Process for visually measuring a microbial activity state during real time

A method of measuring microbial activity of microbes in real time includes preparing a medium to enable microbial activity and placing the medium in operative contact with an appropriate sensor. The sensor is scanned to provide measurement electrical signals representative of position and amount of microbial activity, the sensor being responsive to a by-product of the microbial activity, such as a change in pH. The measurement signals can then be processed to provide a visual image display, and the display can visually distinguish between levels of pH activity and coordinate positions of the microbial activity.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a process for measuring the state of microbial 
activities and more particularly a real time measurement with displayed 
images. 
2. Description of Related Art 
The controlled activities of microorganisms are extensively used in a wide 
variety of industrial fields, such as the brewing of liquors and wines, 
manufacturing of drugs, restoration of environmental pollution, etc. On 
the other hand, undesirable 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 a process (for example, the brewing process) and an 
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. 
However, in any of the above-mentioned cases, evaluations are made on the 
results in which microorganisms have already carried out some activities 
or microorganisms have finished their activities, and a considerable time 
is required to have the results, and it is not possible to evaluate the 
state of microbial activities in real time. Thus, there is still a need to 
improve the measurement of microbial action. 
OBJECTS AND 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 a process for 
measuring the state of microbial activities which can be evaluated in real 
time. 
In order to achieve the above-mentioned object, a process for measuring the 
state of microbial activities according to this invention (hereinafter 
simply called the "measuring method") is characterized by allowing 
microorganisms to work or incubating microorganisms in the process or 
environment in which the microorganisms are expected to work, and 
observing the change of chemical substances varied by the metabolism of 
microorganisms as two-dimensional images, as well as numerically 
identifying the activity state of the microorganisms based on image 
changes. 
In actuality, it has become possible to directly observe the state of 
microbial activities without waiting for the final results by determining 
the change in chemical substances near the microorganism which are caused 
by metabolism 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 
measurement, either of the following can be obtained. 
(1) Ratio of concentration change in chemical substances to time at the 
point in which the chemical substance concentration is either the maximum 
or the minimum; 
(2) Ratio of the change in the area to time by finding the area of the 
portion in which the chemical substance concentration is higher or lower 
than the specified value; 
(3) Ratio of the total change to time by finding the total change in 
chemical substances; and 
(4) Ratio of propagation of one individual microorganism by counting the 
population of the portion in which the chemical substance concentration is 
higher or lower than the specified value. 
These values serve as indices of a large or small metabolism of the 
microorganism, enabling the numerical evaluation of the degree of 
microbial activities. 
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 the 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 "Improvement of 
Spatial Resolution of a Laser-Scanning pH-Imaging Sensor", Japan Journal 
of Applied Physics, Vol. 33 (1994), pages L394-L397, by Nakao et al. and 
incorporated herein by reference. 
The distribution of concentration of the dissolved substance is measured by 
inserting the sensor surface of the 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 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 two-dimensional concentration 
distribution image with the electromagnetic wave image and to determine 
the correlationship between the concentration distribution of the chemical 
substance caused by the microorganism and the actual existing position of 
the microorganism.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The following description is provided to enable any person skilled in the 
art to make and use the invention and sets forth the best modes 
contemplated by the inventors of carrying out their invention. Various 
modifications, however, will remain readily apparent to those skilled in 
the art, since the generic principles of the present invention have been 
defined herein specifically to provide a process for measuring a microbial 
activity state. 
Referring now to the drawings, the present invention will be described in 
detail hereinafter. 
Embodiment 1 
Referring now to FIGS. 1 and 2, an example in which the measuring process 
according to this invention is applied to a food manufacturing process, 
such as fermentation and brewing, is described. In the process in which 
microbial capabilities are utilized, the microorganism best suited 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 a microorganism is the hydrogen ion, and an 
optical scanning two-dimensional concentration distribution measuring 
apparatus, which is sensitive to pH, is used for the 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 a SiO.sub.2 layer 3 and a Si.sub.3 N.sub.4 layer 4 successively 
formed on one surface (top surface in the illustrated example) of the 
substrate 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.sub.3 N.sub.4 layer 4, 
which is the sensor surface, and are connected to the potentiostat 18, to 
be discussed later, to apply a voltage. OC is an ohmic electrode or 
working electrode installed on the semiconductor substrate 2 for taking 
out current signals, and is connected to a potentiostat 18 which includes 
a stabilizing bias circuit 10, a current-voltage converter 11 and an 
operational amplifier circuit 12, to be discussed later. 
Numeral 5 is a light irradiating portion for irradiating the semiconductor 
substrate 2 of the 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 sensor portion 1 and the 
light irradiating portion 5 and comprises the potentiostat 18 for applying 
suitable bias voltage to the semiconductor substrate 2, the stabilizing 
bias circuit 10, 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 unit 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. As noted on the display 15, the actual images 
16 representative of the position and concentration of the pH distribution 
is generated from the current flow contemporaneously with the X, Y 
position of the scanning laser beam in real time. The rings are indicative 
of different levels of pH and, for example, could be color coded. Display 
15 also provides a pH scale to correlate the level of pH relative to the 
video images. Thus, the content and position of the visual images, as 
displayed in real time, enable the operator to practice different methods 
of evaluating the activity of specific microorganisms by observing the 
changes in pH rather than counting the number of microorganisms. By 
visualizing two-dimensional concentration distribution images of a 
chemical concentration (for example, pH), the operator can readily 
determine, in an economical manner, the state of microbial activities. 
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 a condition suitable 
for microbial activities in a suitable container 21, is housed in a Petri 
dish 24 and is designated as gel 25. On the other hand, as shown FIG. 
2(B), 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 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 the 
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 potentiostat 18 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 specified bias voltage is applied to the 
semiconductor substrate 2. The semiconductor substrate 2 with is 
irradiated with a probe light 6 intermittently at given intervals (for 
example, 10 kHz) so that an alternate current optical current is generated 
in the semiconductor substrate 2. The intermittent frequency of the light 
scan can remove background noise from the signal. 
In addition, by moving the light irradiating portion 7 in an 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 can be 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 the 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 logarithmical 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 until then be T.sub.4 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.sub.4 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 a smaller T.sub.4 and a 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.sub.4 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 may be a case in which the pH value 
change first occurs at 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 point 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. As in the 
case of Embodiment 1, an incubation of microorganism B is allowed to take 
place and two-dimensional concentration distribution images of a chemical 
substance discharged are successively obtained. The chemical substance 
measured is a hydrogen ion, as that in Embodiment 1, but may be any other 
chemical substances. 
Microorganism B increases in population as incubation takes place for a 
specified time period and forms a colony 29 of 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 the 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 
of 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 FIG. 4(B). Again, as in the method described 
above, it is fixed to the sensor surface 4 of the optical scanning sensor 
portion 1 of the 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 a microorganism increases 
the population as time passes by carrying out the initial microorganism 
incubation and the 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.sub.b '), and letting the logarithmic value of the then-increment be S', 
the microorganism with a smaller T.sub.b ' and a larger S' can be regarded 
as the microorganism which provides the capabilities to efficiently carry 
out the intended process, and therefore, using these values, the screening 
and optimum activity conditions of microorganism B can be obtained. 
Embodiment 3 
In this embodiment, a 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 a microbial agent. The contamination varies in forms and requires a 
more suitable screening of microorganism B and a 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 hydrogen ion, 
but 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 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, 4c, . . . , 4n so that several 
types of candidate microorganisms B can be simultaneously evaluated, and 
in the relevant measuring zones 4a-4n, different microorganisms B.sub.1, 
B.sub.2, B.sub.3, B.sub.4, . . . B.sub.n (not illustrated) are incubated, 
and their activity state is observed. After each of the microorganisms 
B.sub.1 -B.sub.n is diluted with a suitable solution, it is applied to 
each of the measuring zones 4a-n and thereafter, the placement of the soil 
32 to be decontaminated on each zone, as shown in FIG. 5(B), can fix the 
relevant microorganisms B.sub.i -B.sub.n. Any measurement after this stage 
should be carried out in the same manner as in Embodiment 1. 
The measurement can be made in the same manner not only in screening of the 
optimum microorganism B, but also in the process of determining the 
conditions in which the screened microorganism B can perform from being 
inactive to having the maximum activity. However, in this event, the 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. 
Instead of placing the soil 32 which is to be decontaminated on the sensor 
surface 4, screening is possible by directly embedding the sensor portion 
1 with each of the microorganisms B.sub.1 -B.sub.n applied to the soil to 
be decontaminated. 
Embodiment 4 
All of the above-mentioned embodiments describe the manner in which the 
chemical substance to be changed by the metabolism of microorganism B is 
hydrogen ion, but instead of this substance, 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 case, the sensor surface 40 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, the manner in which the existence of harmful 
microorganisms is found is described. The sample used in this method is 
generally a used sample, 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 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 substances 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 a chemical substance differs 
around this colony from that at the start of the 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 a 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 a chemical substance 
is generated, that is, to identify the correlationship between the 
chemical information and the 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 research on vital 
reactions, etc. 
Additionally, in the above-mentioned optical scanning two-dimensional 
concentration distribution measuring apparatus, a 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 an easy, quick, 
and real-time evaluation of the activity state of the microorganisms, 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 the screening the an optimum microorganism in various processes 
in which microorganisms are utilized, as well as to determine the optimum 
activity conditions of the microorganism. This also enables a quick 
determination of the existence of harmful microorganisms. 
Those skilled in the art will appreciate that various adaptations and 
modifications of the just-described preferred embodiment can be configured 
without departing from the scope and spirit of the invention. Therefore, 
it is to be understood that, within the scope of the appended claims, the 
invention may be practiced other than as specifically described herein.