JP2017060451A - Method and apparatus for sterilization testing - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test method and apparatus capable of quantitatively evaluating the bactericidal effect of a bactericidal agent that fumigates within a facility.SOLUTION: The present invention provides a method for a bactericidal effect evaluation, the method comprising: dropping dilutions S0 to S7 of respective predetermined dilution series D0 to D 7 of microorganisms resistant to drying onto a substrate 11 and drying and attaching them thereon to provide a test substrate 10 having the initial bacteria number No; placing the test substrate 10 in a facility 1 that fumigates it with a f bactericidal agent M for a predetermined time, followed by incubation with addition of a culture medium C and a sterilization neutralizer E; and determining the number N of surviving bacteria from the microbial growth state at each dilution stage D0 to D7 on the test substrate 10 to evaluate a bactericidal effect. Preferably, the present invention provides a method for a bactericidal effect evaluation, the method comprising: repeating a plurality of times a cycle of dropping dilutions S0 to S7 of the respective dilution series D0 to D7 in rows onto a substrate 11 and drying and attaching them thereon to provide a test substrate 10; calculating the number of surviving bacteria by the MPN method from the number of positive (+) microbial growth in the row of each of the dilution series D0 to D7 on the test substrate 10 to which a culture medium C and a sterilization neutralizer M are added after being placed in a facility 1 to evaluate a bactericidal effect.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a sterilization test method and apparatus, and more particularly to a test method and apparatus for evaluating the sterilization effect in a facility fumigated with a disinfectant.

  In facilities such as hospitals, dispensing pharmacies, pharmaceutical manufacturing factories, food manufacturing factories, research laboratories that use laboratory animals, etc. where maintaining a sanitary environment is important, facilities such as clean rooms with controlled microbial concentrations may be established. . Such facilities are designed so that they are less likely to be contaminated with microorganisms. However, it is required to completely sterilize the facilities at the time of new start-up, at the end of the experiment, at the time of periodic cleaning, etc. For example, formalin gas Fumigation sterilization in which a facility is filled with a bactericide such as (formaldehyde gas) and left for a required time is performed (see Patent Document 1). Formalin gas has a sterilizing power that kills not only general bacteria but also viruses, microbes that make spores, etc., and it can also be sterilized to every corner of the facility by entering small gaps and ceilings. Has advantages.

In fumigation sterilization, for example, after opening an opening communicating with the outside of the facility, the sterilizing agent is introduced into the facility in the form of gas or mist (fog) and filled, and the microbial concentration in the entire facility is sufficiently reduced. Just leave it alone. The fumigation time is set so that it can be sterilized to every corner of the facility (for example, 24 hours or more), but if necessary, a base material (for example, 10 ³ to 10 ⁸ ) of microorganism spores retained (for example, The bactericidal effect can be confirmed using a bioindicator (BI) that combines a filter paper or the like and an ampoule with a medium (see Non-Patent Document 1). That is, the bioindicator is placed in a place where the disinfectant in the facility before fumigation is most difficult to reach, and after fumigation, the bioindicator base material is immersed in an ampoule containing a medium and cultured. If the sterilization in the facility is complete, the microorganisms will not grow, so the color of the bioindicator medium will not change, but if the sterilization is incomplete, the microorganisms will grow and the color of the medium will change. By this, it can be confirmed whether or not the entire sterilization in the facility has been completed (whether or not a predetermined number of microorganisms have been annihilated).

  However, formalin gas is toxic (irritant to the skin, etc.) and has recently been designated as a regulated subject due to carcinogenicity (see Non-Patent Document 2), so it requires careful handling and fumigation. It is necessary to take measures against the remaining residue. For this reason, switching from formalin gas to alternative disinfectant in fumigation sterilization has been promoted, a method using chlorine dioxide gas (see Non-Patent Document 3), a method using hydrogen peroxide gas, a method using ozone gas (Non-Patent Document) 4), a method of spraying a diluted solution of peracetic acid water in a mist form (see Non-Patent Document 5), a method using hypochlorous acid (see Non-Patent Document 6), a method using methanol gas (Non-Patent Document 7) ) Etc. have been proposed.

JP-A-2005-323809

Cosmo Bio Co., Ltd. “ACEtest Biological Indicator (for high-pressure steam sterilization) specification manual” August 2015 search, Internet <http: // search. cosmobio. co. jp / cosmo_search_p / search_gate2 / docs / FUK_ / H372.2014140121. pdf> Ministry of Health, Labor and Welfare “2008 Report of Risk Assessment Study Group on Prevention of Workers' Health Injuries Caused by Chemical Substances (About Formaldehyde at Medical Sites)” November 2008, Internet <http: // www. mhlw. go. jp / bunya / roudukijun / anzenisei17 /> Ikari disinfection Co., Ltd. “Chlorine dioxide gas generator Minidox-M Gas Generator” August 2015 search, Internet <https: // www. ikari. co. jp / service / pdf / microbe_nisankatanso. pdf> Taisei Corporation "I want to introduce a more effective method for aseptic manufacturing room fumigation" Search in August 2015, Internet <http: // librytaisei. jp / library / mono / medicine / fujiyakuhin / index_02. html> Ozu Sangyo Co., Ltd. “Peracetic acid disinfectant MINNCARE ACTIL” search in August 2015, Internet <http: // www. ozu. co. jp / products / pdf / mincare_01. pdf> Peace Tech Co., Ltd. “Hypochlorous acid Microtect” search in August 2015, Internet <http: // peacetech-co. com / items / steri / index. html> Wing Turf Co., Ltd. “Environmental sterilization of MRG system” August 2015 search, Internet <http: // www. winturf. com / pdf / MRG1. pdf> Mitsuo Takano / Rio Yokoyama "Food Disinfection-Science and Technology" fortunate bookstore, published on June 25, 1998, p63 Halvorson, H.M. O. and Ziegler, N .; R. “Application of statistics to problems in bacteriology: I. A means of deterministic population by the distilling method”, Journal of Bactolol. 25 No. 2, February 1933, pp 101-121

  However, the conventional fumigation sterilization described above reduces the microorganism concentration in the facility, but has a problem that the sterilization effect is not quantitatively evaluated. In other words, the conventional bioindicator used in formalin fumigation evaluates the bactericidal effect only by endotoin, which is the annihilation of the predetermined number of retained microorganisms, and even if the number of microorganisms surviving after fumigation is one digit, it grows. If the color of the medium changes, it must be evaluated as incomplete sterilization. For this reason, the conventional method tends to be excessively sterilized, and the fumigation time increases, the residue after fumigation also increases, and the building material deteriorates. From the viewpoint of efficiently sterilizing the inside of a facility with the minimum fumigation time, development of a test technique that can quantitatively evaluate the sterilization effect is required.

  In addition, quantitative evaluation of the sterilizing effect in the facility is important even when the above-described alternative disinfectant of formalin gas is used. That is, these are methods of filling the facility with an alternative disinfectant instead of formalin gas, but the fumigation time and fumigation method are not necessarily the same as formalin gas. It is necessary to set a fumigation time for each disinfectant that does not cause excessive sterilization within the range that can be sterilized in every corner of the facility, and quantitative evaluation of the bactericidal effect is necessary for designing the fumigation time. In addition, when spraying the sterilizing agent in a mist rather than in a gaseous state, it is assumed that the propagation in the facility (the manner of entering the gap) and the sterilizing action are different from those of formalin gas. In order to design an appropriate fumigation method, quantitative evaluation of the bactericidal effect is important.

  Accordingly, an object of the present invention is to provide a test method and apparatus capable of quantitatively evaluating the bactericidal effect of a bactericide that is fumigated in a facility.

  Referring to the embodiment of FIG. 1, the sterilization test method according to the present invention drops each of the dilutions S0 to S7 of a predetermined dilution series D0 to D7 of a microorganism resistant to drying onto the base material 11 and attaches them to dryness. The test substrate 10 having the initial bacterial count Np (= N0 to N7) is used (see FIGS. 1A and 1B), and the test substrate 10 (11 + S0 to S7) is fumigated with the disinfectant M in the predetermined facility 1 After setting for time t, culture medium C and bactericidal neutralizing agent E are added and cultured (see FIGS. 1C and 1D). From the microbial growth state at each of the dilution stages D0 to D7 on the test substrate 10 The number of surviving bacteria Nt or the survival rate Nt / Np is obtained to evaluate the bactericidal effect (see FIGS. 1E and 1F). The microorganism that is resistant to drying can be, for example, a microorganism in which spores are formed during drying.

  Referring also to the embodiment of FIG. 1, the sterilization test apparatus according to the present invention drops each of the dilutions S0 to S7 of the predetermined dilution series D0 to D7 of microorganisms that can withstand drying onto the base material 11 and causes them to dry and adhere. The initial substrate number Np (= N0 to N7) of the test substrate 10 (see FIG. 1 (B)), and the medium C and bactericidal neutralizer E (see FIG. 1 (D)) added to the test substrate 10 It is provided, and after the test substrate 10 is fumigated with the fungicide M in the facility 1 for a predetermined time t, the medium C and the fungicide neutralizer E are added and cultured (FIGS. 1C and 1D). 1), the number of surviving bacteria Nt or the survival rate Nt / Np is determined from the microbial growth state at each of the dilution stages D0 to D7 on the test substrate 10, and the bactericidal effect is evaluated (FIG. 1 (E ) And (F)). The microorganism that is resistant to drying can be, for example, a microorganism in which spores are formed during drying.

In a preferred embodiment, as shown in FIG. 2, each of the dilutions S0 to S7 of the dilution series D0 to D7 is dropped on the substrate 12a in a line and dried and adhered in a matrix by repeating a plurality of times. 10 (12a + S0 to S7), and the number of microorganisms in each dilution stage D0 to D7 on the test substrate 10 to which medium C and bactericidal neutralizer M were added after installation in the facility 1 was positive (+) by the MPN method. Determine the number of surviving bacteria. For example, as shown in FIG. 3, the dilutions S0 to S7 of D0 to D7 are dropped on the base material 12a in a row and attached in a matrix by repeating N times (N is an integer of 6 or more). In the dilution stage including both the positive (+) and negative (−) microorganism growth on the test base material 10 to which the base material 10 (12a + S0 to S7) is set and the medium C and the bactericidal neutralizing agent M are added after the installation in the facility 1 From the number P (N>P> 0) of positive (+) microbial growth, the number of surviving bacteria Nt is obtained by the following equation (1).
Number of surviving bacteria Nt = ln (N / (NP)) ………………………… (1)

  In a further preferred embodiment, as shown in FIGS. 4 to 6, a sealing covering material 15 for the test substrate 10 loaded with the medium C and the sterilizing neutralizer E is provided. The medium C and the bactericidal neutralizer E are added simultaneously with sealing with the covering material 15. For example, as shown in FIG. 5, the base material 11 is a microplate 12a with a lid 12b in which a matrix of depressions is formed, and the dilutions S0 to S7 of the dilution series D0 to D7 are used as the lid 12b or the microplate 12a. The test substrate 10 ((12b + S0 to S7) or (12a + S0 to S7)) was dropped on the substrate in a row and dried and adhered in a matrix by repeated multiple times, and the medium C and the bactericidal neutralizer E were added to the microplate 12a. Alternatively, it is loaded on the lid member 12b, and after being installed in the facility 1, the microplate 12a and the lid member 12b are brought into close contact and cultured.

  In the sterilization test method and apparatus according to the present invention, each of the dilutions S0 to S7 of a predetermined dilution series D0 to D7 of a microorganism that is resistant to drying is dropped on the base material 11 and attached to the substrate 11 by dry adhesion. N7) is used as the test base material 10 and after the test base material 10 is fumigated with the bactericide M for a predetermined time t, the medium C and the bactericidal neutralizer E are added and cultured. Since the bactericidal effect is evaluated by obtaining the survival cell count Nt or the cell viability Nt / Np from the microorganism growth state in each of the above dilution stages D0 to D7, the following advantageous effects are obtained.

(A) Since the survival cell count Nt or the cell viability Nt / Np is obtained from the microbial growth state after the fumigation sterilization of the test substrate 10 having the initial cell count Np to which the dilution series D0 to D7 are attached, the cell viability The bactericidal effect can be quantitatively evaluated from Nt / Np or the number of killed digits (= log (Nt / Np)).
(B) Further, the number Nt of surviving bacteria is obtained by the MPN method from the microorganism growth state after the fumigation sterilization using the test substrate 10 in which the dilution series D0 to D7 are attached in a plurality of rows (matrix) instead of one row. Thus, the detection accuracy of the survival cell count Nt can be increased, and the accuracy of quantitative evaluation of the bactericidal effect can be improved.
(C) The test substrate 10 can be as small as a palm, and a plurality of test substrates 10 are installed in the facility 1 fumigated with the bactericide M, and the number of surviving bacteria Nt is determined every predetermined time. By detecting continuously, the optimal fumigation time for every disinfectant M can be designed.

(D) In addition, the evaluation of sterilization effect at multiple locations in the facility can be detected as a quantitative distribution of the survival rate of the bacteria, rather than the evaluation using only the end points as in the conventional bioindicator. It is also possible to design a fumigation method according to the difference from the mist-like fungicide M.
(E) The culture medium C and the bactericidal neutralizing agent E are loaded on the covering material 15 for sealing the test base material 10, and the medium C and the bactericidal neutralizing agent E are added at the same time that the test base material 10 is sealed with the coating material 15. Thus, the sterilization time for each dilution series D0 to D7 of the test substrate 10 can be made uniform.
(F) In addition, by combining a system that automatically seals the test substrate 10 with the covering material 15, for example, in a facility that is filled with a strong disinfectant M and cannot be accessed by humans, It becomes possible to detect the number Nt of bacteria with high accuracy.

Hereinafter, embodiments and examples for carrying out the present invention will be described with reference to the accompanying drawings.
These are explanatory drawings of one Example of the sterilization test apparatus of this invention. These are explanatory drawings of the other Example of the sterilization test apparatus of this invention. These are explanatory drawings of other Example of the sterilization test apparatus of this invention. These are explanatory drawings of the Example of the bactericidal test apparatus of this invention which loaded the culture medium and the bactericidal neutralizer in the sealing coating material of the test | inspection base material. These are explanatory drawings of the other Example of the bactericidal test apparatus of this invention which loaded the culture medium and the bactericidal neutralizer in the sealing coating material of the test | inspection base material. These are explanatory drawings of other Example of the sterilization test apparatus of this invention which loaded the culture medium and the sterilization neutralizer in the sealing coating material of the test | inspection base material. These are explanatory drawings of the Example of the sterilization test apparatus of this invention using the culture | cultivation unit type | mold test | inspection base material.

  FIG. 1 shows an embodiment in which the sterilizing effect of the facility 1 fumigated with the sterilizing agent M is evaluated using the sterilization test apparatus of the present invention. As shown in FIG. 1 (B), the sterilization test apparatus of the illustrated example includes an assay base material 10 having an initial bacterial count Np in which a predetermined dilution series D0 to D7 of microorganisms is attached to the base material 11, and FIG. 1 (D). As shown in Fig. 5, the medium C and the bactericidal neutralizer E added to the test substrate 10 are included. As shown in FIG. 1 (C), after setting the test substrate 10 for a predetermined time t in the facility 1 fumigated with the bactericidal agent M, the medium C and the bactericidal neutralizing agent E are added and shown in FIG. 1 (E). Thus, the survival bacteria count Nt or the bacteria survival rate Nt / Np is obtained from the microorganism growth state of each dilution stage D0 to D7 of the test substrate 10, and the bactericidal effect in the facility 1 is evaluated.

1A and 1B show a method for producing the test substrate 10. First, as shown in FIG. 1 (A), a microorganism resistant to drying is diluted with 10 milliliters of sterilized distilled water to prepare a sample D0 having a predetermined concentration per unit amount, and 9 milliliters of sterilized distilled water is added to each of seven test tubes. Dispense one by one, and dilute 1 ml of sample D0 10 times with sterile distilled water into 7 stages to prepare diluted solutions S0 to S7 of 8-stage dilution series D0 to D7. Each dilution series D1-D7 contains 10 ⁻¹ to 10 ⁻⁷ times as many microorganisms as the dilution series D0 per unit amount. Next, as shown in FIG. 1 (B), each of the dilutions S0 to S7 of the dilution series D0 to D7 is dropped on the substrate 11 by d milliliters (for example, 0.005 milliliters (5 microliters)) and dried. (For example, it is air-dried), and the test | inspection base material 10 to which the microbe of the initial bacterial count Np adhered is produced. It is not practical to specify the assay substrate 10 (and the initial bacterial count Np) directly from the structure or characteristics, and the assay substrate 10 can be identified by the dilution series D0 to D7 to be attached by drying.

  In the present invention, the initial bacterial count Np means the number of microorganisms adhering to the test substrate 10 when the dilutions S0 to S7 of the dilution series D0 to D7 are dropped. It means the original number of bacteria when it is not installed inside. As shown in the example, when the dilutions S0 to S7 of the 8-stage dilution series D0 to D7 are dropped on the test substrate 10, microorganisms with different numbers N0 to N7 contained in each dilution S0 to S7 are tested. It adheres to the base material 10, and the initial bacterial count Np at each dilution stage is N0 to N7. In counting the number of bacteria after installation in the facility, the survival cell count Nt or the cell survival rate Nt / Np is calculated from the microbial growth state at any dilution stage.

  The reason for using microorganisms that can withstand drying is that the microorganisms of each of the dilution series D0 to D7 are brought into contact with the disinfectant M in the air in the facility 1 in a state where moisture has been lost. This is because the diluted solutions S0 to S7 dropped on the base material 11 are dried to obtain the test base material 10 in order to avoid the influence on the base material 11. Microorganisms resistant to drying are, for example, aerobic bacteria (for example, Bacillus genus, Geobacillus genus, Alicyclobacillus genus, etc.), anaerobic bacteria (for example, Clostridium genus, etc.), fungi in which spores are formed during environmental stress such as dryness and starvation. It may be a spores of certain fungi (for example, Aspergillus genus, Penicillium, etc.), spores of protozoa and algae, cysts, dormant cells, and the like. However, it is possible to use microorganisms that can withstand drying for a relatively short period of time without forming spores. When dry cells of microorganisms can be used, or when cells cannot be completely dried. E. coli (Escherichia coli NBRC3972) or the like can also be used.

  Further, in the illustrated example, a small synthetic resin plate of the size of a palm having eight depressions (wells) formed in a row as the substrate 11 is used, and the dilution solutions S0 to S7 of the eight-stage dilution series D0 to D7 are arranged in a row. The test substrate 10 is attached. By reading the microbial growth state of the plurality of dilution stages D0 to D7 from the test base material 11, the survival cell count Nt at the installation position of the test base material 11 can be obtained. However, for example, as shown in FIG. 6 (B), the dilution solutions S0 to S7 of the respective dilution series D0 to D7 may be attached to separate base materials 11 to form the test base material 11. In this case, it is necessary to use the test substrate 10 of each dilution stage D0 to D7 as a set so that the sterilization position and sterilization time of the test substrate 10 of each dilution stage D0 to D7 do not shift. is there. Note that the number of dilution series may be less than 8 or more than 8.

  After the prepared test substrate 10 is installed at a required position in the facility 1, as shown in FIG. 1 (C), the facility 1 is filled with the disinfectant M in the form of gas or mist as in the conventional fumigation sterilization. Let The installation position of the test substrate 10 can be a plurality of locations in the facility 1 as well as the location where the disinfectant M is hard to reach. Unlike the conventional detection of end point, which is the annihilation of a predetermined number of bacteria such as a bioindicator, the assay base material 10 in FIG. 1 (B) can detect the quantitative bacteria survival rate at the installation position. The bactericidal effect can be evaluated as the distribution of the bacterial survival rate in the facility 1 by installing the test substrate 10 at the position and detecting the bacterial survival rate at each point.

  FIG. 1 (D) shows a treatment in which the test substrate 10 installed in the facility 1 for a predetermined time t is taken out of the facility 1 and added with the medium C and the bactericidal neutralizer (bactericidal reaction terminator) E and cultured. Is shown. As the medium C, a medium suitable for each microorganism can be appropriately selected, but a general bouillon medium (NB medium) can also be used. In addition, the sterilizing neutralizer E is filled with the sterilizing agent M in the test base material 10 installed in the facility 1, and if the test base material 10 is cultured while the sterilizing agent M is left, the number of accurate survival bacteria Since Nt cannot be obtained, the effect of the remaining fungicide M is eliminated. The sterilizing neutralizer E can be appropriately selected and used according to the type of the sterilizing agent M (see Table 1). In addition, since the NB medium contains peptone (protein), the NB medium can function as the medium C and the neutralizing agent E for the bactericidal agent M in which the protein functions as a neutralizing agent.

FIG. 1 (E) shows an example of the culture result of the assay substrate 10 to which the medium C is added, and the growth of microorganisms is detected (positive) at the dilution stages D0 to D4 (10 ⁰ to 10 ⁻⁴ ). This shows that the growth of microorganisms is not detected (negative) in the dilution stages D5 to D7 (10 ⁻⁵ to 10 ⁻⁷ ). Since the dilution stage D4 is the initial bacterial count N4 and the dilution stage D5 is the initial bacterial count N5, the survival rate Nt / Np after the fumigation sterilization treatment from the microbial growth state in FIG. 1 (E) is (1 / N5 ) Or more and less than (1 / N4), and the bactericidal effect in the facility 1 can be quantitatively evaluated by the bacteria survival rate Nt / Np.

  FIG. 1 (F) shows an evaluation apparatus 20 that calculates the bactericidal effect in the facility 1, that is, the survival rate of microorganisms, from the microbial growth state at each of the dilution stages D0 to D7 of the test substrate 10 shown in FIG. 1 (E). Examples of An example of the evaluation device 20 is a computer having an input device 21, an output device 22, and a storage unit 23. The storage means 23 stores the above-mentioned initial bacterial count Np (= N0 to N7) and the like. In addition, the computer 20 in the illustrated example has, as a built-in program, a survival rate calculation unit 25 that calculates the bacterial survival rate from the microorganism growth state of the test substrate 10 and an output unit 26 that appropriately outputs the calculated bacterial survival rate to the output device 15. And have.

  In the evaluation apparatus 20 of FIG. 1 (F), the microorganism growth state (dilution stage in which the microorganism growth of FIG. 1 (E) is detected) of each dilution stage D0 to D7 of the test substrate 10 is survived via the input device 21. When input to the rate calculation means 25, the survival rate calculation means 25 calculates the bacteria survival rate Nt / Np and displays it on the output device 22 via the output means 26. By installing the test base material 10 at a plurality of positions in the facility 1 and storing each installation position in the storage means 23 and inputting the microbial growth state of the test base material 10 at the plurality of positions, the output means 26 sets the facility. The distribution of the survival rate of bacteria within 1 can also be illustrated on the output device 22. In addition, a plurality of test substrates 10 are installed at each position in the facility 1, and the test substrate 10 is taken out every predetermined time and the microbial growth state is input. Changes in survival over time can also be evaluated.

  FIG. 2 shows another embodiment of the sterilization test apparatus of the present invention for evaluating the sterilization effect of the facility 1 fumigated with the sterilizing agent M. The sterilization test apparatus of FIG. 2 is also composed of the test substrate 10 (see FIG. 2 (B)), the medium C, and the sterilization neutralizer E (see FIG. 2 (D)). Using the formed microplate as a base material 12a, each dilution solution S0 to S7 of the eight-stage dilution series D0 to D7 is dropped in a line and repeated multiple times and dried (for example, air-dried), thereby performing an assay. Dilution solutions S0 to S7 of dilution series D0 to D7 are attached to the substrate 10 in a matrix.

  As described above, the survival rate Nt / Np after fumigation sterilization can be detected using the assay substrate 10 of FIG. 1 (B) with the dilution series D0 to D7 attached in a row, but the number of dead digits can be detected. It is difficult to improve the detection accuracy. On the other hand, the most probable method (MPN method) for culturing the dilution series of the sample N times instead of in a single line, and determining the number of survival in the sample from the microbial growth state at each dilution stage in each of the N times of repetition Has been developed (see Non-Patent Document 8). When the dilution is repeated, the probability that the number of viable bacteria becomes 0 comes out, but the probability that the number of K viable bacteria becomes 0 after repeating a certain dilution stage N times is calculated from the binomial distribution. The MPN method is a method for estimating the number of inoculated microorganisms from the probability of the presence or absence of growth when a sufficiently dilute microorganism suspension is repeatedly inoculated and cultured in a fresh medium.

  Specifically, the number of repetitions N is set to 3 times, and the number K1 of growth detected at the lowest dilution stage in which all or most of the N times had grown microorganisms, and the growth at the next dilution (10-fold dilution) stage. The number of detected cells K2 and the number of detected proliferations K3 in the next dilution (100-fold dilution) stage are respectively obtained, and the number of viable bacteria in the stock solution is estimated from the code [K1, K2, K3] in which the three numbers are arranged. . The estimated value from this code [K1, K2, K3] is called the most probable number (MPN). If an MPN table as shown in Table 2 is prepared in advance, the code [K1, K2 is compared with the MPN table. , K3] can be converted into the survival cell count Nt. In the system with 3 iterations N, the number of MPN tables is 64. However, the number of iterations can be further increased to improve accuracy, and in the system with 5 iterations N, there are 216 MPN tables.

  2 (A) and 2 (B) use a microplate in which eight depressions (wells) are formed in three rows as a base material 12a, and use dilution liquids S0 to S7 of an eight-stage dilution series D0 to D7 in a matrix. A method for producing the test substrate 10 by attaching to (3 × 8) is shown. First, as in the case of FIG. 1, dilution liquids S0 to S7 of a dilution series D0 to D7 of microorganisms that can withstand drying are prepared. Next, each of the dilutions S0 to S7 is dropped into each well on the substrate 12a in the form of d milliliters (for example, 5 microliters) in a row, repeated three times, and further dried (for example, air-dried) to be assayed substrate. 10 (12a + S0 to S7) is produced. However, the number of repetitions is not limited to three, and for example, a microplate to which five or more repetitions can be attached can be used.

  After the test substrate 10 produced as shown in FIG. 2 (C) is placed in the facility 1 where the fumigant M is fumigated for a predetermined time t, each well of the test substrate 10 as shown in FIG. 2 (D). Medium C and bactericidal neutralizer E are added to the culture medium. FIG. 2 (E) shows an example of the culture result of the assay substrate 10 to which the medium C has been added. The lowest dilution stage D3 in which microbial growth was detected in all three repetitions (positive). The code [3, 2] is calculated from the number K1 (= 3) of growth detection, the number K2 (= 2) of growth detection in the next dilution stage D4, and the number K3 (= 0) of growth detection in the next dilution stage D5. 0] is obtained. Referring to Table 2, since the MPN corresponding to this code is 0.93, the survival cell count Nt (= 0.93) of the dilution stage D4 at the center of the code can be obtained. The bacterial survival rate Nt (= 0.93 / N4) can be calculated from the initial bacterial count Np = N4.

  FIG. 2 (F) shows an embodiment of the evaluation device 20 that calculates a quantitative bacterial survival rate as a bactericidal effect in the facility 1 from the codes [K1, K2, K3] obtained in FIG. 2 (E). The evaluation device 20 of FIG. 2 is also configured by a computer, and the MPN table T as shown in Table 2 is stored in the storage unit 23 together with the initial number of microorganisms Np. Further, as a built-in program, in addition to the survival rate calculation means 25 and the output means 26, there is a survival bacteria count calculation means 24 for calculating the survival bacteria count Nt from the microorganism growth state of the test substrate 10. When the code [K1, K2, K3] is input from the input device 21 to the evaluation device 20, the survival cell count calculation means 24 calculates the survival cell count from the code [K1, K2, K3] and the MPN table T as described above. Nt is calculated, and the survival rate calculation means 25 calculates the bacteria survival rate Nt / Np and displays it on the output device 22 via the output means 26. As shown in FIG. 2, by using the assay base material 10 in which the dilution series D0 to D7 are attached in a plurality of rows (matrix) instead of one row, the number of surviving bacteria is obtained by the MPN method, thereby detecting the survival rate of the bacteria. The accuracy of quantitative evaluation of the sterilizing effect in the facility 1 can be improved as compared with the case of FIG.

Preferably, as shown in FIG. 3, the number of iterations N of the MPN method is set to 6 times or more, and each dilution solution S0 to S7 of the dilution series D0 to D7 is dropped in a line shape and adhered in a matrix form by repeating 6 times or more. Thus, the test substrate 10 is produced. Compared with the case where the number of iterations N is 3 as shown in FIG. 2, the detection accuracy of the bacteria survival rate can be further increased by making the number of iterations N 6 or more. However, the MPN table of the system having the number of iterations N of 6 or more is very large, and although it is not impossible to create, it is complicated. On the other hand, as an MPN method that does not require an MPN table, there is a method for obtaining the survival cell count Nt by the following equation (1) from the state of the dilution stage in which the growth of microorganisms includes both positive (+) and negative (−). It has been developed (see Non-Patent Document 9). In formula (1), N represents the number of repetitions, and P represents the number of positive (+) microorganism growth (N>P> 0) among the number of repetitions N.
Number of surviving bacteria Nt = ln (N / (NP)) ………………………… (1)

  In FIG. 3A, a microplate in which eight depressions (wells) are formed in six rows is used as the base material 12a, and, as in FIGS. 2A and 2B, an eight-stage dilution series D0 to D7. The test substrate 10 with the number of repetitions N = 6 produced by attaching the dilution solutions S0 to S7 in a matrix (6 × 8) is shown. After the prepared test substrate 10 is placed in the facility 1 where the fungicide M is fumigated for a predetermined time t, the medium C and the bactericidal neutralizer E are added to each well and cultured. FIG. 3 (B) shows an example of the culture result of the assay substrate 10 to which the medium C is added, and shows that the dilution stage D4 includes both positive (+) and negative (−) microorganism growth. . In the dilution stage D4, the number of positive (+) microorganism growth is 1 among the number of repetitions N = 6. Therefore, the number of surviving bacteria Nt (= ln (6 / (6-1) in the dilution stage D4 according to the equation (1). )) ≈0.182), and the bacterial survival rate Nt (= 0.182 / N4) can be calculated from the initial bacterial count Np = N4 at the dilution stage D4.

FIG. 3 (C) shows an example of the result of culturing the test substrate 10 of FIG. 3 (A) without fumigation sterilization treatment. The initial bacterial count Np of the test substrate 10 can be calculated from the dilution series D0 to D7 when the test substrate 10 is produced as described above, but as shown in FIG. 3 (B) in consideration of force majeure during the experiment. It can be confirmed by culturing a non-sterilized untreated assay substrate 10. Specifically, in the illustrated example, the dilution stage D6 includes both positive (+) and negative (−) microorganism growth, and in the dilution stage D6, the number of microbial growth positive (+) out of the number of repetitions N = 6. Therefore, the survival cell count N (= ln (6 / (6-2)) ≈0.405) at the dilution stage D6 can be obtained from the equation (1). The number of bacteria Np (= 0.405 × 10 ³ ) can be calculated. Accordingly, by simultaneously culturing the test substrate 10 after sterilization treatment shown in FIG. 3B and the test substrate 10 not treated with sterilization shown in FIG. 3C, the survival rate Nt (= 0.182) /0.405×10 ³ ) can be obtained to evaluate the bactericidal effect.

  For example, in the evaluation apparatus 20 of FIG. 2 (F), the number of iterations N = 6 is stored in the storage means 23 instead of the MPN table T, and the test substrate 10 after the sterilization treatment as shown in FIG. By inputting the number P of microbial growth positive (+) at the dilution stage D4 from the input device 21 to the evaluation device 20, the survival cell count calculation means 24 calculates the survival cell count Nt by the equation (1), and the survival The rate calculating means 25 can calculate the bacteria survival rate Nt / Np. Further, by inputting the number P of microbial growth positive (+) at the dilution stage D6 of the sterilization-untreated test substrate 10 as shown in FIG. 3C from the input device 21 to the evaluation device 20, the survival rate calculating means 25 can also calculate the bacterial viability Nt / Np by calculating the initial bacterial count Np at the dilution stage D4.

  Thus, it is possible to achieve the “test method and apparatus capable of quantitatively evaluating the bactericidal effect of a bactericide that is fumigated in a facility”, which is an object of the present invention.

  In FIG. 4, a coating material 15 for sealing the test substrate 10 is provided, the culture material C and the sterilizing neutralizer E are loaded on the coating material 15, and the test substrate 10 is sealed with the coating material 15. 10 shows an example in which medium C and bactericidal neutralizer E are added. 1 to 3, the test substrate 10 installed in the facility 1 is taken out after a lapse of a predetermined time t, and the medium C and the bactericidal neutralizer E are added. There was a possibility that some deviation was caused in the addition timing of the medium C and the bactericidal neutralizing agent E for each of the dilution series D0 to D7. As shown in FIG. 4, the medium 15 and the bactericidal neutralizing agent E are loaded into the covering material 15 that seals the test base material 10, so that the test base material 10 is sealed with the covering material 15 and at the same time the dilution series D0 to D7. The medium C and the bactericidal neutralizing agent E can be added, and the detection error of the survival cell count Nt due to the shift in the addition timing can be avoided.

  FIG. 4A shows an assay substrate 10 produced using a synthetic resin plate 11 in which eight depressions (wells) are formed in a row, and FIG. 4B shows the same type for sealing the assay substrate 10. The sheet-like coating material 15 is shown. The coating material 15 in the illustrated example includes a coating material sheet 15a having a medium C and a bactericidal neutralizing agent E applied on one side surface, and a pollution prevention film 15b that is detachably attached to the surface of the medium C and the bactericidal neutralizing agent E. It is comprised by. For example, after the test substrate 10 of FIG. 4 (A) is installed in the facility 1 for a predetermined time t, the contamination prevention film 15b is peeled off from the surface of the coating material sheet 15a of FIG. 4 (B), and FIG. As shown in FIG. 2, the medium application surface of the coating material sheet 15a is pressed against the test substrate 10 to be coated.

  According to the method of sealing the verification base material 10 with the covering material 15 as shown in FIG. 4, the trouble of taking out the verification base material 10 installed in the facility 1 to the outside is saved, and the covering material 15 is held when a predetermined time t has elapsed. A worker who has entered the facility 1 can easily add the medium C and the sterilizing neutralizer E. In addition, if a base material sealing system 30 as shown in FIG. 4D is installed in the facility 1, for example, in the facility 1 where a strong disinfectant M is filled and a person cannot enter, It is also possible to automatically seal the test substrate 10 with the covering material 15 at the time when the predetermined time t elapses and add the medium C and the bactericidal neutralizer E.

  The substrate sealing system 30 shown in FIG. 4 (D) is coated so that the annular belt on which the test substrate 10 is placed is driven when a predetermined time t has passed, and the test substrate 10 is opposed to the substrate transport roller 31. The belt conveyor apparatus includes a covering material conveying roller 32 that conveys the material 15 and a film peeling roller 33 that peels the contamination prevention film 15b from the covering material 15. The test substrate 10 placed on the annular belt is fumigated as it is until a predetermined time t. When the predetermined time t has elapsed, the base material transport roller 31, the coating material transport roller 32, and the film peeling roller 33 are simultaneously driven. The film peeling roller 33 peels the contamination prevention film 15 b from the coating material 15, and the coating material transport roller 32. As a result, the medium-coated surface of the coating material sheet 15a is pressed against the test substrate 10 on the annular belt for coating.

  FIG. 5 shows an example in which a microplate 12a with a lid member 12b in which a matrix of depressions is formed and each dilution S0 to S7 of a predetermined dilution series D0 to D7 is attached in a matrix on the lid member 12b. An example is shown in which the material 10 (12b + S0 to S7) is formed, the medium C and the sterilizing neutralizing agent E are loaded on the microplate 12a, and the lid material 12b and the microplate 12a are brought into close contact after the fumigation sterilization. In this embodiment, the microplate 12a functions as a covering material 15 that seals the lid member 12b that is the test base material 10, and by sealing the test base material 10 with the cover material 15 as in the case of FIG. Medium C and bactericidal neutralizer E can be added to the assay substrate 10.

  FIG. 5A shows a matrix (3 × 8) of dilute solutions S0 to S7 of the 8-stage dilution series D0 to D7 on the lid 12b of the microplate 12a, as in FIG. 2B. 1 shows an assay substrate 10 made by adhering. FIG. 5 (B) shows the coating material 15 produced by loading the medium C and the bactericidal neutralizing agent E into the matrix (3 × 8) wells of the microplate 12a. For example, after the test base material (lid material) 10 of FIG. 5 (A) is installed in the facility 1 for a predetermined time t, the test base material is covered with a covering material (microplate) 15 as shown in FIG. 5 (C). By sealing 10, the medium C and the bactericidal neutralizing agent E are simultaneously added to each dilution series D0 to D7 of the test substrate 10.

  As shown in FIG. 5, the diluents S0 to S7 are attached on the flat lid member 12b to form the test substrate 10, so that the diluents S0 to S7 are attached to the depressions (wells) as shown in FIG. Compared with the case where the test substrate 10 is used, the microorganism can be brought into contact with the bactericide M evenly when the test substrate 10 is installed in the facility 1 and fumigated. That is, for example, when the disinfectant M is in the form of mist, the microorganisms attached in the well (well) may not contact the mist evenly, whereas the microorganisms attached on the flat lid 12b If there is, it can be evenly contacted with the mist. For this reason, the improvement of the sterilization effect evaluation accuracy can be expected by using the smooth test substrate 10 as shown in FIG. However, for example, when the bactericidal agent M is gaseous, the microplate 12a and the lid member 12b are exchanged, the test substrate 10 is produced from the microplate 12a, and the medium C and the bactericidal neutralizer E are produced from the lid member 12b. It is also possible to produce a coating material 15 loaded with (see Example 3 in FIG. 6).

  FIG. 6 shows an embodiment in which an assay base material 10 with a lid is used, the culture medium C and the sterilizing neutralizer E are loaded on the lid, and the lid is closed and closely adhered after fumigation sterilization. In this embodiment, the lid of the test base material 10 functions as the coating material 15, and the test base material 10 is sealed with the coating material 15 in the same manner as in FIGS. C and bactericidal neutralizer E can be added. FIG. 6 (A) shows an assay substrate 10 with a lid 15 made using a synthetic resin plate 11 in which a plurality of depressions (wells) are formed in a row, and FIG. 6 (B) shows one depression ( The test base material 10 with the lid | cover material 15 formed using the synthetic resin plate 11 in which the well) was formed is shown. When each of the dilution stages D0 to D7 is an individual test substrate 10 as shown in FIG. 6 (B), for example, one set of the test substrate 10 of each dilution stage D0 to D7 is shown in FIG. 6 (C). Can be used so that there is no deviation in the sterilization position and sterilization time of the test substrate 10 in each of the dilution stages D0 to D7.

  The base material sealing system 30 in FIG. 6C includes a base material transport roller 31 that drives an annular belt on which the test base material 10 is placed when a predetermined time t has elapsed, and a test base material 10 that is transported on the conveyor belt. It is comprised by the belt conveyor apparatus which has the polygon roller 34 which closes and close | closes the cover material 15 of this. The verification base material 10 placed on the annular belt is sterilized by fumigation with the lid 15 open until a predetermined time t, and the base material transport roller 31 is driven and moved when the predetermined time t elapses. The test substrate 10 is covered by the lid member 15 coming into contact with and closing the polygonal roller 34 on the path. As shown in FIG. 6 (C), covering the covering material 15 and sealing the test base material 10, the medium C and the bactericidal neutralizing agent E can be simultaneously added to each dilution series D0 to D7 of the test base material 10. it can. Note that the substrate sealing system 30 in FIG. 5C is not limited to the assay substrate 10 in which the dilution stages D0 to D7 are individually attached as shown in FIG. 6B, but also as shown in FIG. 6A. It is also applicable to the assay substrate 10 in which the dilution stages D0 to D7 are attached in a row.

  FIG. 7 shows an embodiment of a culture unit type base material 40 in which a granular test base material 10 is used and a medium C and a bactericidal neutralizing agent E are incorporated in a cylindrical container 41 that houses the test base material 10. FIG. 7 (A) shows a process for producing the test substrate 10 by attaching the dilutions S0 to S7 of the dilution series D0 to D7 to the granular substrate 13. The produced granular test | inspection base material 10 is accommodated in the cylindrical container 41 as shown in FIG.7 (B). A diaphragm 43 is stretched in the intermediate portion in the depth direction of the cylindrical container 41, and the inside of the cylindrical container 41 is partitioned by the diaphragm 43 into a region on the opening side and a region on the bottom wall 42 side. A medium C and a sterilizing neutralizing agent E are loaded in the side region of the bottom wall 42, and the granular test substrate 10 is stored in the region on the opening side. For example, the plurality of depressions (wells) of the microplate 12a as shown in FIG. 5B are replaced with cylindrical containers 41 as shown in FIG. 7B, respectively, and the medium C and the bactericidal neutralizing agent E are placed in each well. It can be set as the culture | cultivation unit type base material 40 incorporated.

  FIG. 7C shows a lid member 44 that is closely attached to the cylindrical container 41. The lid member 44 in the illustrated example is provided with a needle 45 that protrudes inward when covered with the cylindrical container 41. When the lid member 44 is closed as shown in FIG. By breaking through the diaphragm 43, the medium C and the bactericidal neutralizing agent E built in the bottom wall 42 can be permeated to the opening side. The needle 45 is preferably provided with a permeation promoting groove 46 that promotes permeation of the medium C and the bactericidal neutralizer E. The lid member 44 is preferably provided with a scatter prevention skirt 47 that covers the periphery of the needle 45 so that the medium C and the sterilizing neutralizer E do not scatter when the needle 45 breaks through the diaphragm 43. For example, the needle 34 and the skirt 47 can be attached to the positions opposite to the recesses of the microplate lid member 12b as shown in FIG.

  The culture unit type base material 40 of FIG. 7 is sterilized by fumigation of the test base material 10 stored in a cylindrical container (for example, a well of a microplate) 41 with the lid material 44 open until a predetermined time t, and the predetermined time t At the time of elapse of time, the cover material (for example, the cover material of the microplate) 44 is closed to seal the test substrate 10. Simultaneously with closing of the lid member 44, the needle 45 of the lid member 44 breaks through the diaphragm 43 of the container 41, so that the medium C and the sterilizing neutralizer E can be permeated and added to the assay substrate 10.

DESCRIPTION OF SYMBOLS 1 ... Sterilization facility 10 ... Test | inspection base material 11 ... Base material 12a ... Base material (microplate) 12b ... Base material (cover material of a microplate)
DESCRIPTION OF SYMBOLS 13 ... Base material (granular base material) 15 ... Coating material 15a ... Coating material sheet 15b ... Antifouling film 15c ... Hinge 20 ... Evaluation apparatus (computer) 21 ... Input device 22 ... Output device 23 ... Storage means 24 ... Surviving bacteria Number calculation means 25 ... fungus survival rate calculation means 26 ... output means 30 ... base material sealing system 31 ... base material transport roller 32 ... coating material transport roller 33 ... film peeling roller 34 ... polygon roller 40 ... culture unit type base material 41 ... Cylindrical container 42 ... Bottom wall 43 ... Diaphragm 44 ... Lid 45 ... Needle 46 ... Permeation promoting groove 47 ... Anti-scattering skirt C ... Medium E ... Neutralizing agent D ... Dilution series M ... Disinfectant N ... Repeat number Np ... First Number of bacteria Nt ... Surviving bacteria number P ... Positive number S ... Diluent T ... MPN table

Claims (12)

Each dilution of a predetermined dilution series of microorganisms that can withstand drying is dropped onto the base material and dried to adhere to the test base material for the initial bacterial count. The test base material is placed in a facility that is fumigated with a bactericide for a predetermined time. And then culturing with the addition of a culture medium and a sterilizing neutralizer, and determining the number of surviving bacteria or viability from the microbial growth state at each dilution stage on the test substrate, and evaluating the sterilization effect. . 2. The sterilization test method according to claim 1, wherein the microorganism that is resistant to drying is a microorganism in which spores are formed during drying. 3. The method according to claim 1 or 2, wherein each dilution solution of the dilution series is dropped in a row on the substrate and dried and adhered in a matrix by repeating a plurality of times to form an assay substrate, and the medium after installation in the facility And a sterilization test method in which the number of surviving bacteria is determined by the MPN method from the number of positive microbial growth at each dilution stage on the test substrate to which a sterilizing neutralizer is added. The method according to claim 3, wherein each dilution of the dilution series is dropped on the substrate in a row and attached in a matrix by repeating N times (N is an integer of 6 or more) to form an assay substrate, Sterilization by obtaining the number of surviving bacteria from the number P of positive microbial growth at the dilution stage including both positive and negative of microbial growth on the test substrate to which medium and bactericidal neutralizer are added after installation in the facility by the following formula Test method.
Number of surviving bacteria = ln (N / (N−P)) 5. The method according to claim 1, wherein the covering material for sealing the test base material is loaded with the culture medium and the bactericidal neutralizing agent, and the test base material after installation in the facility is sealed with the covering material. A sterilization test method comprising adding a culture medium and a sterilization neutralizer. 6. The method according to claim 5, wherein the base material is a microplate with a lid member in which a matrix of depressions is formed, and each dilution liquid in the dilution series is dropped in a row on the lid member or microplate and repeated a plurality of times. Sterilized by drying and adhering in a matrix form as a test substrate, loading the medium and bactericidal neutralizer on a microplate or lid, and incubating the microplate and lid after intimately installing in the facility Test method. A test substrate of the initial number of bacteria that has been dripped onto the substrate and each diluted solution of a predetermined dilution series of microorganisms resistant to drying is attached to the substrate, and a medium and a bactericidal neutralizing agent to be added to the test substrate. After the test substrate is fumigated with a bactericidal agent for a predetermined time, the medium and a bactericidal neutralizing agent are added and cultured. A sterilization test device that evaluates the sterilization effect by determining the number of bacteria or the survival rate of bacteria. 8. The sterilization test apparatus according to claim 7, wherein the microorganism that is resistant to drying is a microorganism in which spores are formed during drying. 9. The apparatus according to claim 7 or 8, wherein each dilution solution of the dilution series is dropped in a row on the substrate and dried and attached in a matrix by repeating a plurality of times to form an assay substrate, and the medium after installation in the facility. And a sterilization test apparatus for determining the number of surviving bacteria by the MPN method from the number of positive microbial growth at each dilution stage on the test substrate to which a sterilizing neutralizer is added. The apparatus according to claim 9, wherein the dilution series of the dilution series is dropped on the base material in a row and attached in a matrix by repeating N times (N is an integer of 6 or more) to form a test base material, Sterilization by obtaining the number of surviving bacteria from the number P of positive microbial growth at the dilution stage including both positive and negative of microbial growth on the test substrate to which medium and bactericidal neutralizer are added after installation in the facility by the following formula Test equipment.
Number of surviving bacteria = ln (N / (N−P)) The apparatus according to any one of claims 7 to 10, wherein a sealing covering material for the test base material loaded with the culture medium and a bactericidal neutralizing agent is provided, and the test base material after installation in the facility is sealed with the covering material. At the same time, a sterilization test device to which a culture medium and a sterilization neutralizer are added. 12. The apparatus according to claim 11, wherein the base material is a microplate with a lid member formed with a matrix of depressions, and the dilutions of the dilution series are dropped in a row on the lid member or microplate and repeated a plurality of times. Sterilized by drying and adhering in a matrix form as a test substrate, loading the medium and bactericidal neutralizer on a microplate or lid, and incubating the microplate and lid after intimately installing in the facility Test equipment.