JP5947476B1 - Method for counting Bacillus bacteria - Google Patents

Abstract

Provided is a method for counting Bacillus bacteria, which uses a hydrophobic grid filter and can easily and easily grasp the count of activated sludge contained in sewage and Bacillus bacteria contained in commercially available Bacillus preparations. . When a hydrophobic lattice filter holding a sample containing a Bacillus bacterium is placed on an agar medium and cultured at a predetermined temperature for a predetermined time, a color-changed colony is formed and formed in the lattice compartment of the hydrophobic lattice filter. From the colony morphology, the number of positive compartments based on Bacillus bacteria is counted. [Selection] Figure 15

Description

  The present invention relates to a method for counting Bacillus bacteria.

  In the human waste treatment plant, high-load processing of human waste is performed, and an operation state (so-called high-load / odorless processing of human waste) that does not have a human waste odor is realized. When the activated sludge of this treatment plant was investigated, the priority of Bacillus bacteria was recognized in the activated sludge by microscopic observation, and the involvement of mineral components such as silicic acid and magnesium was necessary for the priority of Bacillus. It has been known.

Based on such research background, utilization of Bacillus bacteria in sewage treatment, that is, “Bacillus prioritization movement” in which Bacillus bacteria are prioritized in activated sludge to perform sewage treatment has been proposed. For example, in a study group such as A society for study of New Activated Sludge Technology (NAST, secretariat: Osaka Institute of Technology Co., Ltd., Eco Solution Net), an activated sludge method using Bacillus bacteria is used. A symposium is held every year, including functional improvements. For this “Bacillus priority movement”, “Bacillus preparations” prepared from isolates of Bacillus bacteria and “Mineral agents such as silicic acid and magnesium” which are growth promoters of Bacillus bacteria are sold. For example, after adding a Bacillus preparation to activated sludge, a growth promoter for Bacillus bacteria is continuously added.By such activated sludge method with priority to Bacillus, the amount of activated sludge generated can be reduced efficiently. This is thought to be inferred from the results of efficient degradation of starch and cooked meat by in vitro degradation tests performed using Bacillus isolates, but efficiently decomposes organic matter and proteins. The results of the in vitro tests only show how Bacillus bacteria are involved in the organic matter removal mechanism and sludge measurement mechanism in actual activated sludge. It is unknown.

When Bacillus bacteria are cultured on a plate agar medium etc., colonies spreading around the Bacillus are formed and colony counting is difficult, and colonies of other bacteria may be hidden in the Bacillus colonies . In the first place, there is no separation medium common to Bacillus bacteria. Therefore, in the case of “prioritization” of Bacillus bacteria, (1) Microbial observation of activated sludge and the presence of a large amount of microorganisms like Bacillus bacteria (2) The current situation is that the activated sludge is cultivated on an agar medium and the Bacillus-specific colonies are prioritized and so on are referred to as “priority”.
Thus, despite the fact that accurate counts, which are quantification data of the genus Bacillus, have not been grasped, the fact that Bacillus preferential movement is being carried out by the use of Bacillus preparations means that `` high load of human waste ・This is based on the phenomenology of “activated sludge prioritized by Bacillus in odorless treatment facilities” and in vitro degradation test results.

In addition, as a method for treating muddy water, a plurality of flat filter bodies having uniform holes are disposed in the housing so as to be opposed to each other with a space therebetween, and the filter body utilizes a water head difference to store the filter bodies. The turbid water containing the neutralized cement is poured in between and filtered so that the turbidity reduction rate by the water test method is not less than a predetermined value. A processing method has been proposed in which the paper is further extracted (see Patent Document 1).
In addition, as a method for counting dark colonies such as coliforms cultured on a standard agar medium, a method of measuring the number of colonies by attaching and removing a white light scattering plate and enhancing contrast (see Patent Document 2), a medium A method has been proposed in which a dye compound (staining agent) is added in advance and cultured, and the number of bacteria stained with a fluorescence microscope is counted (see Patent Document 3).

JP 2003-144816 A JP 2000-270840 A Japanese Patent Laid-Open No. 10-201496

  In order to clarify the involvement of Bacillus in the evaluation of sewage treatment of Bacillus bacteria and the mechanism of sewage treatment, that is, for the proper evaluation of Bacillus bacteria in sewage treatment, the power of activated sludge using Bacillus bacteria It is necessary to grasp the chemical characteristics (BOD (Biochemical Oxygen Demand) removal rate, yield coefficient, self-degradation coefficient). In addition to grasping the yield coefficient and self-degradation coefficient, the Bacillus bacteria in the activated sludge are accurately quantified. Need to be evaluated. At least in the activated sludge with the addition of Bacillus and minerals, the situation is that the number of Bacillus bacteria increases, the BOD removal rate is high, the yield factor decreases, and the self-degradation factor increases compared to the additive-free series Otherwise, it cannot be claimed that Bacillus bacteria or Bacillus preparations are effective in activated sludge treatment.

  Regarding the kinetic analysis of Bacillus bacteria and the quantification of Bacillus bacteria in activated sludge, there are studies on the kinetic constant calculation using a bench scale model and the quantification of Bacillus bacteria by real-time PCR (Polymerase Chain Reaction). can give. In this study, dynamic constants were obtained by laboratory experiments using synthetic sewage, and in the future, dynamic analysis in actual treatment plants will also be required. In addition, real-time PCR used for Bacillus quantification requires measurement skill, expensive equipment, and large analysis costs. Even from the viewpoint of cost effectiveness, it is extremely difficult to implement as a measurement item in daily maintenance work of activated sludge treatment facilities.

  In addition, no effective method has yet been found for counting Bacillus bacteria in activated sludge contained in sewage, and a Bacillus preparation diluted in a sterile petri dish by the pour method is added to form a flat plate. When culturing using the agar medium, the medium spreads in a planar shape and falls into a state where it is impossible to count Bacillus bacteria.

  Therefore, the purpose of the present invention is to use a hydrophobic grid filter to quantify the priority of Bacillus bacteria in activated sludge without using expensive equipment and analytical reagents, It is an object of the present invention to provide a method for counting Bacillus bacteria that can be easily and inexpensively counted for Bacillus bacteria contained in the Bacillus preparation.

In order to achieve the above object, the present invention comprises the following arrangement.
A pretreatment step in which a sample containing Bacillus bacteria diluted at a predetermined dilution rate is heated at a predetermined temperature for sterilization, and the pretreated sample is filtered through a hydrophobic lattice filter to remove microorganisms in the sample. A step of holding the cells in the compartment of the hydrophobic lattice, a step of preparing a plate-shaped agar medium hardened in a flat plate shape by adding and mixing a redox indicator having a predetermined concentration, and a hydrophobic lattice filter retaining the microorganisms A culture process in which microorganisms are grown at a predetermined temperature for a predetermined period of time on the agar medium to form color-changed colonies, and from the form of colonies formed in the lattice compartments of the hydrophobic lattice filter, positive based on Bacillus bacteria A step of counting the number of compartments, and a step of calculating an estimated content of Bacillus bacteria contained in the sample from the number of positive compartments.

  When a hydrophobic lattice filter holding a sample containing a Bacillus bacterium is placed on an agar medium and cultured at a predetermined temperature for a predetermined time, a color-changed colony is formed, and from the form of the colony formed in the lattice compartment of the hydrophobic lattice filter The number of positive compartments based on Bacillus bacteria can be easily counted.

The estimated content of Bacillus bacteria contained in the sample is preferably calculated by the most probable number MPN (Most Probable Number) method per 1 mL of the sample using the total number of compartments and the number of positive compartments.
Thereby, the estimated content of the Bacillus genus bacteria contained in a sample can be clarified quantitatively.

In the pretreatment step, it is desirable that the sterilization treatment is performed by heating the sample within a range of 75 ° C. or more and less than 95 ° C. for a predetermined time.
Thereby, Bacillus bacteria and other bacteria are separated in the sample, and only the Bacillus bacteria are allowed to survive and be counted easily.

The redox indicator is triphenyl tetrazolium chloride (TTC), and it is desirable that the addition concentration is 1.25 mg / 100 ml or more and 5.00 mg / 100 ml or less.
Thus, the presence of the Bacillus bacterium in the sample can be easily confirmed by causing the Bacillus bacterium to develop a surface or a point in the hydrophobic lattice compartment with the TTC indicator while appropriately delaying the degree of cultivation of the Bacillus bacterium in the sample.

  By counting the number of planar colonies as the number of positive colonies among the planar colonies that fill the grid sections of the hydrophobic grid filter and the dot colonies scattered in the grid sections, it grows in plane in the grid sections This is caused by a Bacillus bacterium, and the punctate colony can be determined to be other bacteria (for example, Bacillus cereus), and the Bacillus bacterium can be easily and accurately counted.

  By using the method for counting Bacillus bacteria described above, a hydrophobic lattice filter is used, and the activated sludge contained in the sewage sample and the Bacillus bacteria contained in a commercially available Bacillus preparation are inexpensively and easily executed. be able to.

It is a photograph figure which shows an example of a hydrophobic lattice filter. It is a photograph figure which shows the process of mounting | wearing the holder of a bacteria test | inspection apparatus with the hydrophobic lattice filter of FIG. It is a photograph figure which shows the spread of the Bacillus genus bacteria colony on a flat plate culture medium. It is a photograph figure which shows the colony which grew in the division without using an indicator. It is a red-colored principle diagram of TTC (redox indicator). It is a chart which shows the amount of TTC addition to various agar media. It is a contrast diagram of the TTC density | concentration which affects the count value of a Bacillus formulation, a medium kind, and a culture method. It is a chart which shows the comparison result of a lattice culture method and a pour culture method. It is a photograph figure which shows the colony formation condition by the difference in the dilution rate of a Bacillus formulation. It is a graph which shows the count result of the Bacillus cereus adjusted from the glycerol stock. It is a photograph figure of the standard agar medium which carried out the pour culture without adding TTC about a Bacillus formulation. It is a photograph figure of the BHI agar medium culture | cultivated by the lattice F method without TTC addition about a Bacillus formulation. It is a photograph figure of the BHI agar medium for the lattice F method culture | cultivation which confirms the influence of pretreatment conditions. It is a graph which shows the count result of the Bacillus genus bacteria in the lattice F method culture | cultivation which added TTC to the culture medium, and pour culture. It is a photograph explanatory drawing which shows the criteria of the microorganisms growth condition in a hydrophobic lattice division. It is a graph which shows the difference in the count value of a lattice culture method and a pour culture in a TTC addition system. It is a graph which shows the influence of the pre-processing temperature with respect to MPN calculated | required from the lattice F method. It is a graph which shows the analysis result regarding the pretreatment temperature and the kind of culture medium in the lattice F method. It is a graph which shows evaluation of the influence of the pretreatment temperature by Kruskal-Wallis. It is a graph which shows the ANOVA result regarding the kind of pretreatment temperature and culture medium in the pour method. It is a graph which shows the influence of the kind of culture medium with respect to the ratio of the number of planar positive divisions of the lattice F method. It is a photograph figure which shows the confirmation result of Bacillus cereus in the Bacillus formulation by PCR method. It is a photograph figure which shows the detection result of the Bacillus genus bacteria contained in activated sludge. It is a photograph figure which shows the detection result of the Bacillus genus bacteria contained in the activated sludge following FIG. It is explanatory drawing which shows the flow of the simple determination method of a Bacillus genus bacteria.

Hereinafter, a method for counting Bacillus bacteria according to an embodiment of the present invention will be described.
First, the equipment used for the counting method will be described.

  As the hydrophobic lattice filter, for example, an isogrid membrane filter (ISO-GRID manufactured by NEOGEN) having a pore size of 1,600 (40 × 40) sections and having a pore size of 1,600 (40 × 40) is used and sealed one by one aseptically ( (See FIG. 1). When a water sample such as sewage is supplied to the filter after being clamped with a hydrophobic grid filter in a dedicated filter holder provided on the manifold connected to the suction pump, the microorganisms in the water sample are hydrophobic. It is held in a section enclosed by a lattice. When this hydrophobic lattice filter is placed on a plate agar medium and cultured, the microorganisms grow in the compartments to form colonies (see FIG. 2).

As the number of microorganisms in the water sample increases, there is a high possibility that two or more bacteria will enter one compartment, so the counting method using a hydrophobic lattice filter is a positive lattice compartment frame formed by colonies. MPN (Most Probable Number) is calculated from the following formula.
C (MPN / mL) = N ・ log _e (N / (NX) ・ 1 / V
C: The most probable number per mL of sample (MPN / mL)
N: Total number of filter sections (1,600)
X: Number of plots where colonies are growing (number of positive plots)
V: Sample volume (ml)
Therefore, the unit of the counting method using the hydrophobic lattice filter is not the Colony Forming Unit (CFU), but the most probable number (MPN).
In the present embodiment, by using a hydrophobic lattice filter, the effect of preventing the spread of Bacillus bacteria during growth (swarming) (see FIG. 3) by the hydrophobic lattice and preventing the spread of colonies during pour culture. I expected.

Next, pretreatment for separating Bacillus bacteria will be described.
Bacillus and Clostridium bacteria form spores in the cells. These spores are extremely resistant to physicochemical treatment and can withstand heating at 100 ° C. for a considerable amount of time. For this reason, as a method for detecting spore-forming bacteria in food, after heating for a certain period of time as pretreatment conditions to eliminate bacteria other than spore-forming bacteria, surviving spore-forming bacteria are detected after culturing. Examples of heating conditions that kill spores of Bacillus and Clostridium bacteria, which are spore-forming bacteria, are summarized as follows.
Bacillus spores for 30 minutes at 70 ° C
15 minutes at 75 ° C
10 or 30 minutes at 80 ° C
5,10 or 20-30 minutes in boiling water (100 ° C)
Clostridium spore for 30 minutes at 62.5 ℃
5, 10 or 15 minutes at 75 ° C
Therefore, spore-forming bacteria such as Bacillus can be counted for 20 minutes at 70 ° C and by pre-treatment with appropriate temperature and heating time, so that spore-forming bacteria containing Bacillus can be counted. I guessed.

Next, colony discrimination within the compartment of the hydrophobic lattice filter will be described.
If the color of the colony is light, it may be difficult to count the number of compartments with a filter having 1,600 compartments. At the beginning of the experiment, when trying to culture and count with a hydrophobic grid filter using a Bacillus preparation, colonies with a light color were obtained (see FIG. 4).

  In the method for measuring the dehydrogenase activity of activated sludge, red TF (Triphenyl Formazone), which is used as a redox indicator called Triphenyl Tetrasolium Chloride (TTC), which reduces colorless TTC when bound to the hydrogen acceptor. ) Is used (see FIG. 5). Therefore, in order to clearly determine the presence or absence of microbial growth in the compartment of the hydrophobic lattice filter, the optimum amount of TTC added to the medium and its effect were also examined.

  Next, selection of a culture medium will be described. When measuring environmental microorganisms, they may not grow on highly nutrient media. For this reason, in the water supply test method, the number of heterotrophic bacteria (Heterotrophic Plate Counts; HPC) is measured in an R2A medium as an environmental aquatic microorganism that proliferates under malnutrition. As a medium for microorganisms that require high nutrition, a Brain Heart Infusion (BHI) agar medium is also used.

In this embodiment, since nutritional requirements regarding high nutrition and low nutrition of the medium are unknown, BHI medium (Eiken Chemical) is used as a high nutrition medium, and R2A medium (Eiken Chemical Co., Ltd.) is used as a low nutrition medium. A standard agar medium (manufactured by Eiken Chemical Co., Ltd.) was used as an intermediate medium. As described above, the examination items related to the basic examination of Bacillus bacteria using a hydrophobic lattice filter are as follows.
(1) Effect of using hydrophobic lattice filter As a control, comparison with pour culture (2) Heating conditions for measurement of bacteria that formed spores As a control, comparative study with room temperature conditions (3) Reddening colonies Effect of TTC to be compared As a control, comparison with TTC-free condition (4) Comparison of colony formation according to nutrient condition (high nutrition, standard, low nutrition) of medium

Hereinafter, experimental materials and experimental methods will be described.
(A) Test microorganism (1) Bacillus preparation A commercially available Bacillus preparation was used. When using this Bacillus preparation, the following examination was conducted to grasp an appropriate dilution factor. In addition, it may replace with a Bacillus formulation and the sewage containing the activated sludge of a sewage treatment plant may be sufficient.
1) 1 ml of a Bacillus agent was collected in a clean bench, and 10-fold diluted water was prepared in addition to 9 ml of sterilized diluted water (10-fold dilution method). Such a 10-fold dilution step operation was repeated 10 ⁶ times. Up to double dilution Bacillus formulation samples were made.
2) 1 ml of each diluted Bacillus preparation sample diluted 10 ² times to 10 ⁶ times was added to a sterilized petri dish and subjected to pour culture using a standard agar medium. This operation was performed three times for one dilution stage.
3) The culture medium subjected to pour culture was cultured at 35 ° C for 24 hours, and the number of colonies at each dilution stage was determined to obtain a Bacillus colony that could be counted (one that formed colonies that did not spread excessively). The dilution ratio was determined.
(2) Bacillus standard strain Bacillus bacteria detected in human waste treatment, sewage treatment and domestic wastewater treatment are reported to be mainly the following species).
1) Bacillus cereus
2) Bacillus licheniformis
3) Bacillus megaterium
4) Bacillus pumilus
5) Bacillus subtilis
6) Bacillus thuringiensis

In this embodiment, a glycerol stock was prepared according to the following procedure using Bacillus cereus (hereinafter referred to as Bacillus cereus) IFO3466 strain and used as necessary.
1) 100 ml of a liquid medium containing 0.25 g of yeast extract, 0.5 g of casein peptone, and 0.1 g of glucose was prepared, and 10 ml each was dispensed into a medium test tube, and then autoclaved at 121 ° C. for 20 minutes. This liquid medium is a liquid medium obtained by removing agar components from a standard agar medium.
2) A single colony obtained by smear culture (35 ° C., 24 hours) of a stock strain of Bacillus cereus IFO3466 on a standard agar medium was collected with a platinum loop and inoculated into a liquid medium. The culture was performed for 24 hours.
3) After vortexing the liquid medium in which Bacillus cereus IFO3466 was grown, an equal amount of sterilized 80% glycerin and liquid culture solution were mixed to prepare a 40% glycerol stock solution. This glycerol stock solution was stored in a deep freezer at -80 ° C.
In addition, the optimal dilution rate at the time of using a Bacillus cereus solution was performed according to the method at the time of a Bacillus formulation. The bacterial solution at this time was prepared by dispersing three colonies of Bacillus cereus in 10 ml of sterilized diluted water.

(3) Non-spore-forming bacteria As bacteria that do not form spores, E. coli isolated from humans was used. A glycerol stock of E. coli was prepared in the same manner as for Bacillus cereus. In addition, three colonies obtained using glycerol stock were decomposed into 10 ml of sterilized diluted water to prepare a bacterial solution. For confirmation of E. coli, a compact dry EC (E. coli is blue, E. coli group is red colony, manufactured by Nissui), which is an enzyme substrate medium, was used.

(B) Medium The BHI agar medium (Pearl Core (registered trademark) Brain Heart Infusion Agar 'Eiken') is used as the high nutrient medium, and the standard agar medium (Pearl Core (registered trademark) standard agar medium) is used as the standard nutrient medium. “Eiken” [E-MB65]) was used as the low nutrient medium, and R2A agar medium (R2A agar medium “Eiken” [E-MB68]) was used. Each medium was prepared according to the manufacturer's prescription.

(C) Examination of TTC solution amount (preliminary test)
The amount of TTC added to the medium was determined by conducting the following preliminary test.
(1) Preparation of TTC solution 1) 0.25 g of TTC (2.3.5 Triphenyltetazolium Chloride) was weighed with an electronic balance and put into a test tube, and 10 ml of purified water was added and mixed well.
2) The TTC was transferred to a beaker in a clean bench, sucked with a syringe, sterilized by filtration (0.22 μm), and stored in a 15 ml sterilized tube.
(2) Addition of TTC solution to various agar media For BHI agar medium, standard agar medium, and R2A agar medium not solidified on the plate, the addition concentrations shown in the table below (addition amount of TTC (mg) per 100 ml of medium) The above TTC solution was added to each agar medium (before coagulation) and gently mixed. The amount of TTC added is shown in the chart of FIG.

(3) Preliminary test method 1) Difference between counting method using hydrophobic lattice filter and counting method by pour method 2) Difference in TTC addition amount 3) Difference in medium used. The test microorganism and culture conditions were 1 ml of a suitably diluted Bacillus preparation and cultured at 35 ° C. for 24 hours. Specific culture methods by filtration culture using a hydrophobic lattice filter (hereinafter referred to as “lattice F method”) and the pour method were as follows.

[Lattice F method]
1) BHI agar medium, standard agar medium, and R2A agar medium with different TTC addition conditions were prepared in advance.
2) A diluted diluted Bacillus preparation was prepared.
3) A sterilized filtration unit was attached to the filtration manifold, a hydrophobic lattice filter was set on the filtration unit with tweezers, and then fixed with a unit clamp.
4) 1 ml of diluted Bacillus preparation (10 ⁵ fold dilution) was added to 90 ml of sterilized diluted water, mixed well, then poured into a filtration unit and subjected to suction filtration. This is to prevent microorganisms from solidifying in a part of the hydrophobic lattice filter.
5) The hydrophobic filter was taken out with tweezers and placed in close contact with a previously prepared flat plate agar medium and cultured at a predetermined temperature and time.

[Pounding method]
Add 1 ml of diluted Bacillus preparation (diluted 10 ⁵ times) to a sterilized petri dish filled with the necessary information, add about 20 ml to 25 mml of agar medium cooled to the appropriate temperature, and gently and thoroughly mix and harden into a flat plate The cells were cultured under the same conditions as in [Lattice F method].

(D) Counting test of Bacillus preparations (this test: examination of [Lattice F method])
Using a moderately diluted Bacillus preparation, this test was performed under a combination of the following conditions. In this examination experiment, one combination condition (combination of pretreatment warming 4 conditions × medium 3 conditions × TTC presence / absence 2 conditions × culture method 2 conditions) was repeated 3 times, and if counting was possible, 3 The geometric mean of the times was obtained. In addition, as a comparison / target test, a count test of Bacillus cereus (standard strain) and Escherichia coli (non-spore bacterium) was performed under a combination of experimental conditions similar to those of the Bacillus preparation.
1) Pretreatment heating conditions The room temperature conditions were set as controls, and heating conditions of 75 ° C, 85 ° C, and 95 ° C were set. Regardless of the heating conditions, the heating time was unified to 15 minutes. The heating time may be longer or shorter than 15 minutes.
2) Medium
Three types of media were used: BHI agar, standard agar, and R2A agar.
3) Presence / absence of TTC Two conditions were set: an “addition series” in which TTC having an addition concentration set in the preliminary test was added to each medium, and an “no addition series” in which TTC was not added.
4) Culture method The cells were cultured at 35 ° C. for 24 hours using the [Lattice F method] or [Pour method].
In addition, the formation status of colonies in small compartments in [Lattice F method] (small colonies, colonies spread on the surface in the compartments, colonies that crossed the hydrophobic lattice) and colony formation status on the flat plate were also observed.
MPN is calculated from the number of positive compartments obtained by [Lattice F method], CFU is calculated from colonies by [Pour method], and these values are converted to common logarithms as necessary. Processed.

(E) Statistical processing
SPSS STATISTICS ver.22 which is statistical analysis software made by IBM Corporation was used.

[Experimental results and discussion]
(A) Determination of TTC addition concentration At the start of the experiment, the measurement of activated sludge dehydrogenase activity (sewage test method) specifies that TTC should be added as follows when measuring the dehydrogenase activity of activated sludge. ing.
Add 1ml of TTC (5mg / ml) per 10ml of activated sludge
→ Addition of 5 mg of TTC per 10 ml of activated sludge As the above concentration is converted to 100 mg of activated sludge, the concentration of TTC was set based on this concentration at the beginning of the experiment.

However, when the Bacillus preparation was cultured at this TTC addition concentration, colonies did not grow at all, so the TTC concentration, the type of medium, and the culture method (pour method or lattice F method) were used as preliminary factors. Went.
The results of this preliminary test are as shown in Table 1 shown in FIG. 7, but the dilution ratio of the Bacillus preparation is 10 to the fifth power, the sample volume is 1 ml, the culture temperature is 35 ° C., the culture time is 24 hours, In the lattice F method], the number of positive lattice sections is converted to MPN.

It can be seen that when the added concentration of TTC exceeds 25 mg / 100 ml, the growth of microorganisms in the Bacillus preparation is greatly inhibited. In addition, at 12.5 mg / 100 ml, the count value (CFU and MPN) might be affected depending on the culture method and the type of medium. Under conditions where TTC was not added or 0.5 mg / 100 ml, the number of microorganisms could not be measured in the R2A medium. Therefore, it was considered that three conditions of 1.25 mg / 100 ml to 5.00 mg / 100 ml do not affect the numerical value. In this experiment, 2.5 mg / 100 ml, which is an intermediate value in this range, was used as the TTC addition condition to be used in future experiments.

  When the difference between the count values in the lattice F method and the pour culture was performed by Freidman's distribution comparison of the nonparametric test, a significant difference was observed (P = 0.000) (see FIG. 8). Therefore, there was a tendency for the count values to differ between the lattice F method and the pour culture. This is because the MPN conversion is based on the pour method that counts the number of colonies and the number of sections of the positive lattice. There is a possibility that it is caused by the difference in the unit of CFU and MPN.

  On the other hand, the effect of the type of medium on the count value (CFU or MPN) was compared with Kruskal-Wallis, which is a nonparametric test, but no significant difference was found (MPN: P value = 0.958, CFU : P value = 0.890). Therefore, when counting the number of microorganisms in the Bacillus preparation (presumably Bacillus genus bacteria), it was considered that the count value did not change due to the difference in nutrient state (high nutrition, standard, low nutrition) of the medium . In this study, however, the factors of the type of medium were continuously examined.

(B) Determination of Appropriate Dilution Ratio of Test Bacterial Solution (1) Bacillus Preparation As the Bacillus preparation forms colonies spreading around, it is difficult to count colonies as described above. However, the following results were obtained when examining the dilution ratio at which an approximate colony count was possible at the time of pouring, although the colony count was difficult (see the attached photograph Fig. 9).
Therefore, in this test using a Bacillus preparation, a dilution of 10 to the fifth power was performed and used for the experiment. Since this value is the same value as in the preliminary test, it was assumed that the number of microorganisms in the Bacillus preparation was maintained relatively stably.

(2) Bacillus cereus When 3 colonies of Bacillus cereus cultured from glycerol stock were decomposed into 10 ml of sterilized diluted water, the following count values were obtained (see FIG. 10; Table 2).
Therefore, the dilution rate of Bacillus cereus was targeted to the Bacillus cereus solution adjusted so that a count value of around 100 CFU was obtained with 10 5 times dilution as a guide.

(3) Escherichia coli (non-spore bacterium)
When checking the number of colonies in a compact dry EC to decompose the three colonies of E. coli cultured from glycerol stocks into sterile dilution water 10 ml, the count value of 79CFU and 80CFU were obtained in bacterial solution of 10 ⁵ fold dilutions 1ml . Therefore, this dilution rate was used as the dilution rate in this test.

(C) Examination of various factors affecting the count value of the lattice F method (1) Bacillus preparation 1) When TTC is not added to the medium, Bacillus-like bacteria spread throughout the plate agar medium and counting is impossible with the pour method. It became a state. (See FIG. 11).
In addition, even in the lattice F method, if TTC is not added, the Bacillus-like bacteria grow over the hydrophobic lattice, making counting impossible (see FIG. 12).

FIG. 13 shows an example in which the pretreatment temperature condition is changed in the lattice F method culture. As shown in FIG. 13, in each temperature condition, the number of colonies is estimated to be a count value of about 10, but when such a phenomenon occurs in a count state of about several hundreds, the lattice filter method having a partition number of 1,600 is used. It can be easily imagined that the measurement is meaningless. Further, the carry-over phenomenon as shown in FIGS. 11 and 12 was observed in any medium (BHI medium, standard agar medium, R2A medium).
Although counting was not possible in this way, the pretreatment conditions of 75 ° C / 15 minutes, 85 ° C / 15 minutes, and 95 ° C / 15 minutes were the same as the room temperature conditions of the target test. The growth of new Bacillus-like bacteria was observed. Therefore, it was confirmed that the Bacillus preparation contains bacillus spores that are resistant to heating conditions at 75 ° C to 95 ° C for 15 minutes.

2) TTC addition series On the other hand, when TTC is added, as shown in Table 3 of FIG. 14, it is possible to count Bacillus genus bacteria in the Bacillus preparation in both cases of the lattice F method and the pour culture method. I understand that. Table 3 shows a comparison of the count results of Bacillus bacteria under the combined conditions of pretreatment temperature conditions and culture methods (pour method / lattice F method) medium types. However, in this table, a planar colony (symbol: plane) is a planar colony (symbol: plane), and a dot colony (symbol: dot) is a point colony within the grid partition frame. It is called. The determination for distinguishing “surface” or “point” is based on, for example, the form of colonies that develop color in the lattice section shown in the photograph of FIG.

Further, from the data shown in Table 3 of FIG. 14, when the difference between the count values in the lattice F method and the pour culture was performed by Freidman's distribution comparison of the nonparametric test, a significant difference was observed (P = 0.000) ( (See FIG. 16).
Therefore, the [Lattice F method] and the [Powder method] tend to have a difference in the count value in the culture, and the same tendency as the preliminary test result was recognized. This may be due to the difference in units of CFU and MPN in addition to the difference between the [Powder Method] and [Lattice F Method], and further examination is required.

Next, when the two-way analysis of variance without repetition was performed on the common logarithm values of the MPN number calculated from the number of all (spot + plane) positive sections obtained by [Lattice F method], the following dispersion was performed. A figure (see FIG. 17) and a test result (see FIG. 18; Table 4) were obtained.
From these results, it was found that when the pretreatment temperature was 95 ° C., the MPN value calculated from the lattice F method was lower than the room temperature, 75 ° C., and 85 ° C. conditions. From the multiple comparison, the P value of the significant difference between the temperature conditions of 85 ° C and 95 ° C was 0.041.
Further, it was confirmed that the MPN value calculated from the lattice F method was lowered at the pretreatment temperature of 95 ° C. also by comparison with the Kruskal-Wallis test shown in FIG. (P = 0.017).
Therefore, when counting the number of microorganisms in a Bacillus preparation (presumably mainly Bacillus bacteria), it is necessary to fully recognize that the pretreatment conditions at 95 ° C. are different from other pretreatment conditions.

However, as shown in Table 5 of FIG. 20, the common logarithm of the number of colonies in the pour method was subjected to a two-way analysis of variance based on the pretreatment temperature and the type of medium. There was no significant difference in any of the types.
When the ratio of planar positive compartments in the lattice F method was compared with Kruskal-Wallis shown in FIG. 21, it was found that the ratio decreased in R2A medium, that is, the number of dotted positive compartments increased. (P = 0.049).
If the culture time is extended for the punctate positive compartment, it will eventually become a planar positive compartment (data not shown). Therefore, it was found that the growth of Bacillus-like bacteria in the Bacillus preparation is slowed when the R2A medium, which is a low nutrient medium, is used. That is, it is necessary to use a standard agar medium or BHI agar in order to increase the growth rate of microorganisms in the planar positive section and make it easy to read. However, in this experiment, the exact number of Bacillus bacteria in the Bacillus preparation was not measured, so the accuracy is unknown. Because it is easy to read, there is no reason to be accurate, so in the future, it will be necessary to examine not only this accuracy but also precision and select a more appropriate medium.

(2) Bacillus cereus
When pretreatment was performed at 75 ° C. for 15 minutes, 85 ° C. for 15 minutes, and 95 ° C. for 15 minutes, the Bacillus cereus was killed by heating, and neither the lattice F method nor the pour method could obtain count values. . Therefore, even though spore-forming bacteria, those that have become nutrients by plate culture are killed by subsequent heat treatment.
The environmental samples that are considered to be detected include Bacillus bacteria from both companies in the spore state and the nutritional state. Therefore, pretreatment conditions (for example, at about 50 ° C) are used to form spores of the Bacillus genus trophozoites. It is necessary to add (heat for a certain period of time) to the pretreatment conditions of the lattice F method (heating for spore formation → heating for sterilization of non-spore bacteria → measurement).

(3) Escherichia coli (non-spore bacterium)
Whether Escherichia coli can be killed by pretreatment at 75 ° C. for 15 minutes, 85 ° C. for 15 minutes, and 95 ° C. for 15 minutes was examined using E. coli. In the lattice F method, it is possible to inhibit the growth of microorganisms contained in the sample in the hydrophobic lattice filter placed on the upper surface of the three types of media by heating in both the TTC addition series and the TTC non-addition series. did it.
1) Lattice F method
TTC addition series: No colonies detected under warming conditions for all 3 types of media No addition series: No colonies detected under heating conditions for all 3 types of media However, in the case of pour culture, the following A very small number of cases (less than 10 per case) could survive. 2) Pour method
TTC addition series
75 ℃ BHI agar medium 1 of 3 cases Standard agar medium Not detected
R2A agar medium 1 of 3 cases
Not detected in 3 types of medium at 85 ℃
95 ° C BHI agar medium 2 cases in 3 cases Standard agar medium 3 cases in 3 cases
R2A agar medium 1 out of 3 cases
75 ° C Not detected in all three media
85 ℃ BHI agar medium 1 of 3 cases Standard agar medium Not detected
R2A agar medium Not detected
95 ° C BHI agar medium 2 cases in 3 cases Standard agar medium 2 cases in 3 cases
R2A agar medium 1 of 3 cases However, it was confirmed using compact dry EC that these colonies were not E. coli. In the lattice F method, microcolonies are not formed as in the pour method, but in the pretreatment conditions of 95 ° C in the pour method, microcolonies are formed regardless of whether TTC is added or not. In view of this, it was speculated that the detection probability of non-spore bacteria could be further reduced by not using the 95 ° C. condition.

(D) Confirmation of Bacillus cereus in Bacillus preparation In this experiment, a Bacillus bacterium standard strain (IFO3466) was used as one species of Bacillus bacteria constituting Bacillus bacteria. Although not specifically stated in the experimental method, the presence of Bacillus cereus in the Bacillus preparation was detected using the TaKaRa Bacillus cereus (CRSgene) PCR Detection Kit, and the following results were obtained (see FIG. 22). The PCR reaction conditions were the same as described in the kit.
As can be seen from FIG. 22, the band of the Bacillus preparation was only the band resulting from the internal control, as in the negative control, and the same band as the positive control was not detected. Accordingly, it was found that Bacillus cereus was not present in the Bacillus preparation. In future studies, it is considered necessary to identify the species of Bacillus genus bacteria present in the Bacillus preparation.

(E) Estimation of bacteria belonging to the genus Bacillus in activated sludge A detection method using a hydrophobic lattice filter has not been established yet, but an activated sludge treatment facility that conducts the Bacillus priority movement (agriculture in the Chikudaira district of Nagano City, Nagano Prefecture) Detecting Bacillus-like bacteria in activated sludge of the first and second series of village wastewater treatment facilities (hereinafter referred to as “Chikuhei 1 series treatment” and “Chikuhira 2 series treatment”) There was no activated sludge treatment facility (agricultural settlements in the Shinno area of Nagano City, Nagano Prefecture, a wastewater treatment facility, hereinafter abbreviated as “new treatment”) as a control (see FIGS. 23 and 24).

The pretreatment temperature condition for detecting Bacillus-like bacteria was 85 ° C for 15 minutes, and the target temperature condition was room temperature. Moreover, the BHI agar medium was used as the medium, and the amount of microorganisms in the activated sludge was estimated by MLVSS (mg / L). Originally, ultrasonic crushing is performed to disperse particles of activated sludge, but since the influence of ultrasonic crushing has not been studied, the activated sludge sample is thoroughly mixed and diluted 10 ⁴ times. 1 ml was used.
1) Bacillus priority operation processing facility Chikyudaira 1 series processing (MLVSS: 4,660ml / L)
Number of positive compartments with heating treatment: 4 → 4MPN
Number of positive compartments in control treatment: 16 → 16MPN
Bacillus presence rate = 4/16 = 25%
Bacillus amount = 4MPN × 10 ⁴ / ml / 4.66mg / ml = 8,600MPN / mg
Chikuhei 2 series processing (MLVSS: 4500mg / L)
Number of positive compartments with heating treatment: 1 → 1MPN
Number of positive compartments in control treatment: 27 → 27MPN
Bacillus presence rate = 1/27 = 4%
Bacillus amount = 1MPN x 10 ⁴ / ml / 4.5mg / ml = 2,200MPN / mg
2) Normal activated sludge treatment Renewed treatment (MLVSS: 5600mg / L)
Number of positive compartments with heating treatment: 2 → 2MPN
Number of positive compartments in control treatment: 19 → 19MPN
Bacillus presence rate = 2/19 = 11%
Bacillus amount = 2MPN x 10 ⁴ / ml / 5.6mg / ml = 3,600MPN / mg
Therefore, in the Chikuhei 1 series, it was suggested that there was a possibility that Bacillus spp. Existed more than normal activated sludge treatment. Become.

[Summary]
A basic study for estimating the abundance of Bacillus bacteria in a commercially available Bacillus preparation using a hydrophobic lattice filter showed the possibility of simple quantification of Bacillus bacteria. The following is a summary of the findings from this study.
(1) When an appropriate amount of TTC was added, there was an effect of suppressing the spread of colonies unique to the genus Bacillus together with the red color change of the hydrophobic lattice section.
(2) A significant difference was observed between the counting method using a hydrophobic lattice filter and the counting method using pour. However, there may be a difference in the display method of MPN and CFU.
(3) The pretreatment temperature condition at 95 ° C. for 15 minutes tended to show a lower count value than the other temperature conditions. Therefore, it is necessary to consider the conditions of 75 ° C for 15 minutes and 85 ° C for 15 minutes separately from those for 95 ° C for 15 minutes.
(4) When R2A medium was used, the growth rate of bacteria in the compartment of the hydrophobic lattice filter tended to be slower than that of ordinary agar medium or BHI agar medium.
(5) It is thought that the sporeless bacteria can be killed under pretreatment conditions of 75 ° C. and 15 minutes or more. However, since microcolonies were formed under the conditions of 95 ° C for 15 minutes in pour culture, it was assumed that the heating condition was 75 ° C to 85 ° C.
(6) When detecting Bacillus bacteria in activated sludge, it is necessary to examine the conditions for crushing and dispersing activated sludge, and to examine the conditions for spore formation of Bacillus bacteria in the nutrients.
(7) Under heating conditions for spore-forming bacteria, clostridium and the like survive in addition to Bacillus bacteria. In the future, research on separation and identification of whether or not the bacteria present in the positive compartment of the hydrophobic lattice filter are Bacillus is necessary.

Here, with reference to FIG. 25, an example of a simple method for quantifying Bacillus bacteria that detects the ratio of spore cells to the total number of cells contained in the sludge sample will be described.
First, two ice-cooled sludge samples are prepared as samples. As a pretreatment step, the first sample is kept ice-cooled, and the second sample is heated at 85 ° C. for 15 minutes (this kills non-spore cells (vegetative cells)). The first and second samples that have undergone the pretreatment process are each diluted in stages (for example, diluted to the fifth power of 10) while being ice-cooled, and filtered through a hydrophobic lattice filter to remove microorganisms in the sample within the compartment of the hydrophobic lattice. To hold. A standard agar medium to which a TTC reagent has been added is prepared, and the above-described hydrophobic lattice filter holding the first and second samples is placed thereon and cultured, for example, at 37 ° C. for 24 hours to form colonies.

The number of positive lattices is counted from the form of colonies formed in the lattice sections of the hydrophobic lattice filters of the first and second samples described above. The number of positive lattices is counted by the most probable number MPN (Most Probable Number) method. From the difference in the pretreatment process (whether heating is performed), the number of lattices (total cell lattice number) including both vegetative cells and spore cells is detected in the first sample (not heated). Further, in the second sample (heating treatment), the number of lattices (spore cell lattice number) of only spore cells is detected. As a result, the ratio of spore cells contained in all cells can be calculated by a simple quantitative method by dividing the number of spore cell lattices by the number of all cell lattices.
When quantitatively determining the loss on ignition (VSS) of bacteria belonging to the genus Bacillus contained in sludge, the sample is filtered in advance with a membrane filter (MF), which is an organic filtration membrane, and captured by the membrane filter (MF). The dried material is dried at a predetermined temperature, and the mass is measured to determine the amount of suspended solids (SS). After obtaining the amount of suspended solids (SS), for example, a step of igniting at 600 ± 25 ° C. for 30 minutes and measuring the mass of the residue to obtain the loss on ignition (VSS) is required.

  As explained above, using the method for counting Bacillus bacteria, using a hydrophobic grid filter, it is possible to easily and inexpensively grasp the count of activated sludge contained in sewage and Bacillus bacteria contained in commercially available Bacillus preparations. can do.

Claims (5)

A pretreatment step in which a sample containing Bacillus bacteria diluted at a predetermined dilution rate is heated at a predetermined temperature and sterilized;
Filtering the pretreated sample with a hydrophobic lattice filter to retain microorganisms in the sample within the compartment of the hydrophobic lattice;
Preparing a plate-shaped agar medium solidified by adding a redox indicator of a predetermined concentration and mixing, and
A culture step of placing the hydrophobic lattice filter holding the microorganism on the agar medium and growing the microorganism at a predetermined temperature for a predetermined time to form a color-changed colony;
Counting the number of positive compartments based on Bacillus bacteria from the form of colonies formed in the lattice compartments of the hydrophobic lattice filter;
Calculating the estimated content of Bacillus bacteria contained in the sample from the number of positive compartments, and a method for counting Bacillus bacteria.   The estimated content of Bacillus bacteria contained in the sample is calculated by the most probable number MPN (Most Probable Number) method per 1 mL of the sample using the total number of compartments and the number of positive compartments. Counting method.   The method for counting Bacillus bacteria according to claim 1, wherein in the pretreatment step, the sterilization treatment is performed by heating the sample within a range of 75 ° C or more and less than 95 ° C for a predetermined time.   The method for counting Bacillus bacteria according to claim 1, wherein the oxidation-reduction indicator is triphenyl tetrazolium chloride (TTC) and is added at a concentration of 1.25 mg / 100 ml to 5.00 mg / 100 ml.   The counting method of the Bacillus genus bacteria of Claim 1 which counts the number of planar colonies as the number of positive divisions among the planar colonies which fill in the lattice division of the said hydrophobic lattice filter, and the dotted | punctate colony scattered in a lattice division. .