time based on the latest measurement period measured is there.

  A fifth aspect of the method for measuring a microorganism according to the present invention is any one of the first to fourth aspects of the method for measuring the number of microorganisms. In the step (b), the absolute value of the time difference value is determined. When the value (| Knew |) falls within the predetermined range (E2) in the predetermined period (S108b, S109), it is determined that the time difference value is stable.

  A sixth aspect of the microorganism measuring method according to the present invention is any one of the first to fifth aspects of the method for measuring the number of microorganisms, wherein in the step (b), the time of the time difference value is set. When the absolute value of the fluctuation with respect to (| Knew−Kold | / Δt) falls within the predetermined range (E1) for a predetermined period (S108a, S109), it is determined that the time difference value is stable.

Preferably, in the sixth aspect of the method for measuring microorganisms, the predetermined range (E1) is 0.6 nA / mm ² / min ² in terms of current density, and the predetermined number (Z2) is 10 times, The predetermined period is a length in which the measurement of the oxygen current is continuously performed five times (Z1).

  A seventh aspect of the method for measuring a microorganism according to the present invention is any one of the first to sixth aspects of the method for measuring the number of microorganisms, wherein in the step (b), the step (a) Thereafter, after a predetermined dead time has elapsed, it is determined whether or not the time difference value has stabilized.

  An eighth aspect of the microorganism measuring method according to the present invention is the seventh aspect of the microorganism count measuring method, wherein the dead time is set to 200 minutes.

  A ninth aspect of the microorganism measuring method according to the present invention is any one of the first to eighth aspects of the method for measuring the number of microorganisms, wherein (d) the oxygen current is a second threshold value (Id). ) Is further provided for obtaining a second measurement time (tt) that is a time required for the time period to drop beyond the above.

The tenth aspect of the method for measuring microorganisms according to the present invention is the ninth aspect of the method for measuring the number of microorganisms, wherein the second threshold (Id) is 60 nA / mm ² / min in terms of current density. ² .

  An eleventh aspect of the microorganism measuring method according to the present invention is any one of the first to tenth aspects of the method for measuring the number of microorganisms, wherein the initial number of the microorganisms is determined based on a calibration curve. The first measurement time is determined for the medium that is unknown, and the initial number that was unknown is calculated. The calibration curve is obtained for the microorganism and the medium from the relationship between the first measurement time and the known initial number for each of the culture media, the initial numbers of which are different from each other and known. Yes. The culture medium that is the measurement target of the first measurement time in the calibration curve is the same type as the culture medium used when calculating the initial number that was unknown. However, in the eleventh aspect of the method, although the unknown initial bacterial count of the microorganism is calculated based on the calibration curve, it is not required until the calibration curve is created.

  A twelfth aspect of the microorganism measuring method according to the present invention is any one of the first to eleventh aspects of the method for measuring the number of microorganisms, wherein the initial numbers of the microorganisms are different from each other and known. The first measurement time is obtained for each of the same type of culture medium, and the calibration curve for the microorganism and the culture medium is obtained from the relationship between the plurality of the known initial number and the plurality of the first measurement times. Ask.

  According to a first aspect of the microorganism measuring apparatus of the present invention, an oxygen current (Inew) that flows in accordance with the amount of oxygen is sequentially measured in a medium containing microorganisms whose initial number is to be measured (S101, S112). An oxygen current measurement unit (201) that performs the evaluation, and an evaluation unit that obtains a first measurement time (ts) that is a time required for the oxygen current to decrease beyond the first threshold (IP) a predetermined number of times (Z2) ( 202). And the said evaluation part performs the following process (a)-(c). That is, (a) a step of obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA); (b) the step (a) Step (SB) for determining whether or not the time difference value obtained in step (b) is stable; (c) If the determination in step (b) is affirmative, the time difference value measured in the most recent measurement period is measured. A step of setting the first threshold value depending on a value based on the oxygen current (I1 to IZ + 1) and lower than the value (S114a, S114b);

  A second aspect of the microorganism measuring apparatus according to the present invention is the first aspect of the microorganism count measuring apparatus, wherein the first threshold (IP) was measured most recently in the step (c). It is set by subtracting a predetermined threshold value (Δ) from the oxygen current (IZ + 1).

  A third aspect of the microorganism measuring apparatus according to the present invention is the second aspect of the microorganism count measuring apparatus, wherein the first threshold (IP) was measured most recently in the step (c). It is set by multiplying the oxygen current (IZ + 1) by a coefficient (q) greater than 0 and less than 1.

  Preferably, in the third aspect of the microorganism measuring apparatus, the coefficient (q) is set to 0.8.

  According to a fourth aspect of the microorganism measuring apparatus of the present invention, an oxygen current measuring unit that sequentially measures an oxygen current (Inew) that flows according to the amount of oxygen in a medium containing microorganisms whose initial number is to be measured. (201), and an evaluation unit (202) for obtaining a first measurement time (tu) at which the decrease in the oxygen current becomes slower than a threshold value (E3). And the said evaluation part performs the following process (a)-(c). That is, (a) a step of obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA); (b) the step (a) Step (SB) for determining whether or not the time difference value obtained in step (b) is stable; (c) If the determination in step (b) is affirmative, the difference value (Knew) measured thereafter ) Increases in the increment exceeding the threshold with respect to the difference value (Kold) in the step (b), the step (SC) determines the measurement time based on the latest measurement.

  A fifth aspect of the microorganism measuring apparatus according to the present invention is any one of the first to fourth aspects of the microorganism count measuring apparatus, wherein in the step (b), the absolute value of the time difference value is determined. When the value (| Knew |) falls within the predetermined range (E2) in the predetermined period (S108b, S109), it is determined that the time difference value is stable.

  A sixth aspect of the microorganism measuring apparatus according to the present invention is any one of the first to fifth aspects of the microorganism count measuring apparatus, wherein in the step (b), the time of the time difference value is set. When the absolute value of the fluctuation with respect to (| Knew−Kold | / Δt) falls within the predetermined range (E1) for a predetermined period (S108a, S109), it is determined that the time difference value is stable.

Preferably, in the sixth aspect of the microorganism measuring apparatus, the predetermined range (E1) is 0.6 nA / mm ² / min ² in terms of current density, and the predetermined number (Z2) is 10 times, The predetermined period is a length in which the measurement of the oxygen current is continuously performed five times (Z1).

  A seventh aspect of the microorganism measuring apparatus according to the present invention is any one of the first to sixth aspects of the microorganism count measuring apparatus, wherein in the step (b), the step (a) Then, after a predetermined dead time has elapsed, it is determined whether or not the time difference value has stabilized.

  An eighth aspect of the microorganism measuring apparatus according to the present invention is the seventh aspect of the microorganism count measuring apparatus, wherein the dead time is set to 200 minutes.

  A ninth aspect of the microorganism measuring apparatus according to the present invention is any one of the first to eighth aspects of the microorganism count measuring apparatus, wherein the evaluation unit (201) includes (d) the oxygen A step of obtaining a second measurement time (tt), which is a time taken for the current to decrease beyond the second threshold value (Id), is further executed.

A tenth aspect of the microorganism measuring apparatus according to the present invention is the ninth aspect of the microorganism count measuring apparatus, wherein the second threshold value (Id) is 60 nA / mm ² / min in terms of current density. ² .

  An eleventh aspect of the microorganism measuring apparatus according to the present invention is any one of the first to tenth aspects of the microorganism count measuring apparatus, wherein the initial number of the microorganisms is based on a calibration curve. The first measurement time is determined for the medium that is unknown, and the initial number that was unknown is calculated. The calibration curve is obtained for the microorganism and the medium from the relationship between the first measurement time and the known initial number for each of the culture media, the initial numbers of which are different from each other and known. Yes. The culture medium that is the measurement target of the first measurement time in the calibration curve is the same type as the culture medium used when calculating the initial number that was unknown. However, in the eleventh aspect of the apparatus, the unknown initial bacterial count of the microorganism is calculated based on the calibration curve, but it is not required until the calibration curve is created.

  A twelfth aspect of the microorganism measuring apparatus according to the present invention is any one of the first to eleventh aspects of the microorganism count measuring apparatus, wherein the initial numbers of the microorganisms are different from each other and known. The first measurement time is obtained for each of the same type of culture medium, and the calibration curve for the microorganism and the culture medium is obtained from the relationship between the plurality of the known initial number and the plurality of the first measurement times. Ask.

  In any of the first aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, the time required for the method is determined in the method for determining the initial number of microorganisms by measuring the current flowing through the medium according to the amount of oxygen. Shorten.

  Both the second aspects of the microorganism count measuring apparatus and the measuring method according to the present invention set the first threshold value in the step (c).

  In any of the third aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, when the first measurement time is obtained using the first threshold set in each second aspect, the conventional method is used. Increase the agreement rate with the results.

  In any of the fourth aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, the time required for the method is determined in the method for determining the initial number of microorganisms by measuring the current flowing through the medium according to the amount of oxygen. Shorten.

  Any of the fifth aspects of the apparatus and method for measuring the number of microorganisms according to the present invention determines the stability of the time difference value in step (b).

  In any of the sixth aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, when the stability of the time difference value is determined in each of the fifth aspects, the coincidence rate with the result obtained by the conventional method is increased. .

  In any of the seventh aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, the influence of current disturbance at the beginning of measurement on the determination in step (b) is excluded.

  In any of the eighth aspects of the apparatus for measuring the number of microorganisms and the measurement method according to the present invention, when the first measurement time is obtained by using the dead time employed in each of the seventh aspects, the result obtained by the conventional method is used. Increase the match rate.

  In any of the ninth aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, even if the initial number is so large that the determination in step (c) is not possible, the initial number is determined using the second measurement time. Can be sought.

  In any of the tenth aspects of the apparatus and method for measuring the number of microorganisms according to the present invention, when obtaining the second measurement time employed in each of the ninth aspects, the coincidence rate with the result by the conventional method is calculated. Increase.

  In any of the eleventh aspects of the device for measuring the number of microorganisms and the measuring method according to the present invention, in the method for determining the initial number of microorganisms by measuring the current flowing through the medium according to the amount of oxygen, the time required for the method is obtained. Shorten.

  In any of the twelfth aspects of the microorganism count measuring apparatus and the measuring method according to the present invention, a calibration curve used when calculating the initial number of microorganisms in each eleventh aspect is created.

Basic idea of the invention.
Before going into the detailed description of the embodiments of the present invention, the basic concept of the present invention will be described. Of course, this basic concept is also within the scope of the present invention. In the following description, bacteria are used as examples of microorganisms, but other microorganisms can be handled in the same manner. Further, since the value of the oxygen current and the threshold value related thereto are normalized and shown by the area of the oxygen electrode, they should be handled as current density accurately, but will be described below as current for simplicity.

  FIG. 1 is a graph illustrating the relationship between measurement time and oxygen current using the initial number of bacteria as a parameter. Since the enrichment culture is performed during the measurement time, the amount of dissolved oxygen decreases, and the oxygen current decreases accordingly. Graph L0 illustrates the case where the initial number of bacteria is 0, and graphs L1, L2, L3, L4 and L5 illustrate cases where the initial number of bacteria is 101, 102, 103, 104 and 105 (CFU / ml), respectively. .

  Conventionally, the threshold value Ith is set to a low value for the oxygen current. Then, the measurement times (measurement times t10, t20, t30, t40, and t50, respectively) until the graphs L1, L2, L3, L4, and L5 reach the threshold value Ith were obtained. Then, a calibration curve is obtained from the measurement times t10, t20, t30, t40, t50 and the known initial bacterial count 101, 102, 103, 104, 105 (CFU / ml). Therefore, even if the initial bacterial count is unknown, the initial bacterial count can be calculated from the measurement time to reach the threshold value Ith and the calibration curve.

  However, as can be seen from the graphs L1, L2, L3, L4, and L5, the oxygen current decreases as the measurement time elapses, but at the beginning, the inclination of the decrease is small. Then, the slope of decrease increases. Therefore, the measurement time at which the slope of the graph of oxygen current with respect to time (in other words, the difference in oxygen current per unit time: hereinafter simply referred to as “slope of oxygen current”) fluctuates (hereinafter referred to as “slope fluctuation”). "Time": Speaking in accordance with the graphs L1, L2, L3, L4, and L5, the measurement times t11, t21, t31, t41, and t51 when the decrease gradient shifts from small to large, and the decrease gradient is large. Measurement time (t12, t22, t32, t42, t52) for shifting from small to small, and a calibration curve can be created from this and the known initial number of bacteria.

  Then, the initial bacterial count can be calculated from the calibration curve and the slope variation time when the initial bacterial count is unknown. Moreover, the inclination fluctuation times t11 and t12 are shorter than the measurement time t10, the inclination fluctuation times t21 and t22 are shorter than the measurement time t20, the inclination fluctuation times t31 and t32 are shorter than the measurement time t30, and the inclination fluctuation times t41 and t42 are It is shorter than the measurement time t40, and the inclination fluctuation times t51 and t52 are shorter than the measurement time t50. Therefore, if the inclination variation time of the oxygen current is obtained, the initial number of bacteria can be calculated at an early stage. This is the basic idea of the present invention.

  However, visually changing the gradient of the oxygen current hinders work automation. In addition, the inclination variation time cannot be visually confirmed unless the graph after the lapse of the gradient variation time is measured. This makes it difficult to shorten the measurement time.

  Therefore, in the first to third embodiments described below, a convenient method is adopted in place of strictly looking at fluctuations in the gradient of the oxygen current. That is, when the gradient of the oxygen current is stabilized after the gradient fluctuation times t11, t21, t31, t41, and t51, the first threshold value is determined based on the graphs obtained so far. Then, a calibration curve is created based on the measurement time (hereinafter referred to as “first measurement time”) in which the oxygen current has decreased by exceeding the first threshold value at least once and the known initial number of bacteria.

  Further, in the fourth embodiment, when the slope of the oxygen current is stabilized, the absolute value of the slope becomes smaller, so that a bending point of the graph is given (different from the first to third embodiments). A calibration curve is created based on one measurement time (corresponding to the slope fluctuation times t12, t22, t32, t42, and t52) and the known initial number of bacteria.

  After the calibration curve is created, the unknown initial number of bacteria can be calculated from the calibration curve and the first measurement time.

First embodiment.
FIG. 2 is a flowchart showing a part of the microorganism count measuring method according to the first embodiment of the present invention. In step S100, the counters S and F2 used in the processing according to the flowchart and the gradient Kold of the oxygen current are set (reset) to initial values. In step S101, the oxygen current Inew is measured. In step SA, the slope of the oxygen current is obtained. In step SB, it is determined whether or not it is possible to determine that the inclination is stable. In step SC, a first measurement time is determined.

  In order to determine whether the slope is stable in step SB, the oxygen current Inew is sequentially measured, and the slope of the oxygen current is updated until it is determined to be stable. Accordingly, two destinations are designated in step SB. When it is determined that the inclination is stable, the process proceeds from step SB to step SC. When it is determined that the inclination is not, the process returns to step S101 to further measure the oxygen current Inew.

  FIG. 3 is a flowchart illustrating details of step SA. First, in step S104, the counter S is incremented by one. Until step S104 is executed for the first time, the value 0 is set to the counter S by step S100. Therefore, the value of the counter S becomes 1 by executing step S104 first.

  In step S105, oxygen current data sorting is performed. Specifically, the values of the data I2 to IZ + 1 are substituted into the data I1 to IZ, and then the latest oxygen current Inew measured at step S101 is substituted into the data IZ + 1.

  Next, in step S106, it is determined whether or not the counter S is larger than the value Z. If the determination in step S106 is negative, the process returns to step S101 and the next oxygen current Inew is measured. As described above, steps S101 to S105 are repeatedly executed until the determination in step S106 becomes affirmative, whereby Z values of oxygen currents arranged in time series in this order are set in the data I1 to IZ. It will be. As described above, when the counter S is equal to or smaller than the value Z, the process returns from step SA to step S101 as an exception. Such a branch is indicated by a broken line in FIG.

  If the counter S becomes larger than the value Z, the process proceeds to step S107. In step S107, the slope Knew of the regression line for the data I1 to IZ + 1 is obtained. For example, if the measurement of the oxygen current Inew in step S101 is performed at a constant interval T, the measurement time during which step S101 is executed is not particularly necessary for the execution of step S107. The slope Knew can be obtained using, for example, the least square method.

  As a simpler step that can be substituted for step S107, a data difference (IZ + 1-I1) can be obtained and divided by Z · T to obtain a slope. In any case, in step S107, a period (for example, Z · T) in which data I1 to IZ + 1 are obtained is set as a measurement period, and a time that is a difference in oxygen current per unit time from the measured value of oxygen current in the measurement period. A slope Knew is obtained as the difference value.

  After execution of step S107, the process proceeds to step SB. FIG. 4 is a flowchart illustrating details of step SB. First, in step S108a, it is determined whether or not the variation in the slope of the oxygen current falls within a predetermined allowable range. Specifically, the value obtained by dividing the absolute value of the difference between the slope Knew and the slope Kold obtained in step S107 by the time interval Δt from the previous oxygen current measurement time to the current oxygen current measurement time is: It is determined whether or not it is smaller than a predetermined value E1. If the determination is affirmative, the counter F1 is incremented by 1. If the determination is negative, the counter F1 is 0. For example, the above-described constant interval T is adopted as the time interval Δt.

  Until step S108a is executed for the first time, the value ZK is set to the inclination Kold by step S100. The value ZK is larger than the product of the predetermined value E1 and the time interval Δt than the slope Knew obtained by normal measurement. Therefore, the value of the counter F1 is always 0 by executing step S108a first.

  Thereafter, in step S109, it is determined whether or not the counter F1 has reached a predetermined value Z1. First, when step S109 is executed, since the value of the counter F1 is 0 as described above, the determination is negative, and the process proceeds to step S110. In step S110, the inclination Knew obtained at present is adopted as the inclination Kold. Then, the process returns to step S101, and a new slope Knew is obtained again by step SA. Therefore, it is desirable that the above-described interval T is longer than the sum of the processing times of both steps SA and SB.

  From the above, in step S108a, the gradient Knew updated by the latest oxygen current Inew measured sequentially is compared with the gradient Kold obtained immediately before. Further, in view of the processing in steps S108a and S109, the processing advances to step SC when the fluctuation of the gradient of the oxygen current that is sequentially updated is continuously within the range of the predetermined value E1 Z1 times. .

  FIG. 5 is a flowchart illustrating the details of another example of step SB. In this example, a flowchart in which step S108a is replaced with step S108b and step S110 is deleted is employed as compared with the flowchart shown in FIG.

  In step S108b, it is determined whether or not the absolute value of the slope Knew is smaller than a predetermined value E2. If the determination is affirmative, the counter F1 is incremented by 1. If the determination is negative, the counter F1 is 0.

  FIG. 6 is a flowchart illustrating details of step SC. In step SC, first, a first threshold value IP is set in step S111a. Specifically, a value q times (0 <q <1) the data IZ + 1 when proceeding from step SB to step SC is adopted as the first threshold value IP. In step S112, the oxygen current is measured again to obtain the oxygen current Inew.

  Step S112 is easy to execute in the same manner as step S101, and it is desirable to measure the oxygen current at an interval T together with these. In this case, the interval T is preferably longer than the time obtained by adding the processing time of step S111a to the sum of the processing times of both steps SA and SB. In such a desirable mode, a value obtained by multiplying the latest oxygen current Inew by the coefficient q can be adopted as the first threshold value IP in step S111a.

  However, in general, the first threshold value IP may be determined depending on the value based on the oxygen current in the most recent measurement period for measuring the slope. This is because it is determined that the inclination is stable based on the latest calculated inclination. For example, the first threshold value IP can be set based on the average value of the oxygen current in the most recent measurement period. For example, a value obtained by multiplying the average value by a coefficient q can be employed.

  In step S114a, it is determined whether or not the oxygen current Inew measured in step S112 is smaller than the first threshold value IP. If the result of this determination is affirmative, the value of the counter F2 is incremented by one. Since the initial value 0 is set to the counter F2 in step S100, the value of the counter F2 is 0 before the first execution of step S114a.

  Thereafter, the process proceeds to step S115, and it is determined whether or not the value of the counter F2 has reached a predetermined value Z2 (> 1). First, when step S114a is executed, since the value of the counter F1 is 0 or 1, the determination is negative, the process returns to step S112, and the oxygen current Inew is obtained again. Therefore, the interval T is preferably longer than the sum of the processing times of both steps S114a and S115.

  In step S114a, unlike steps S108a and S108b, the counter F2 is not reset when the determination result is negative. Therefore, if the oxygen current Inew that is sequentially updated in step S112 is smaller than the first threshold value IP for Z2 times regardless of whether it is continuous or not, the process proceeds to step S116. In step S116, for example, the measurement time of step S112 executed last is set as the first measurement time.

  FIG. 7 is a flowchart illustrating the details of another example of step SC. Here, steps S111a and S114a are replaced with steps S111b and S114b, respectively. In step S111b, a value obtained by subtracting the current decrease threshold Δ from the data IZ + 1 when proceeding from step SB to step SC is adopted as the first threshold IP. As described above, in general, the first threshold value IP is determined depending on the value based on the oxygen current measured during the most recent measurement period, so the average value of the oxygen current measured during the most recent measurement period. A value obtained by subtracting the current drop threshold Δ from the value may be adopted as the first threshold IP.

  In step S114b, as in the case of the counter F1 in steps S108a and S108b, the counter F2 is reset to 0 unless the condition Inew <IP is satisfied. That is, the process reaches step S116 for the first time when the condition is continuously satisfied Z2 times.

  Since the first measurement time is obtained in this way, when creating a calibration curve, the first measurement time is measured as described above for the case where the initial number of bacteria is known. After the calibration curve is obtained, the initial bacterial count can be calculated based on the calibration curve by measuring the first measurement time even if the initial bacterial count is unknown.

For example, values 30, 5, and 10 can be adopted as values Z, Z1, and Z2, respectively. Further, 1 (min) and 1 (min) can be adopted as the intervals T and Δt, respectively, 0.8 can be adopted as the coefficient q, and 30 nA / mm ² can be adopted as the current drop threshold Δ. Further, 103 (nA / mm ² / min) is adopted as the value ZK, and 0.6 (nA / mm ² / min ² ) and 1.2 (nA / mm ² / min ² ) are respectively set as the predetermined values E1 and E2. Can be adopted. When such specific values are adopted, the above flowchart can be described as follows.

  First, the oxygen current is measured every minute. Then, the slope of the regression line is obtained for the continuous 30-minute oxygen current data. This slope is updated each time the oxygen current is measured, and its stability is judged.

For example, as one method for judging stability (see FIG. 4), when the slope is updated and the absolute value of the fluctuation exceeds 0.6 (nA / mm ² / min ² ), the slope is stabilized. It is not judged to be. It is determined that the slope is stable only when the absolute value of the fluctuation of the slope is continuously reduced to 0.6 (nA / mm ² / min ² ) 5 times.

As another stability determination method (see FIG. 5), when the absolute value of the inclination exceeds 1.2 (nA / mm ² / min), it is not determined that the inclination is stable. It is determined that the slope is stable only when the absolute value of the slope is continuously reduced to 1.2 (nA / mm ² / min) five times.

  Then, the first threshold value is set based on the latest oxygen current when it is determined that the inclination is stable.

For example, as one setting method of the first threshold (see FIG. 6), a value obtained by multiplying the latest oxygen current by 0.8 is set as the first threshold. Alternatively, as another setting method (see FIG. 7), a value obtained by subtracting 30 nA / mm ² from the latest oxygen current is set as the first threshold value.

  After that, if the first threshold value is less than 10 times, it is considered that the inclination variation time has elapsed, and the first measurement time is obtained.

  FIG. 8 is a graph showing an outline of processing for obtaining the first measurement time from the measurement of the oxygen current, and the measurement time is adopted on the horizontal axis and the oxygen current is adopted on the vertical axis. The measured value of the oxygen current is indicated by a white circle, and the value of the data IZ + 1 when step SA is executed last is indicated by a horizontal line. In order to facilitate understanding in the graph, a regression line L for the data I1 to IZ + 1 when the step SA is executed last is shown. Of course, what is required in steps SA and SB is not the regression line L itself but its slope Knew.

  The first threshold value IP is also indicated by a horizontal line. Here, for example, 10 is set as the value of Z2, and the measurement time when the measured value of the oxygen current falls 10 times below the first threshold value IP is defined as the first measurement time ts.

Second embodiment.
As shown in FIG. 8, large fluctuations may occur until the slope stabilizes at the beginning of the measurement time. This not only repeats the processing in step SB unnecessarily but also may be erroneously detected as a stable slope. Therefore, at the beginning of the measurement time, it is desirable to provide a dead time t0 that does not execute step SA and subsequent steps.

  FIG. 9 is a flowchart of step SA in consideration of such dead time setting. Step S103 is added before step S104 in the flowchart shown in FIG. In step S103, it is determined whether or not the dead time has elapsed. If the dead time has not elapsed, the process returns to step S101, and if it has elapsed, the process proceeds to step S104.

  Thereby, the influence of the disturbance of the waveform of the oxygen current at the beginning of the measurement time can be eliminated, and the erroneous determination of the slope of the regression line for the oxygen current data and hence the first measurement time can be avoided.

Third embodiment.
Depending on the measurement result of the oxygen current, the value of the oxygen current may decrease rapidly. 10 to 12 are graphs showing various aspects thereof.

  In the first mode shown in FIG. 10, after the gradient of the oxygen current is stabilized, it decreases until it becomes impossible to measure before it falls below the first threshold value IP by Z2 times. In the second mode shown in FIG. 11, the gradient of the oxygen current is not stabilized and is lowered until measurement becomes impossible. In the third mode shown in FIG. 12, it has been lowered until it becomes impossible to measure in the dead time. Therefore, in these cases, in order to enable the creation of a calibration curve and the calculation of the unknown initial number of bacteria, when the value of the oxygen current falls below the second threshold value Id, the time point is set to the second The measurement time is tt. The second measurement time is handled in the same manner as the first measurement time, and a calibration curve is created and an unknown initial number of bacteria is calculated.

  FIG. 13 is a flowchart showing a part of the microorganism count measuring method according to the third embodiment of the present invention, and corresponds to FIG. 14 and 15 are flowcharts showing details of step SA in the third embodiment of the present invention, which are shown in FIG. 3 shown in the first embodiment and in the second embodiment, respectively. This corresponds to FIG.

In any of FIGS. 13 to 15, immediately after the oxygen current Inew is measured, it is determined whether or not this is larger than the second threshold value Id. Specifically, step S102 is provided immediately after step S101. For example, 60 nA / mm ² is adopted as the second threshold Id.

  If the determination in step S102 is affirmative, step S104 is performed as shown in the first embodiment (see FIGS. 3 and 14), or step S103 is performed as described in the second embodiment. (See FIG. 9 and FIG. 15), respectively. However, if the determination result in step S102 is negative, the process proceeds to step S117, and the time when such an oxygen current is measured is adopted as the second measurement time.

  FIG. 16 is a flowchart showing details of step SC in the third embodiment of the present invention, and corresponds to FIG. 6 or FIG. 7 shown in the first embodiment. Also in this flowchart, immediately after the oxygen current Inew is measured, it is determined whether or not it is larger than the second threshold value Id. Specifically, step S113 is provided immediately after step S112. In the figure, step S112 shows step S112a (FIG. 6) and step S112b (FIG. 7) in an integrated manner, and either one of them is adopted.

  If the determination in step S113 is affirmative, step S114a (see FIG. 6) or step S114b (see FIG. 7) is executed as shown in the first embodiment (see FIG. 7). Step S112 shows step S112a (FIG. 6) and step S112b (FIG. 7) in an integrated manner, either of which is adopted). However, if the determination result of step S113 is negative, the process proceeds to step S117, and the time when such an oxygen current is measured is adopted as the second measurement time.

  In this embodiment, since there is a case where the process branches from step SC to step S117, such a branch is indicated by a broken line in FIG.

  In the first mode shown in FIG. 10, a positive result is not obtained in the determination in step S115 (see FIG. 16), and the process branches from step SC to step S117 to obtain the second measurement time tt. . In the second mode shown in FIG. 11, a positive result is not obtained in the determination in step S106 (see FIGS. 14 and 15), and the process does not proceed from step SA to step SB, but from step S102 to step S102. The process proceeds to S117, and the process proceeds along a branch indicated by a broken line from step SA in FIG. In the third mode shown in FIG. 12, a positive result is not obtained in the determination in step S103 of FIG. 15, and the process proceeds from step S102 to step S117 without proceeding to step S104.

Fourth embodiment.
In the present embodiment, the first measurement time (referred to as “first measurement” in the first to third embodiments) described in “Basic idea of the invention.” T12, t22, t32, t42, and t52 are obtained. As with the first measurement time and the second measurement time described in the first to third embodiments, the first measurement time obtained in the fourth embodiment is also used for creating a calibration curve and calculating the unknown initial number of bacteria. Provide.

  Of course, also in the present embodiment, the dead time shown in the second embodiment may be introduced, or the second measurement time shown in the third embodiment may be introduced. In order to distinguish from the first measurement time described in the first to third embodiments, “tu” is used as a code indicating the first measurement time in this embodiment.

  FIG. 17 is a flowchart showing details of step SC used in the present embodiment. First, after it is determined in step SB that the inclination is stable, the latest inclination Knew is temporarily adopted as the inclination Kold. In step S302, the oxygen current Inew is newly measured. In step S303, the value of the counter F2 is incremented by one. Since the initial value 0 is set to the counter F2 in step S100, the value of the counter F2 indicates the number of times step S302 has been executed.

  As in step S105, the oxygen current data is sorted in step S304. In step S305, it is determined whether the number of executions of step S302 has been repeated a desired number of times (Y + 1) in the same manner as in step S106.

  These operations are the same as those described with reference to FIG. In other words, if the result of the determination in step S305 is affirmative, Y values of oxygen currents arranged in time order in this order are set in the data I1 to IY. In step S306, as in step S107, the slope Knew of the new regression line for the data I1 to IY + 1 is obtained as the difference in oxygen current per unit time.

  Since the oxygen currents I1 to IZ + 1 have already been obtained so far in step S105 of step SA, the result of step S105 can be used in the data sort in step S304.

  In step S307, the inclination Kold set in step S301 is compared with the new inclination Knew obtained in step S306. Both the slopes Kold and Knew are slopes at which the oxygen current decreases and are negative values. Therefore, the time when the slope Knew becomes larger than the slope Kold in increments exceeding a predetermined threshold E3 is the first measurement time tu ( According to FIG. 1, it can be adopted as times t22, t32, t42, t52).

  In step S307, for convenience of determination, a case where it is determined whether or not the absolute value of the inclination Kold is larger than the absolute value of the inclination Knew by an increment exceeding the threshold E3 is illustrated. However, it is clear that it is not necessary to adopt an absolute value in this determination.

  If the result of determination in step S307 is affirmative, the process proceeds to step S307. For example, the last measurement time of step S302 is set as the first measurement time tu.

  If the result of the determination is negative, the value of the counter F2 is reset (adopts a value of 0) in step S308, and then the process returns to step S302 to obtain a new slope.

  Step S302 is set as the return destination of step S308. That is, to determine whether or not the slope of the oxygen current has become gentle, the slope of the oxygen current when determined to be stable in step SB is used as a reference.

  It is also conceivable that step S301 is set as the return destination of step S308, and the first measurement time tu is obtained based on the change in the gradient of the oxygen current. However, since the slope of the oxygen current changes gradually, the first measurement time tu becomes longer than the case where the slope of the oxygen current when determined to be stable in step SB is used as a reference, and reaches the threshold value Ith. The effect of measuring earlier than the measurement time (see FIG. 1) is small. Therefore, from the viewpoint of measuring at an early stage, it is preferable to adopt step S302 as a return destination of step S308 rather than step S301.

  Alternatively, the first measurement time tu may be determined based on the measurement period in which the latest slope Knew is obtained. This is because it is determined that the slope Knew has become larger than the previously obtained slope Kold by an increment exceeding the predetermined threshold E3 due to the slope obtained during the most recent measurement period. For example, the first measurement time tu may be determined as the median value of the latest measurement period.

  The first measurement time and the second measurement time obtained in the various embodiments described above are used to create a calibration curve when the initial number of bacteria is known. Then, based on the calibration curve obtained in this way, the initial bacterial count can be calculated by measuring the oxygen current for a medium whose initial bacterial count is unknown.

  Of course, in order to increase the accuracy of measurement, when creating a calibration curve, the type of microorganism whose initial bacterial count is known is the same as the type of microorganism whose initial bacterial count is unknown, and a calibration curve is created. In this case, it is desirable that the type of the medium containing the microorganism whose initial number of bacteria is known is the same as the type of the medium containing the microorganism whose initial number of bacteria is unknown.

  FIG. 18 is a block diagram showing a configuration of a microorganism count calculation apparatus 200 applicable to a technique for creating a calibration curve or calculating an initial bacterial count as described above. The microorganism count calculation apparatus 200 includes an oxygen current measurement unit 201 that measures the oxygen current in the culture medium, and a control / evaluation unit 202 that controls the operation of the oxygen current measurement unit 201 and evaluates the oxygen current.

  The control / evaluation unit 202 gives a command D for instructing the timing for measuring the oxygen current to the oxygen current measurement unit 201. For example, the oxygen current is measured every predetermined period T, the oxygen current is measured after the dead time has elapsed, or the oxygen current is measured after the first measurement time ts, tu or the second measurement time tt is obtained. Stop it. The oxygen current measurement unit 201 mainly executes steps S101 and S112, and the control / evaluation unit 202 executes steps other than steps S101 and S112 in the flowchart described above.

Other:
It is also conceivable to store a plurality of relationships between the measurement time and oxygen current in advance using the initial number of bacteria as a parameter, and to determine the initial number of bacteria using a parameter that gives the smallest difference from the measured value among these relationships. .

  For example, the graphs L1, L2, L3, L4, and L5 shown in FIG. 1 are stored in advance as current data or as a function form. Then, the current data that minimizes the difference between the measured value as shown by the white circles in FIGS. 8 and 10 to 12 and the current data is specified. For example, regarding the square of the difference between the measured value for each measurement time and the current data, the result obtained by summing at least part of the measurement time is adopted as the difference. Then, the initial bacterial count corresponding to the current data that minimizes the difference is selected as the initial bacterial count obtained from the measured value.

  However, since this method may greatly distort the graph depending on the specimen, it is more accurate to obtain the initial bacterial count by the method shown in the above embodiment.

  Hereinafter, examples of the present invention will be described together with techniques to be compared. Table 1 is a table illustrating the techniques to be contrasted.

  In each of detection conditions # 201 to # 206, the oxygen current is measured every minute, and the oxygen current at a certain point falls five times lower than the set current value with respect to the oxygen current before 30 minutes (time interval). The detected time is used as the detection time. This detection time is also considered to depend on the initial number of bacteria. However, the dead time is set for any of the detection conditions # 021 to # 026.

For example, in the detecting condition # 201, since the set value of the set current to 30 nA / mm ^2, after the dead time 100 minutes have passed, the slope of the oxygen current is ^{-30 (nA / mm 2) /} 30 (min) = - The measurement time is employed as the detection time when it becomes smaller (rapidly) than 1.0 (nA / mm ² / min) five times in succession.

  Table 2 is a table for explaining a technique for determining that the gradient of the oxygen current is stable by determining that the absolute value of the gradient of the oxygen current is within a predetermined range. Such a determination corresponds to step SB shown in FIG.

Detection conditions # 311 to # 319 measure the oxygen current every minute (this interval corresponds to the aforementioned constant interval T). The result of subtracting the oxygen current 10 minutes ago (or 30 minutes ago) (this period corresponds to the product of the value Z for the counter S and the constant interval T) from the current oxygen current is 10 minutes (or 30 minutes). Divide by (minutes) to get the slope. This inclination is different from the above-described inclination Knew in that it is not obtained by the method of least squares. When the absolute value of this inclination is continuous five times (corresponding to the above-mentioned predetermined value Z1) and becomes smaller than 1.2 (nA / mm ² / min) (corresponding to the above-mentioned predetermined value E2), the oxygen current It is judged that the slope of the is stable. Then, the oxygen current having a value smaller than the oxygen current at this time (corresponding to the above-described IZ + 1) by a current decrease threshold (corresponding to the above-described current decrease threshold Δ) is continuously applied 5 times (corresponding to the above-described predetermined value Z2). Then, the time point observed is adopted as the first measurement time. Such a determination corresponds to step SC shown in FIG. The first measurement time set in this way is also considered to depend on the initial number of bacteria. However, the dead time is set for any of the detection conditions # 311 to # 319.

For example, in the detection condition # 311, after the dead time of 100 minutes has elapsed, when the absolute value of the gradient of the oxygen current has become smaller (flat) than 1.2 (nA / mm ² ) for five consecutive times Thus, it is determined that the inclination is stable. Then, a value that is 30 (nA / mm ² ) lower than the value of the oxygen current at the stable time point is adopted as the first threshold value IP, and the time point when the oxygen current smaller than this value is continuously observed five times is the first measurement. Adopt as time.

  Table 3 is a table for explaining a technique for determining that the slope of the oxygen current is stable by determining that the absolute value of the change in the slope of the oxygen current is within a predetermined range. Such a determination corresponds to step SB shown in FIG.

Detection conditions # 321 to # 329 determine the inclination of the oxygen current in the same manner as detection conditions # 321 to # 329. The absolute value of the change amount of the inclination (corresponding to | Knew−Kold | / Δt in step S108a in FIG. 4) is continuously repeated 5 times (corresponding to the predetermined value Z1), 3 (nA / mm ² / min). It is determined that the gradient of the oxygen current is stable when the value becomes smaller. Then, the oxygen current having a value smaller than the oxygen current at this time (corresponding to the above-described IZ + 1) by a current decrease threshold (corresponding to the above-described current decrease threshold Δ) is continuously applied 5 times (corresponding to the above-described predetermined value Z2). Then, the time point observed is adopted as the first measurement time. Such a determination corresponds to step SC shown in FIG. This first measurement time set in this way is also considered to depend on the initial number of bacteria. However, the dead time is set for any of the detection conditions # 321 to # 329.

For example, in detection condition # 321, after the dead time of 100 minutes has elapsed, the absolute value of the change amount of the oxygen current gradient is smaller (flattened) than 3 (nA / mm ² / min) for five consecutive times. When it occurs, it is determined that the inclination is stable. Then, a value that is 30 (nA / mm ² ) lower than the value of the oxygen current at the stable time point is adopted as the first threshold value IP, and the time point when the oxygen current smaller than this value is continuously observed five times is the first measurement. Adopt as time.

Tables 4 to 6 show the above-described detection time (detection conditions # 201 to # 206) and the first measurement time (detection conditions # 311 to # 319, # 321 to # 329), and the change in the inclination of the oxygen current visually. (The technology that automatically performed this corresponds to the fourth embodiment: the result is expressed as “reference point” in the table). Condition # 101 in Table 4 is a case where the measurement time to reach the threshold value Ith is measured using a conventional technique. As the threshold value Ith, 180 nA / mm ² is adopted, and the time when the threshold value Ith falls below three times is adopted as the detection time. Further, the second measurement time based on the second threshold value Id described in the third embodiment is not adopted.

  For each condition, the slope variation time and the measurement result (detection time or first measurement time) of each detection condition are regarded as a pair of samples, and a significant difference test is performed by one-sample t-test for both times (significance level). 1%). Therefore, the degree of freedom is a value obtained by subtracting 1 from the number of observations. The reference point as a sample paired with each detection condition is described in the left column of each detection condition. Therefore, each detection condition and the reference point that are adjacent via the vertical double line are not compared.

  However, the total number of specimens (medium to be measured) is 358. The number of observations of the measurement results for each detection condition is smaller than the total number of specimens because the measurement results (detection time and first measurement time) obtained using each detection condition and the slope fluctuation time obtained from the reference point This is because there is a case where at least one of them exhibits a very large value. In other words, this is a case where the sample is determined to be negative by any measurement method. In these cases, the slope fluctuation time and the measurement results cannot be statistically compared using numerical values, and are excluded from the number of observations.

  As a result of the t test with the detection condition # 101 in Table 4 and its reference point, the rejection threshold probability is smaller than 1% (= 0.01). The absolute value of the t value is larger than the t boundary value. Therefore, it can be seen that there is a significant difference between the average value of the slope fluctuation time and the average value of the measurement time until the conventional oxygen current reaches the threshold value Ith. In other words, as described in “A. Basic idea of the invention”, the initial bacterial count can be calculated at an early stage by obtaining the slope variation time of the oxygen current.

  On the other hand, from Tables 5 and 6, there is no significant difference between the average value of the first measurement time obtained under the detection conditions # 319, # 326, and # 329 and the average value of the slope variation time paired with each. No hypothesis is rejected. On the other hand, except for these three detection conditions, it can be concluded that the null hypothesis can be rejected and there is a significant difference. Strictly speaking, the fact that the null hypothesis cannot be rejected does not mean that the null hypothesis is valid, but in general, such a conclusion is often used. Therefore, among the various detection conditions shown in Table 1, Table 2, and Table 3, only the average value of the first measurement time obtained under the detection conditions # 319, # 326, and # 329 is an inclination obtained by visual observation. Judge that there is no significant difference from the average value of the variation time.

  Now, when the detection conditions # 319, # 326, and # 329 are employed in this way, the work is automated as shown in the first embodiment and the second embodiment with respect to the inclination variation time obtained by visual observation. It seems possible to substitute the first measurement time. Therefore, next, the first measurement time of detection conditions # 319, # 326, and # 329 and the measurement time of condition # 101 were compared. Table 7 shows the result of t-test (significance level 1%) of the average value of measurement time, and Table 8 shows the result of t-test (significance level 5%) of the average value of CV value (Coefficient of Variation) of measurement time. Show. Similar to Table 4, Table 5, and Table 6, these results were also subjected to a significant difference test using a one-sample t-test.

  From Table 7, there is a significant difference between the average value of the first measurement time under the detection conditions # 319, # 326, and # 329 and the average value of the measurement time under the condition # 101. Therefore, the first measurement time of the detection conditions # 319, # 326, and # 329 has the effect of reducing the time with respect to the conventional measurement time until the oxygen current reaches the threshold value Ith, similarly to the visual tilt fluctuation time. I know that there is.

  Further, from Table 8, the null hypothesis that the detection conditions # 319 and # 329 and the condition # 101 have no significant difference in CV values cannot be rejected. From this, it was determined that the variation in the first measurement time was comparable to the variation in the conventional measurement time. The detection condition # 326 was determined to be ineffective from the viewpoint of variation.

  Therefore, based on these four detection conditions, positive / negative bacteria were determined for all 358 specimens. The result was compared with the result of judging the positive / negative of the bacteria by the enrichment culture using an agar medium (hereinafter referred to as “agar technique”). Table 9 shows the comparison results.

  Here, “false positive” refers to a case where it is determined as positive according to each detection condition but is determined as negative according to the agar technique. “False negative” refers to a case in which it is determined to be negative according to each detection condition but is determined to be positive in the agar technique. Therefore, both cases are different from the results of the agar technique. In Table 9, the number of specimens determined to be “false positive” or “false negative” is described for each detection condition, and the coincidence rate with the agar technique is also described. For example, in the detection condition # 101, the number of specimens having “false positive” and “false negative” is 5 and 25, respectively, and the coincidence rate is (358-5-25) /358×100=91.6 ( %).

From the results in Table 9, the coincidence rate with the agar technique is slightly lower than that in the detection condition # 101 which is the conventional technique. Therefore, while following the basic detection method of detection conditions # 326 and # 329 (adopting the flowchart of FIG. 4 as step SB), a device for further increasing the coincidence rate with the agar method was devised. Specifically, the flowchart of FIG. 6 is used as step SC instead of the flowchart of FIG. Here, 0.8 is adopted as the coefficient q. Further, 0.6 (nA / mm ² / min ² ) was adopted as the predetermined value E1. The predetermined values Z1 and Z2 were 5 and 10, respectively. This detection condition is set to # 331. The dead time was 200 minutes. The gradient of the oxygen current is obtained by the least square method, and corresponds to the gradient Knew in step S107 (see FIG. 3).

Furthermore, in addition to the detection condition # 331, the detection condition # 332 is set by adopting the second threshold value Id described in the third embodiment. That is, in the detection condition # 332, the flowcharts shown in FIGS. 6, 13, 14, and 16 (however, steps S111a and S114a are employed) are employed. Here, the second threshold Id is 60 nA / mm ^2, and the measurement time obtained under the detection condition # 332 indicates both the first measurement time and the second measurement time (referred to in the third embodiment).

  The detection conditions # 331 and # 332 were compared with the condition # 101. Table 10 shows the result of the t-test (significance level 1%) of the average value of the measurement time, and Table 11 shows the result of the t-test (significance level 5%) of the average value of the CV value of the measurement time. Similarly to Tables 7 and 8, these results were also subjected to a significant difference test using a one-sample t-test.

  From Table 10, there is a significant difference between the average value of measurement time under detection conditions # 331 and # 332 and the average value of measurement time under condition # 101. Therefore, the measurement time for the detection conditions # 331 and # 332 is shortened with respect to the conventional measurement time for the oxygen current to reach the threshold value Ith, similarly to the measurement times for the detection conditions # 319, # 326, and # 329. It turns out that there is an effect to do.

  Further, it can be seen from Table 11 that the detection conditions # 331, # 332 and the condition # 101 have a significant difference in CV values. That is, it can be seen that if the detection conditions # 331 and # 332 are employed, the variation in the measurement time can be improved over the conventional variation in the measurement time.

  Therefore, next, based on these two detection conditions, 358 positive / negative bacteria were determined for all 358 specimens. The result was compared with the result of judging the positive / negative of the bacteria by the agar method. Table 12 shows the comparison results.

  As can be seen from the comparison with Table 9, the coincidence rate with the agar method is improved even to the detection condition # 331 where the second measurement time is not introduced, and is 88.3%. Furthermore, in the detection condition # 332 in which the second measurement time is introduced, the coincidence rate with the agar method is 91.6%, which is equal to the coincidence rate according to the conventional technique. In addition, the number of false negatives is smaller than that of the conventional condition # 101, which is preferable for safety.

  From the above, it is desirable to adopt the flowchart shown in FIG. 4 rather than the one shown in FIG. 5 as step SB, and in step SA, compared to the case where steps S111b and S114b are used (see FIG. 7). It can be seen that the case where steps S111a and S114a (see FIG. 6) are used is more desirable, and the case where a dead time is provided (see FIG. 13) is desirable. Furthermore, it can be seen that it is more desirable to introduce the second measurement time as shown in the third embodiment (see FIG. 16).

It is a graph which illustrates the relationship between measurement time and oxygen current, using the initial number of bacteria as a parameter. It is a flowchart which shows a part of microorganisms number measuring method concerning the 1st Embodiment of this invention. It is a flowchart which illustrates the detail of step SA. It is a flowchart which illustrates the detail of step SB. It is a flowchart which illustrates the detail about the other example of step SB. It is a flowchart which illustrates the detail of step SC. It is a flowchart which illustrates the detail about the other example of step SC. It is a graph which shows the outline | summary of the process which calculates | requires 1st measurement time from the measurement of oxygen current. It is a flowchart of step SA in the 2nd Embodiment of this invention. It is a graph which shows the 1st aspect from which the value of oxygen current falls rapidly. It is a graph which shows the 2nd aspect from which the value of oxygen current falls rapidly. It is a graph which shows the 3rd aspect from which the value of oxygen current falls rapidly. It is a flowchart which shows a part of microorganisms number measuring method concerning the 3rd Embodiment of this invention. It is a flowchart which shows the detail of step SA in the 3rd Embodiment of this invention. It is a flowchart which illustrates the detail of step SA in the 3rd Embodiment of this invention. It is a flowchart which illustrates the detail of step SC in the 3rd Embodiment of this invention. It is a flowchart which illustrates the detail of step SC in the 4th Embodiment of this invention. It is a block diagram which shows the structure of the microorganisms number calculation apparatus which can apply this invention.

Explanation of symbols

I1 to IZ + 1, Inew oxygen current data Id second threshold value IP first threshold value Knew oxygen current slope q coefficient ts first measurement time tt second measurement time Z1, Z2 predetermined value

Claims (28)

In the medium containing the microorganism whose initial number is to be measured, the oxygen current (Inew), which is the current that flows in accordance with the amount of oxygen, is sequentially measured (S101, S112), and the oxygen current sets the first threshold (IP) as a predetermined value. A method for obtaining a first measurement time (ts), which is a time taken to decrease beyond the number of times (Z2),
(A) obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA);
(B) a step (SB) of determining whether or not the time difference value obtained in the step (a) is stable;
(C) When the determination in the step (b) is affirmative, it depends on a value based on the oxygen current (I1 to IZ + 1) measured in the most recent measurement period and is lower than the value. A method of measuring the number of microorganisms comprising a step of setting a first threshold (S114a, S114b).   2. The microorganism according to claim 1, wherein in the step (c), the first threshold (IP) is set by subtracting a predetermined threshold (Δ) from the oxygen current (IZ + 1) measured most recently. Number measurement method.   The first threshold value (IP) in the step (c) is set by multiplying the most recently measured oxygen current (IZ + 1) by a coefficient (q) greater than 0 and less than 1. 2. The method for measuring the number of microorganisms according to 1.   The method according to claim 3, wherein the coefficient (q) is set to 0.8. In the medium containing the microorganisms whose initial number is to be measured, the oxygen current (Inew), which is the current that flows according to the amount of oxygen, is sequentially measured (S101, S112), and the decrease in the oxygen current exceeds the threshold (E3). A method for obtaining a first measurement time (tu) that is gradual,
(A) obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA);
(B) a step (SB) of determining whether or not the time difference value obtained in the step (a) is stable;
(C) When the determination in step (b) is affirmative, the difference value (Knew) measured thereafter is an increment that exceeds the threshold value with respect to the difference value (Kold) in step (b). A step of determining the measurement time based on the measurement period measured most recently (SC).   In the step (b), it is determined that the time difference value is stable because the absolute value (| Knew |) of the time difference value falls within a predetermined range (E2) within a predetermined period (S108b, S109). The method for measuring the number of microorganisms according to any one of claims 1 to 5.   In the step (b), the absolute value of the variation of the time difference value with respect to time (| Knew−Kold | / Δt) falls within the predetermined range (E1) for a predetermined period (S108a, S109), thereby the time difference value. The method for measuring the number of microorganisms according to any one of claims 1 to 5, wherein it is determined that is stable. The predetermined range (E1) is 0.6 nA / mm ² / min ² in terms of current density,
The predetermined number of times (Z2) is 10 times,
The method for measuring the number of microorganisms according to claim 7, wherein the predetermined period is a length in which the measurement of the oxygen current is continuously performed five times (Z1).   9. The method according to claim 1, wherein, in the step (b), it is determined whether or not the time difference value is stable after a predetermined dead time has elapsed after the step (a). The method for measuring the number of microorganisms according to one.   The method for measuring the number of microorganisms according to claim 9, wherein the dead time is set to 200 minutes. 11. The method according to claim 1, further comprising a step of obtaining a second measurement time (tt) that is a time required for the oxygen current to decrease beyond the second threshold (Id). The method for measuring the number of microorganisms according to one. The method for measuring the number of microorganisms according to claim 11, wherein the second threshold value (Id) is 60 nA / mm ² / min ² in terms of current density. Calibration obtained for the microorganism and the culture medium from the relationship between the first measurement time and the known initial quantity for each of the same type of culture media whose known initial numbers are different from each other and known. Based on the line
The initial number that was unknown is calculated by obtaining the first measurement time for the culture medium of the same species in which the initial number of the microorganism is unknown. The method for measuring the number of microorganisms described in 1. Determining the first measurement time for each of the same types of media in which the initial numbers for the microorganisms are different and known;
The microorganism according to any one of claims 1 to 12, wherein the calibration curve for the microorganism and the culture medium is obtained from a relationship between a plurality of the known initial number and a plurality of the first measurement times. Number measurement method. An oxygen current measuring unit (201) that sequentially measures (S101, S112) an oxygen current (Inew) as a current that flows in accordance with the amount of oxygen in a medium containing microorganisms whose initial number is to be measured;
An evaluation unit (202) for obtaining a first measurement time (ts), which is a time required for the oxygen current to decrease beyond the first threshold (IP) by a predetermined number (Z2),
The evaluation unit is
(A) obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA);
(B) a step (SB) of determining whether or not the time difference value obtained in the step (a) is stable;
(C) When the determination in the step (b) is affirmative, it depends on a value based on the oxygen current (I1 to IZ + 1) measured in the most recent measurement period and is lower than the value. An apparatus for measuring the number of microorganisms that executes the step of setting a first threshold (S114a, S114b).   16. The microorganism according to claim 15, wherein in the step (c), the first threshold (IP) is set by subtracting a predetermined threshold (Δ) from the oxygen current (IZ + 1) measured most recently. Number measuring device.   The first threshold value (IP) in the step (c) is set by multiplying the most recently measured oxygen current (IZ + 1) by a coefficient (q) greater than 0 and less than 1. 15. The apparatus for measuring the number of microorganisms according to 15.   The apparatus for measuring the number of microorganisms according to claim 17, wherein the coefficient (q) is set to 0.8. An oxygen current measuring unit (201) for sequentially measuring an oxygen current (Inew) as a current flowing according to the amount of oxygen in a medium containing microorganisms whose initial number is to be measured;
An evaluation unit (202) for obtaining a first measurement time (tu) in which the decrease in the oxygen current becomes gentler than a threshold value (E3),
The evaluation unit is
(A) obtaining a time difference value (Knew) that is a difference per unit time of the oxygen current from a measured value of the oxygen current in a predetermined measurement period (SA);
(B) a step (SB) of determining whether or not the time difference value obtained in the step (a) is stable;
(C) When the determination in step (b) is affirmative, the difference value (Knew) measured thereafter is an increment that exceeds the threshold value with respect to the difference value (Kold) in step (b). When the number of microorganisms increases, the step of determining the measurement time based on the latest measurement period (SC) is executed.   In the step (b), it is determined that the time difference value is stable because the absolute value (| Knew |) of the time difference value falls within a predetermined range (E2) within a predetermined period (S108b, S109). The apparatus for measuring the number of microorganisms according to any one of claims 15 to 19.   In the step (b), the absolute value of the variation of the time difference value with respect to time (| Knew−Kold | / Δt) falls within the predetermined range (E1) for a predetermined period (S108a, S109), thereby the time difference value. The apparatus for measuring the number of microorganisms according to any one of claims 15 to 19, which determines that is stable. The predetermined range (E1) is 0.6 nA / mm ² / min ² in terms of current density,
The predetermined number of times (Z2) is 10 times,
The apparatus for measuring the number of microorganisms according to claim 21, wherein the predetermined period is a length in which the measurement of the oxygen current is continuously performed five times (Z1).   20. In the step (b), it is determined whether or not the time difference value is stable after a predetermined dead time has elapsed after the step (a). The apparatus for measuring the number of microorganisms according to one.   The apparatus for measuring the number of microorganisms according to claim 23, wherein the dead time is set to 200 minutes. The evaluation unit (201)
25. The method of any one of claims 15 to 24, further comprising the step of: (d) obtaining a second measurement time (tt) that is a time required for the oxygen current to decrease beyond the second threshold value (Id). Measuring device for the number of microorganisms described in 1. 26. The apparatus for measuring the number of microorganisms according to claim 25, wherein the second threshold value (Id) is 60 nA / mm ² / min ² in terms of current density. Calibration obtained for the microorganism and the culture medium from the relationship between the first measurement time and the known initial quantity for each of the same type of culture media whose known initial numbers are different from each other and known. Based on the line
27. The initial number that is unknown is calculated by obtaining the first measurement time for the medium of the same species in which the initial number of the microorganism is unknown. The apparatus for measuring the number of microorganisms described in 1. Determining the first measurement time for each of the same types of media in which the initial numbers for the microorganisms are different and known;
The microorganism according to any one of claims 15 to 26, wherein the calibration curve for the microorganism and the culture medium is obtained from a relationship between a plurality of the known initial number and a plurality of the first measurement times. Number measuring device.

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