JPS615798A - Method of counting living cells and device therefor - Google Patents

Abstract

PURPOSE:The culture mixture is transferred in order using a diluter and, every operation of transfer, the diluter is washed and sterilized to effect high-accuracy measurement of living cell counts of a microorganism. CONSTITUTION:A sample containing a microorganism is poured into the sample cell 6b in the culture plate 6 and the culture mixture containing an indicator is poured into another culture cell 6a'. The driving means 4 is operated to move table 5 so that the diluter 2 comes right above the water jetter 7 and sterile water is poured into the diluter. Table is moved so that the diluter 2 comes right above cell 6b to mix the sterile water with the sample, then the sample is transferred to the first row of culture cells. Then, the diluter is sterilized using a sink 7, wiper 8 and heater 8 and then carries strerile water to the culture cell and the culture mixture is transferred and mixed in the second row of cells.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for measuring the number of viable bacteria such as microorganisms and a device for measuring the number of viable bacteria used in this method.

(Prior art) As a means of counting the number of microorganisms present in a culture solution containing a large number of microorganisms taken out from a microorganism culture tank, an equal amount of culture solution containing no microorganisms is poured into multiple test tubes. A predetermined amount of sample solution containing microorganisms is injected into this prepared test tube, and then this test tube is pipetted with the same amount of culture solution as the sample solution injected above. Then, the sample solution is taken out using a PIT and injected into another prepared test tube, and then injected from the test tube newly injected with the microorganism into another test tube without the microorganism injected in the same way using a pit. The culture solution containing the diluted sample solution at an appropriate f@ rate is transferred to the af7'' rate, after which the microorganisms contained in this culture solution are cultured, and the colonies generated by this are cultured. The most commonly used method is to estimate the a of live microorganisms in the sample solution by counting the number of colonies and multiplying the calculated number of colonies by a dilution factor.

(Problems to be Solved by the Invention) The above-mentioned conventional method for measuring the number of viable bacteria is performed entirely manually, and the operation is extremely complicated.

Furthermore, since the liquid is collected and injected using Viit, dilution cannot be performed at an accurate desired magnification. In other words, human error easily intervenes.Furthermore, -
Biit that has undergone the transfer operation of variable-density nutrient solution cannot be used again unless it is completely washed and sterilized, and for this reason, washing and sterilization is usually performed. It was necessary to prepare il number of bits. Additionally, the dilution operation had to be carried out in a clean room or clean pliers to prevent bacteria from entering the culture solution from outside, which required a large amount of capital investment. Since a tube is used, there is a problem in that a relatively large amount of sample liquid and culture medium are required.

It should be noted that a device is known that automatically serially dilutes a solution using a small piece for dilution (usually called a diluter) having a minute recess at the raw end for holding a liquid and a series of liquid holding cells. However, when attempting to dilute a sample liquid containing live bacteria using this device, the live bacteria that adhered to the diluter during the previous dilution operation are transferred to the subsequent liquid holding cell during the subsequent dilution operation, and the sample liquid is It is not possible to dilute the sample solution simply according to probability theory, and it is not possible to accurately estimate the number of viable bacteria contained in the sample solution.

The object of the present invention is to provide a method for measuring the number of viable bacteria and a device for measuring the number of viable bacteria used in this method, which does not have the problems of the conventional methods and devices described above. That is, an object of the present invention is to provide an apparatus that can automatically dilute a sample liquid containing live microorganisms without human intervention. Another object of the present invention is to provide an apparatus that can dilute a viable microorganism sample solution at an accurate desired magnification.Furthermore, another object of the present invention is to provide an apparatus that can dilute a viable microorganism sample solution at an accurate desired magnification. The objective is to provide a method for measuring the number of viable bacteria that accurately estimates the number of viable bacteria.Also,
Another object of the present invention is to provide a system 4 in which the same parts can be used repeatedly in a regular manner in a device. Furthermore, another object of the present invention is to obtain a viable bacterial count that does not require a clean room. An object of the present invention is to provide an n11 constant device f. Also.

Another object of the present invention is to provide a viable bacteria count measuring method and a viable bacteria count measuring device that make it possible to accurately estimate the viable count of microorganisms contained in a small sample liquid using a small amount of sample liquid. It is about providing.

(Means for Solving the Problem) The method for measuring the number of viable bacteria of the present invention involves inserting and stirring a diluter holding sterile water into a sample liquid containing all viable bacteria held in a sample cell, and stirring the diluter containing sterile water. , a plurality of culture cells are arranged in 0 rows and m columns integrally in an IJ rack shape &[
The culture plate is inserted into a predetermined amount of culture solution containing the indicator held in the culture cells in the first row of the culture plate and stirred, and a predetermined amount of sample solution is transferred from the sample cells to the first row. Transfer to an eye culture cell, wash and sterilize the diluter.

Sterile water is held in this diluter, and this diluter is inserted into the culture solution held in the culture cells in the first row and stirred; this diluter is held in the culture cells in the second row. inserting the diluter into a predetermined amount of culture solution containing an indicator and infusing it, transferring a predetermined amount of the culture solution from the culture cell in the first row to the culture cell in the second row; washing the diluter; The diluter is made to hold sterile water, and the same operation is performed between adjacent cells up to the mth row of culture cells to dilute the sample liquid. Leave it in the incubator, count the number of discolored culture cells in each column, find the x that maximizes , and take the value of x as the number of viable bacteria contained in the sample liquid. Measurement method (however.

rk and Sk indicate the number of discolored cells and the number of non-colored cells in the culture cells in the first column, respectively, and ck satisfies """"s' (I P,)k (1p,) . Here, the volume of the culture solution that does not contain viable bacteria injected into each culture cell is V, the volume of the culture solution transferred by the diluter is vl, and the volume of sterile water transferred into the culture cell by the diluter is vl. Then, ps=□, V+v2 pp==□. ) V + v 1 The first viable cell count measuring device of the present invention includes a casing that isolates the inside from the outside air, a table, a drive device that moves the table left and right, and a diluter that rotates the table and a drive device that moves the table left and right. A culture plate comprising a diluter head for performing migration, a culture plate having a plurality of culture cells arranged in a matrix on the cheagle, a washing water reservoir, a wiper for wiping off the washing water, a heater for drying and sterilizing the diluter, and a diluter for the diluter. A fountain that injects sterile water,
The side wall of the casing on which the culture grate is placed is provided with an empty port provided with an air filter, and a pipe for transporting sterile water to the water fountain. is elastically modified to intermittent the supply of sterile water to the water fountain, and to make the water t-f1ir
A peristaltic pump for ejecting water from the water fountain is provided outside the casing, and the driving device, the diluter head, and the front peristaltic pump are controlled by a control means, so that the peristaltic pump containing live microorganisms is controlled by a control means. Dilute the sample solution.

The second viable cell count measuring device of the present invention includes a casing that isolates the inside from the outside air, a table inside the casing, a drive device that moves the table from side to side, and a diluter that rotates the table fully and moves it up and down. A culture plate having a head and having a plurality of culture cells arranged in a matrix on the table, a washing water reservoir, a wiper for wiping off the washing water, a heater for drying and sterilizing the diluter, an alcohol solution reservoir, and a wiper for wiping off the alcohol. A wiper and a sterile water reservoir are placed in this order, and an air port having an air filter is provided on the side wall of the casing on the side where the culture plate is placed, and the drive device and the die are provided with an air port having an air filter. By controlling the router head using a control means, the sample solution containing viable microorganisms is diluted.

A sixth viable cell count measuring device of the present invention includes a casing that isolates the inside from an outer package, a table in the casing, a ground motion device that moves the table from side to side, and a diluter that rotates the table and moves it up and down. A culture plate having a head, a culture plate having a plurality of culture cells arranged in a matrix on the table, a washing water reservoir, and a washing water supply. :, 'Fiver, alcohol solution reservoir and sterile water reservoir in this order a! The culture grate is placed on the side wall of the casing on which the culture grate is placed.
An air port having a filter is provided, and the sample solution containing live microorganisms is diluted by controlling the drive unit and the diluter head by a control means.

(Effects of the Invention) In the method of the present invention, when the sample solution is completely diluted by successively transferring a desired amount of culture waves to adjacent culture cells using a culture wave sonic diluter, washing is performed every time the transfer operation is completed. Since sterilization is done in a natural manner, viable microorganisms attached to the diluter from the previous operation will not be transferred to the culture cell on the low concentration side. That is, even in a critical culture cell where the transfer of viable bacteria occurs or not, the transfer of viable bacteria is achieved in a completely stochastic manner.

Therefore, based on the result of whether viable microorganisms were transferred or not transferred to each culture cell as a result of the dilution operation, conversely, based on probability theory, the total accuracy of the number of viable microorganisms present in the sample liquid is determined. can be estimated well. Also,
When transferring the culture solution to the adjacent culture cell, the culture solution is transferred after completely injecting non-killed water into the liquid holding recesses of the diluter, which prevents bubbles from forming in the four liquid holding parts of the diluter. However, there is no case where a predetermined amount of culture solution is not transferred to the adjacent culture cell, and a predetermined amount of culture solution is always transferred to the adjacent culture cell, and as a result, viable microorganisms present in the sample solution do not occur. It is possible to estimate the number with high accuracy. Since it is possible to perform the same dilution operation in parallel in the equations, by using these multiple results, it is possible to estimate the number of viable bacteria present in the sample liquid with extremely high accuracy. can.

The device of the present invention can completely automatically dilute a sample solution containing live bacteria without any manual intervention. It is also possible to dilute the sample liquid at an accurate desired magnification. Furthermore, the same parts other than the culture grate can be used repeatedly in a device that is always incorporated into the device. Additionally, since the device of the present invention has a casing, it does not require a clean room or clean pliers. Furthermore, it is possible to accurately estimate the number of viable microorganisms contained in a small sample liquid from a small amount of sample liquid.

(Example) Hereinafter, the present invention will be explained in detail based on examples.

(First Example) FIG. 1 is a schematic overall view showing an example of the first viable cell count measuring device of the present invention.

The viable bacteria count measuring device of the present invention has a casing 1, and the inside of the casing 1 is isolated from the outside. Inside this casing l, there is a diluter head 3 which has minute recesses for liquid retention and which moves and rotates a plurality of diluters 2 equal to the number of rows of culture cells, which will be described later, and a drive device 4 as shown in the figure. A table 5 that can be moved from side to side is arranged. The diluter 2 is shown in Fig. 2A and Fig. 2.
A device having a liquid holding recess 2a at the tip as shown in Figure B can be used. This table 5 includes, from the right side of the figure, a culture deletator in which a plurality of culture cells are integrally arranged in a matrix, a liquid reservoir 7 that holds washing water for washing the diluter 2, and a washing water that adheres to the diluter 2. A wiper 8 for wiping off water, a heater 9 for drying and sterilizing the diluter 2, and a fountain 10 for injecting liquid into the four liquid holding parts of the diluter 2 are provided in this order.

An air inlet 12 provided with an air filter 11 is provided in the side wall of the casing l on the side where the grate 4 is provided, so that sterile air can flow into the interior of the casing l. Air eater 13 is installed on the top of casing 1.
'f, a water storage tank 14 is placed. A silicone tube 15 is extended from this water storage tank 14. This silicone tube 1.5 is divided into two silicone tubes 15' and 15' from the middle layer.
One silicone tube 15' extends to supply water to the liquid reservoir 7, and the other silicone tube 15' extends to supply water to the liquid reservoir 7.
There is a silicon tube f15' on the outside of the casing 1 that supplies water to the fountain lO.

On the side wall of the casing l, there is a pallet f16 that clamps the silicone tube 15' and controls the entire flow of liquid, and a fountain 61' that compresses and elastically deforms the silicone tube 151. A peristaltic pong 17 is provided for spouting water from zero.

As shown in FIG. 5A, the culture grade 6 used in the present invention has a culture cell 6a in row 0 and column m (row 6 and column 9 in the figure) into which a culture solution containing no live microorganisms is injected. It consists of one row of sample cells 6b into which a sample liquid containing live microorganisms is injected. The sample cells 6b do not need to be divided into rows, but may be long and thin grooves as shown in FIG. 5B.
may be placed on the table 5 separately from the culture plate 6.

Next, a dilution operation using a viable cell count measuring device configured as in 1 or more, and a method for estimating the number of viable bacteria contained in a sample solution' f - using Fig. 1 and schematic diagram Fig. 4. explain.

First, a sample liquid containing live microorganisms is injected into the leftmost sandal cell 6b of the culture plate 6, and each culture cell 6 e' e located on the right side of this sample cell 6b is injected.
6 B'... does not contain live microorganisms.

When microorganisms proliferate, metabolites are released into the culture solution. When the pH of the culture solution changes, a predetermined amount of the culture solution containing an indicator that indicates this pH change by a change in color is uniformly injected. In this way, the sample solution and culture medium%
The culture plate 6 into which the sample liquid was injected was placed on the table 5 on the right side of the liquid reservoir 7 so that it was positioned to the left in the figure. When the grate 6 is placed on the table 5 in this way, the culture cell 6a becomes the sample cell 6b with respect to the inlet 12 of the casing l.
If the dilution operation is performed so that the concentration of viable microorganisms in the culture solution in the culture cell decreases as it approaches the upstream side, the air flow from the air inlet 12 will decrease. As a result, viable microorganisms are not blown away from the culture cells on the high concentration side to the culture cells on the low concentration side, so stochastic treatment is not disturbed. In addition, since the diluter that holds the culture solution containing live microorganisms does not transfer over culture cells where the culture solution is not being transferred, live microorganisms may be inadvertently transferred during the dilution operation using the diluter. Since it does not enter the culture cell, this is also extremely advantageous in terms of probabilistic handling.In addition, one row of culture cells in which a culture solution containing no live microorganisms is injected is inserted into the first row of culture cells from the left. , second row of cultured cells, etc.
..., the 0th row culture cell.

By driving the driving means 4 by a control means such as a microcomputer installed inside or outside the apparatus, the table 5vil is moved to the right in the figure, and the diluter 2 is positioned directly above the water fountain 7, and the peristaltic pump is moved. 17 to inject sterile water into the liquid holding recess of the diluter 2. Next, the table 5 is moved so that the sample cell 6b into which the sample liquid is injected is located directly below the /ilutor 2.

The diluter bed 3f is operated to lower the diluter 2, insert the tip of the diluter 2 provided with the liquid holding recess into the liquid, and rotate to mix sterile water, and the liquid in the diluter 2 is mixed with sterile water. Transfer sandal liquid to the four holding parts, then move the diluter 2 upward and move the table 5 to the right in the figure.

Each culture cell in the first row is positioned directly below the Guiluta 2. In this state, the diluter 2 is hung down, the tip of the diluter 2 is inserted into each culture cell in the first row, and the sample liquid held in the liquid holding recess of the diluter 2 is rotated in the culture solution. 'iJf! Stir and mix into the culture solution of each culture cell in one row. Move the stirred and mixed diluter 2 upwards, and then move the table 5 to the right in the figure on the right. Wash it with unsterilized water in the reservoir 7, then wipe off the water adhering to the diluter 2 with the Waino 4-8, and then sterilize and dry it with the heater 9 on all of the diluter 2. Aseptically inject water with a fountain lO,
The diluter 2 heated by the heater 9 is cooled down. Clean in this way.

The diluter, which has been sterilized and has sterile water injected into its liquid holding recess, is again introduced into the first row of culture cells, and the sterile solution held by the diluter is mixed with the culture solution in the first row of culture cells. After that, a predetermined amount of the culture solution from the first row of culture cells is transferred and mixed into the culture solution of the second row of culture cells. After this operation is completed, the diluter 2 is washed, sterilized, and sterile water is injected in the same manner as described above, and the culture is transferred from the second row of culture cells 68' to the fifth row of culture cells 6a'. The liquid transfer operation is performed.

Thereafter, the operation for transferring the culture solution is carried out in the same manner as described above up to the culture cell in the n-th column. In addition, in the dilution operation described above, a predetermined amount of culture solution containing no live bacteria was injected into each culture cell 6a', 6a'+, etc. at once. A culture solution containing an indicator but not containing live bacteria may be injected into an adjacent culture cell, and in this way, the time used for the dilution operation can be shortened. In order to achieve this, it is desirable to provide a dripping means in the apparatus for dripping the culture solution into each culture cell. A material wrapped in graph wool 8b is preferred because it can be used repeatedly. Also heater 9
However, instead of heating only the liquid holding recess of the diluter 2, at least three heaters 9a + 9b and 9ce are installed as shown in FIG.
It is preferable that the sides of the wafer are also heated and dried.

Furthermore, when the diluter is introduced into the culture solution history, stochastic processing is also hindered by defining the movement range of the diluter head so that parts other than those sterilized by the heater 9 do not enter the culture solution. In addition, a surfactant (such as Tyeen 20) that is harmless to the growth of viable microorganisms in the culture solution is added to the culture solution. Since it enters the holding recess smoothly without creating bubbles, it is possible to take out and transfer the exact desired amount of culture solution or sterile water.In this way, it is possible to easily remove and transfer the desired amount of culture solution or sterile water. After transferring a predetermined amount of culture solution from the culture cells, the culture solution injected into each culture cell of culture grade 6 is left in an incubator (for example, at 37° C. for 1 day).

The microorganisms in the culture solution are grown. When microorganisms grow, the organic acid released by the microorganisms lowers the pH, which causes the color of the indicator in the culture solution to change. (The live bacteria in the small part of the body that are counted are B. ido bac.
teria - and Bromc as an indicator
resol-green 2! l (when used,
Culture cells containing viable microorganisms turn green. )
Count the number of nine cultured cells that change color in each row,
The number of viable microorganisms Φ present in the sample liquid is estimated, and the method for estimating the number of viable microorganisms present in the sample liquid will be explained below.

X number of live microorganisms and V- of the same size as this microorganism
If one particle is taken from this culture liquid, two microorganisms will be present in this one particle. The probability of containing live bacteria is determined by hypergeometric probability.

(B) It is expressed as

Since V and V are extremely large, the Starling formula can be applied, and equation (1) can be approximated as .

The culture solution dilution operation of the present invention is composed of a combination of five basic operations, and the probability of extracting viable bacteria in these five basic operations is shown below.

(a) As shown in Figure 7a, when a culture solution K with a volume V containing 8 live microorganisms and sterile water with a volume v2 are injected and mixed, and then a culture solution with a volume v2 is taken out. Then, what is the probability that b viable microorganisms are extracted from this culture solution?

P1=(:)Psb(1-Ps)ab(3).

(However, p5 = □.) V + V2 (b) As shown in Figure 7b, the volume v1 containing live microorganisms in % of the culture of body f#v that does not contain live microorganisms. The culture solution was transferred and mixed, and then a volume v1 of the culture solution containing i viable microorganisms was extracted, and then a volume v2 of sterile water was transferred and mixed into i.
The probability of taking out a volume v1 of culture solution containing viable microorganisms can be expressed as follows.

(However, % Pp - 100) Equation (4) is expressed as p2 : [P
2+Ps (1-Pp)] k"'.

(C) As shown in FIG. 7C, a volume v1 of culture solution containing viable microorganisms is transferred and mixed into a culture volume V containing no viable microorganisms, and then i Take out a volume v1 of culture solution containing viable microorganisms 1
Next, after introducing and mixing volume v2 of sterile water, what is the probability that a volume v2 of culture solution containing m viable microorganisms will be taken out?

This equation (5) can be simplified as follows by using the binomial formula.

Furthermore, if there is a phase boundary between the method of holding the culture solution and sterile water in the diluter, the size of the cell that holds the liquid, and the viscosity of the culture solution and sterile water, there will be a difference in the amount of liquid transported by the diluter. , generally vithi
-v2.

From the above formulas (3), (4), and (5), the probability that microorganisms were transferred to the culture cell in the 1st column is given by CI (=
1-Ps"(1-'ρ'p>' (1-Ps)
(It can be expressed as 61.

Therefore, let the number of cells in which viable microorganisms exist in the culture cell group in the %th column be ``k'', and the number of cells in which viable microorganisms do not exist be Sk.

The total probability P7 is It can be expressed as

By determining the value of X that maximizes P7, the number of viable microorganisms in the sample liquid can be estimated.

The value of a that maximizes P7 can be derived by differentiating equation (7) and finding the value of X that satisfies this differential equation, but it is impossible to solve it analytically. In reality, the X value is determined by numerical solution such as the Newton-Raphson method. As can be seen from equation (7), it goes without saying that increasing the number of rows of culture cells improves the probability of the obtained value of X.

(Second Embodiment) FIG. 8 is a diagram schematically showing each element placed on a table in an embodiment of the second viable cell count measuring device of the present invention.

Comparing this embodiment with the seventh embodiment, a liquid reservoir 21 that simply holds alcohol is placed in place of the fountain 1'l that injects sterile water into the diluter.

The difference is that on the left side of this liquid reservoir 21, a liquid reservoir 23 that holds sterile water and a wine 4-22 are arranged.

As shown in the figure, the diluter 2 that has completed the operation of transferring the culture medium to the adjacent culture cell is cleaned in the cleaning liquid reservoir 7, water is wiped off with the wiper 8, and the heater 9 After drying and sterilization, alcohol is applied to the tip of the diluter 2 in the alcohol reservoir 21.
After that, sterilized water is injected into the liquid holding recess of the diluter in the liquid reservoir 23 by a dipstick. After the injection of sterilized water into the diluter 2 is completed, the culture solution is transferred to the next adjacent culture cell. In this way, when the diluter is attached to the diluter, the hydrophilicity of the inner wall of the liquid holding recess of the diluter is distorted and becomes ineffective.

By dipping, a predetermined amount of sterilized water is injected into the liquid holding recess of the diluter.As shown in FIGS. By ejecting the alcohol from the bottom of the reservoir 21 so that the alcohol adheres to all the inner walls of the four liquid holding parts of the diluter 2, it is possible to more reliably transfer a predetermined amount of the culture liquid. The alcohol overflowing from the liquid reservoir 21 is
It is discharged to the outside via the ray 721bft.

(Third Embodiment) FIG. 10 is a diagram schematically showing each element placed on a table in an embodiment of the fifth viable cell count measuring device of the present invention.

This example will be compared with the first example.

In order, Waino F-8K is located adjacent to it.

In accordance with this, the control-mounted sea fence that controls the operation of the device has also been changed, and as shown in the figure, the diluter that has completed the operation of transferring the culture medium to the adjacent culture cell is similar to the first embodiment. , is washed in a cleaning liquid reservoir 7, and water is wiped off with a wiper 8. Next, the tip of the diluter is sterilized with alcohol in an alcohol reservoir 21 as shown in FIGS. 9A and 9B, and then a sterile water reservoir 23 is used.
At , sterile water is injected into the reservoir recess of the diluter. After sterile water is injected into the diluter, the culture solution is transferred to the next adjacent culture cell, similarly to each of the above embodiments.

In this example, the diluter is sterilized by the sterilizing power of alcohol without using a heater, so the four liquid holding parts of the diluter can be coated with a polymeric material, such as silicon coating. This allows a predetermined amount of sterile water to be smoothly transferred to the liquid holding recess of the diluter.

(Fourth Example) Next, a series of systems for determining the number of viable bacteria in sandals using the above-described apparatus and according to the above-described method will be described.

FIG. 11 is a schematic overall diagram showing an example of this system.

The culture grate 6, which has culture cells arranged integrally in a matrix, first enters an automatic droplet device 30, where a culture solution containing an indicator is injected into the culture cells. Next, this culture grade 6 advances to an automatic dilution device 31, where a sample solution containing a large amount of microorganisms is injected into a sample solution reservoir installed in the sample cell or dilution device 31 of the culture grade. The dilution operation described above is performed. After this dilution operation is completed, the grade 6
is transferred to incubator 32, where it is incubated at 57°C for 2
The culture solution is left for 44 hours to allow microorganisms in the culture solution to grow. When microorganisms grow, the organic acid released by the microorganisms lowers the pH of the culture solution, and the indicator in the culture solution changes color in response to this decrease in pH. In this way, the culture plate, in which the culture cells change color depending on the presence or absence of live microorganisms, is processed by the automatic reader 3.
3, where the number of discolored culture cells for each row is optically read and counted. Once the number of discolored culture cells in each row, that is, the number of culture cells to which microorganisms have been transferred and the number of culture cells to which microorganisms have not been transferred in each row, is determined, this data is sent to the computer 34, and is sent to the computer 34 as described above. The number of microorganisms contained in the sample liquid can be determined using the calculated method. The obtained number of microorganisms is output and displayed from the output device 35 in the form of a total number of microorganisms or the number of microorganisms per unit volume, that is, density. This output device includes liquid crystal, LED
You can use any of the photo booth printers. The culture plate after reading is either fixed and stored, or washed and sterilized and used again for fF measurement of the number of viable bacteria.

(Fifth Example) FIG. 12 is a schematic diagram of a circulation system for determining the number of microorganisms in a sample according to the above-described apparatus and method.

A plurality of culture sheets are stacked on a sheet stacker 40, and culture plates 6 are transferred one by one from this sheet stacker 40 to an automatic sample culture liquid dropping and diluting device 41. In this automatic sample culture solution dropping and dilution device 41, a culture solution and a sample containing microorganisms are placed on a culture plate 6.
The culture solution is injected into a predetermined culture cell, and the culture solution is diluted in the same manner as described above. After the dilution operation has been completed, the culture plate is sent to the six-pack incubator 42 and left for a predetermined period of time under predetermined conditions. Microorganisms are grown and the number of discolored cells is counted for each row in an automatic counting device 43, and the number of viable microorganisms in the sample is determined in this automatic counting device 43 based on the method described above. be done. The number of viable microorganisms thus obtained is output from the automatic counting/computing device 43 to an output device such as a printer.

The plate 6 whose viable number of microorganisms has been counted in this way is sent to the plate washer 44 and the culture plate 6
After being cleaned and sterilized, they are again stacked on the stacker 40 and used for circulation.

(Sixth Embodiment) FIG. 13 is a schematic diagram of a system in which the rfi system and the circulation system shown in FIG. 12 are further integrated.

In this embodiment, an automatic diluter 45 is placed on an automatic reader 46 that counts the number of discolored culture cells in each row, and a grate feeder 47 that transfers the grate to each device 45, 46 and these devices. Receive the plate ejected from 45.46.

j [The plate stocker 48 is
An automatic diluter 45° automatic reading device Wt46, a grate feeder 47, and a grate stocker 48 are integrated. great feeder 47
The culture reed plates deposited on the
5, where the sample dilution operation described above is performed. After this dilution operation is completed, the plates are once deposited on the plate stocker 48 and then sent to the incubator 49 for incubation, and then transferred to the grate feeder 47 again. The gray after incubation is then sent to an automatic reader 46,
After the number of discolored cultured cells is counted, they are transferred to the plate stocker 48 again.

The culture plate that has undergone a series of cycles for measuring the number of viable bacteria is transferred from the grate stocker 48 to the washer 50.
After the plates are washed and sterilized,
It is sent again to the great feeder 47 and used for measuring the number of viable bacteria in the next sample. In this embodiment, a computer can be built-in to estimate the number of viable microorganisms in a sample from the number of culture cells in each row obtained by the automatic reader 46. can also be shared with the control means of the diluter,
The whole system can be made very coherent.

[Brief explanation of drawings]

FIG. 1 is a schematic overall view showing an embodiment of the first apparatus of the present invention used in a method for measuring the number of viable bacteria. FIGS. 2A and 2B are a side view and a cross-sectional view, respectively, of a diluter used in the present invention. FIG. 3A and FIG. 5B are schematic diagrams each showing an example of a culture grade. FIG. 4 is a schematic diagram schematically showing the parts placed on the table and the dilution operation t-m in the first apparatus of the present invention, and FIG. 5 is a diagram of the wiper used in the present invention. A schematic diagram showing an example; FIG. 6 is a schematic diagram showing an example of a heater used in the present invention; FIGS. FIG. 8 shows the second embodiment of the present invention.
FIG. 2 is a schematic diagram schematically showing parts placed on a table and a dilution operation in the apparatus. FIG. 98 and FIG. 9B are a plan view and a side view, respectively, showing an example of an alcohol reservoir used in the apparatus of the present invention. FIG. 1a shows a table 1 in a fifth apparatus of the invention.
FIG. 2 is a schematic diagram schematically showing parts placed in a trap and a dilution operation. Figures 11, 12 and 13 are schematic diagrams showing a series of systems for determining the number of viable bacteria in a sample using the apparatus and method of the present invention, respectively. 1...Casing, 2...Diluter, 3...
Diluter head, 4... Drive device, 5... Table, 6-... Culture gray Fs6m... Culture liquid cell,
6b...Sample cell, 7...Washing water reservoir, 8.
... Rai/Coo, 9... Heater, 10... Fountain,
11...Air filter, 12...Empty inlet, 14.
...Water tank, 15...7IJ Contube, 1
6...Pulp, 17...Peristalsis 4nde, 21...
Alcohol liquid reservoir, 22...W/4-12°3...
・Sterile water reservoir, 30...Automatic droplet device, 31.45
...Automatic droplet device, 32.42.49...Ink knee beta, 33.46...Automatic reader, 34...Calculator, 35...Output device, 40... stunker,
41...Automatic sample culture liquid dropping and diluting device, 43...
・Automatic counting device, 44, 50...Plate washer. 47...Plate feeder, 48...Plate tracker: 4 Fig. 2A Fig. 3A Fig. 38 Fig. 4 Fig. 5 Fig. 6 MVa Fig. 8 Fig. 19 AWi Fig. 98

Claims (1)

[Scope of Claims] (1) A diluter holding sterile water is inserted and stirred into a sample solution containing live bacteria held in a sample cell, and this diluter is connected to a plurality of culture cells in n rows and m columns. The first part of the culture plate is arranged integrally in a matrix of
Inserting it into a predetermined amount of culture solution containing the indicator held in the culture cell in the column and stirring, transferring a predetermined amount of the sample solution from the sample cell to the culture cell in the first column, The diluter is washed and sterilized, the diluter is made to hold sterile water, the diluter is inserted into the culture solution held in the culture cell in the first row and stirred, and the diluter is added to the culture solution in the second row. Insert into a predetermined amount of culture solution containing the indicator held in the culture cell and stir to transfer a predetermined amount of culture solution from the culture cell in the first row to the culture cell in the second row. , wash and sterilize the diluter, make the diluter hold sterile water, and then perform the same operation between adjacent cells up to each culture cell in the mth column to dilute the sample liquid; The culture plate was left in an incubator for a predetermined period of time, the number of discolored culture cells in each row was counted, and P = (r_
2+S_1)! /[(r_1)! (S_2)! ](C_
1^x)^r2・(1-C_1^x)^S1x(r_2
+S_2)! [(r_2)! (S_2)! ](C_2^
x)^r2・(1-C_2^x)^S2…x(r_k+
Sk)! /[(r_k)! (S_k)! ](C_k^x
)^rk・(1-C_k^x)^Sk…x(r_m+S
＿m)! /[(r_m)! (S_m)! ](C_m^x
) ^rm・(1-C_m^x)^ A viable cell count measurement method in which x that maximizes Sm is determined and the value of x is the number of viable bacteria contained in the sample liquid (however, r_k and S_k are respectively The number of discolored cells and the number of non-discolored cells in the culture cells in the kth column are shown, and C_k is Ck=1-P_S^k(1-P_p)^k(1-P_S
) is satisfied. Here, the volume of the culture solution that does not contain live bacteria injected into each culture cell is V, the volume of the culture solution transferred by the diluter is v_1, and the volume of sterile water transferred into the culture cell by the diluter is v_2. Then, P_S=(v_1)/(V+v_2)Pp=(V
_1)/(V+v_1). ) (2) A casing that isolates the inside from the outside air, a table,
The table is equipped with a drive device that moves the table from side to side, and a diluter head that rotates the diluter and moves it up and down, and on the table, there is a culture plate having a plurality of culture cells arranged in a matrix, and a washing water reservoir. , a wiper for wiping off washing water, a heater for drying and sterilizing the diluter, and a fountain for injecting sterile water into the diluter are placed in this order, and on the side wall of the casing on which the culture plate is placed, An air port provided with an air filter is provided, and a pipe for transporting sterile water to the water fountain is elastically modified to intermittent the supply of sterile water to the water fountain and to supply water to the water fountain. A peristaltic pump is provided outside the casing to eject live bacteria from the container, and the drive device, the diluter head, and the peristaltic pump are controlled by a control means to dilute the sample solution containing live bacteria. Number measuring device. (3) A casing that isolates the inside from the outside air, and in this casing, a table, a drive device that moves the table left and right, and a diluter head that rotates the diluter and moves it up and down, and a matrix on the table. A culture plate with a plurality of culture cells arranged in a shape, a washing water reservoir, a wiper for wiping off the washing water, a heater for drying and sterilizing the diluter, an alcohol solution reservoir, a wiper for wiping off the alcohol, and a sterile water reservoir are arranged in this order. An air port having an air filter is provided on the side wall of the casing on the side on which the culture plate is placed, and the driving device and the diluter head are controlled by a control means. ,
A viable bacteria count measuring device that dilutes a sample solution containing viable bacteria. (4) A casing that isolates the inside from the outside air, and in this casing, a table, a drive device that moves the table left and right, and a diluter head that rotates the diluter and moves it up and down, and a matrix is placed on the table. A culture plate having a plurality of culture cells arranged in a shape, a washing water reservoir, a wiper for wiping off the washing water, an alcohol solution reservoir, and a sterile water reservoir are placed in this order, and the culture plate of the casing is placed thereon. An air port having an air filter is provided on the side wall where the cell is placed, and the number of viable bacteria is measured by diluting a sample solution containing viable bacteria by controlling the drive device and the diluter head with a control means. Appearance. (5) A culture system having a plurality of culture cells arranged in a matrix, which are used to dilute a sample liquid, at a predetermined location of a viable bacteria count measuring device that performs a dilution operation of a sample liquid containing viable bacteria. A viable bacteria count measuring device comprising a plate feeder that automatically feeds plates according to the sequence of the measuring device.