JPH0773512B2 - Quantitative method and quantification device for microorganisms - Google Patents

Description

TECHNICAL FIELD The present invention relates to a quantification method and quantification device for microorganisms and the like.
More specifically, the present invention relates to a quantification method and a quantification device that can easily and reliably quantify microorganisms and pyrogens in a sample solution.

(B) Conventional technology The measurement (quantification) of the amount of microorganisms in various media can be performed not only in fields such as fermentation and brewing where microorganisms are actively used, but also in medical treatment.
It is important for public health and quality control in various fields such as food and analysis.

The following methods have been generally known as methods for quantifying such microorganisms.

Visual measurement method: After dyeing the sample solution containing microorganisms,
For example, a method of counting the number of bacteria in the visual field with a microscope placed on a slide glass and quantifying the amount of microorganisms as the number of bacteria based on this value.

Optical metering method: A method of irradiating a sample solution containing microorganisms with light to measure the turbidity and quantifying the amount of microorganisms based on this value.

Dilution culture method: A method in which a predetermined dilution series of a microorganism-containing sample solution is prepared, inoculated into a culture tube and cultured, and the amount of microorganisms is statistically quantified based on the growth state.

On the other hand, pyrogenic substances (virogens) are known as substances that are not microorganisms but cause damage in various media like microorganisms, and their quantitative determination is also desired in public health and quality control. Quantification of pyrogens in distilled water such as distilled water for use and distilled water for dialysis is important for public health.

As a method for quantifying or testing such a pyrogen, a method based on an increase in the body temperature of a rabbit caused by injecting a target sample into a rabbit has been widely performed (Japanese Pharmacopoeia; “Pyrogenic test method”). However, there is a disadvantage that the results are easily influenced by individual differences in rabbit susceptibility and that long-term changes in body temperature are observed and animals are raised. Therefore, recently, by utilizing the fact that the blood cell extract of horseshoe crab selectively reacts with a pyrogen, the gelling degree due to it and the P-nitroaniline which can be generated by the above reaction in the presence of a chromogenic synthetic substrate are diazotized. The so-called limulus test, which measures pyrogenic substances based on the absorbance after conversion, has attracted attention as a method for quantifying and evaluating pyrogenic substances (hepatobiliary pancreas 12
(4): 523-528, 1986).

(C) Problem to be Solved by the Invention However, in the conventional method for quantifying microorganisms, there is a problem that the measurement operation takes time, the operation is complicated and requires skill, and the quantification range of the amount of microorganisms is narrow and There is a problem that the reliability of measurement is lacking. Also,
There is also a problem that it cannot be applied to continuous measurement.

On the other hand, even in the conventional method for quantifying and evaluating an exothermic substance, the operation is time-consuming and costly in terms of using an expensive horseshoe crab extract, and the operation is complicated and requires skill, and continuous measurement is required. There was a problem that it was not suitable for.

The present invention has been made in view of the above circumstances, and particularly to provide a method and a device that can easily and reliably quantify the amounts of microorganisms and pyrogenic substances in a short time, and that are also suitable for continuous measurement. To do.

(D) Means and Actions for Solving the Problems Thus, according to the present invention, the TC (total carbon) value A is measured by directly introducing a predetermined amount of a sample liquid containing a microorganism (or a pyrogen) into a total carbon analyzer. Introducing the decarbonization (or depyrogenic substance) sample liquid obtained by treating the process and the same sample liquid containing microorganism (or pyrogenic substance) into the total carbon analyzer
It consists of the process of measuring TC value B, and from TC value A to TC value B
A method for quantifying microorganisms and the like is provided, which is characterized by quantifying the microorganisms (or pyrogens) in the sample solution based on the value obtained by subtracting.

Further, according to the present invention, the sample liquid supply path I extending from the sample liquid supply unit containing the microorganism (pyrogenic substance) and the sample liquid supply path I extending from the sample liquid supply unit containing the microorganism (or exothermic substance) A sample liquid supply path II having a means for removing microorganisms (or a pyrogen), and a sample solution supply path I which is switchably connected to any one of the sample solution supply path I and the sample solution supply path II. There is provided an apparatus for quantifying microorganisms and the like, which comprises a measuring flow path which can be supplied to a chamber and a total carbon analysis type which is connected so as to introduce a predetermined amount of sample solution from a solution measuring means.

The most characteristic feature of the present invention is that a total carbon analyzer is used to quantify microorganisms and pyrogens. As such a total carbon analyzer, a carbon-containing substance is oxidized to generate carbon dioxide, and the amount of this carbon dioxide is detected by a non-dispersive infrared analyzer, a conductivity meter, a thermal conductivity detector, etc. A carbon analysis type can be used, and various commercially available so-called TOC meters and TC meters are applicable. Here, as the oxidation method of the carbon-containing substance, there are a humidity oxidation method and a combustion oxidation method by UV oxidation or oxidizing agent such as potassium peroxosulfate solution.
It is preferable to use the combustion oxidation method because the oxidation is strongest and surely performed. At this time, an oxidation catalyst may be appropriately used, and examples of this include platinum black. Further, it is suitable to carry out the combustion in a combustion tube heated to 680 to 950 ° C. in an atmosphere of high-purity air or oxygen gas.
On the other hand, it is suitable to detect carbon dioxide with a non-dispersive infrared detector (NDIR) in terms of reliability and operability.

On the other hand, the other feature of the present invention is that a de-microbiological sample liquid or a depyrogenic substance sample liquid obtained by subjecting a sample liquid containing a microorganism (or a pyrogen) to a treatment of a microorganism or a pyrogenic substance is used. It is used for quantification.

As means for removing microorganisms, all means for removing microorganisms such as membrane filtration method, adsorption treatment method and absorption treatment method can be applied.
The filtration process or the adsorption process is suitably performed by passing the microorganism-containing sample liquid through the filtration removal means or the adsorption removal means. Examples of the filter remover include a paper filter, a membrane filter, a glass filter, and the like.The pore size of these filters depends on the size of the microorganisms that can be present in the test liquid, and the microorganisms that are intended to be measured. It may be of a size that can capture the smallest microorganisms in the group. Usually, a pore size equivalent to an ultrafiltration membrane is suitable.

On the other hand, a charged membrane or an ion exchange resin is used as the adsorption / removal means, but an insoluble pyridinium type resin such as a crosslinked poly-N-alkyl-4-vinylpyridinium halide is effective.

On the other hand, as a means for removing the exothermic substance, a filter paper used for filtration, a reverse osmosis membrane, an ultrafiltration membrane, or the like is suitable. The removal means is simple and preferable.

In the sample liquid containing microorganisms, not only microorganisms but also organic substances (sugars such as glucose) derived from the culture liquid, contaminant organic substances (organic amine compounds and soluble polymers) or inorganic substances such as dissolved CO ₂ , carbonates, carbonate ions, etc. Since carbon compounds and the like often coexist, it is difficult to accurately quantify the amount of microorganisms from the value obtained by directly introducing this into the total carbon analyzer, the TC (total carbon) value A. In addition, exothermic substances are usually present in the sample in trace amounts, and other organic components can often coexist in the sample.Therefore, the value obtained by directly introducing this sample into the total carbon analyzer described above. It is difficult to accurately quantify the amount of pyrogenic substances from the TC value A.

In the present invention, the TC value B of the demicrobial (or depyrogenic substance) sample solution that has been filtered or adsorbed is further measured, and the value obtained by subtracting the TC value B from the TC value A is It is calculated. Microorganisms (or pyrogens) are substantially removed by the above-mentioned filtration treatment or adsorption treatment, but other coexisting organic substances and inorganic carbon compounds are not removed, and demicrobialization (or depyrogenation) sample liquid Remains inside. Therefore, the value obtained by subtracting the TC value B from the TC value A substantially corresponds to the amount of microorganisms and the amount of pyrogen in the original sample solution,
Microorganisms and pyrogens can be accurately quantified based on this value.

In the practice of the present invention, the sample liquid supply path I having no means for removing microorganisms and exothermic substances and the sample liquid supply path II having means for removing microorganisms and exothermic substances can be switched to liquid measuring means. It is preferable to use an apparatus having a channel structure and configured so that a predetermined amount of the sample liquid can be introduced into the total carbon analyzer from the liquid measuring means, because the quantification can be performed more easily, and automation, continuity, and the like can be achieved. Furthermore, as a liquid measuring means,
There is also an automatic measuring means for a microsyringe, but it is preferable to use a valve switching type in terms of automation and continuity.

However, a perfect continuous measurement method may be performed in which two NDIR units are used to simultaneously measure the TC value A and the TC value B, respectively.

(E) Example 1 FIG. 1 is an explanatory diagram showing an example of an apparatus for quantifying microorganisms for carrying out the method of the present invention. In the figure, a sample liquid supply path I is branched from a sample liquid supply unit 2 containing a microorganism (or a heat generating substance), which comprises a sample tank 2a for storing a sample liquid containing a microorganism (or a heat generating substance) and a liquid feed pump 2b. And the sample liquid supply path II are connected by a pipeline. The sample solution supply path II is provided with a filter (3) for removing microorganisms (or heat-generating substances) composed of an ultrafilter. Downstream of the sample liquid supply paths I and II are assembled into a three-way valve 4, and by switching the three-way valve 4, the sample liquid is supplied to the measurement flow path 5 through either one of the sample liquid supply paths I and II. A high-pressure hexagonal switching valve 6 is connected to the measurement flow path 5, and by switching the sample solution, the sample solution is transferred to either a drainage tank 8 or a solution measuring means 7 composed of a conduit of a certain length. To do. The sample liquid introduced into the liquid measuring means 7 is introduced into the total carbon analyzer through the introduction pipe 9 by switching the valve 6.

The total carbon analyzer 11 consists of a quartz glass combustion tube 12, an electric heater 13, and a drain separator, which contains a catalyst carrier layer (inner diameter 12 mm, length 90 mm) in which platinum black is coated on quartz wool.
14, an electronic cooler 16, and a non-dispersion type infrared detector 18 are connected in this order, and a high-purity air cylinder 21 (HC, CO, and CO ₂ content of impurities is 1 ppm or less for carrier gas supply). Storage). In the figure, 10 is a carrier gas supply passage, 15 and 17 are drain pots, 19 is a flow meter, 20 is a calculation unit, and 22 is a flow control valve.

The quantification operation of microorganisms using the quantification device 1 will be described below.

First, with the three-way valve 4 set on the supply path I side, by driving the liquid feeding pump 2b, the microorganism-containing sample liquid is directly introduced into the measuring flow path 5. Here, the high-pressure hexagonal switching valve 6 is switched from the solid line side to the broken line side, the sample liquid is introduced into the liquid measuring means, and the liquid measuring means 7 is filled with the sample liquid after a predetermined time. Here, the valve 6 is switched to the solid line side. As a result, the sample liquid filled in the liquid measuring means by the carrier gas consisting of the air supplied from the cylinder 21 is pressure-fed and introduced into the combustion tube 12 heated at a high temperature, where the microorganisms present in the sample liquid are present. In addition, the contaminant organic components are burnt and oxidized and converted into carbon dioxide. Water produced as a by-product of combustion oxidation is drain separator 14 and electronic cooler 16 (cooled to 2 ° C)
Are removed by gas-liquid separation. Carbon dioxide produced by combustion oxidation is transferred to the non-dispersion infrared analyzer 18 together with carbon dioxide produced by thermal decomposition of carbonates and carbon dioxide dissolved in the sample, where the absorbance of carbon oxide is The TC value A is detected, the TC value A is calculated by the calculation unit 20 based on the detected output, and the TC value A is recorded and displayed.

Next, the three-way valve 4 is switched to the supply path II side, and the demicrobialized sample solution obtained by passing the microorganism-containing sample solution through the filtration removing means 3 is introduced into the measurement channel 5, and the same as the above. Then, a predetermined amount thereof is introduced into the combustion tube 12. Here, substantially no microorganisms are present in the sample solution, but since the contaminating organic components are not removed by the filtration removal means, the organic components are burnt and oxidized to be converted into carbon dioxide, and the absorbance is absorbed together with other carbon dioxide. The TC value B is detected, and the TC value B is calculated based on the detected value and recorded and displayed.

Here, the value obtained by subtracting the TC value B from the TC value A corresponds to the amount of microorganisms. Therefore, the microorganisms can be directly quantified as the amount of carbon by this value, and by preparing a calibration curve of the amount of carbon and the concentration of microorganisms (weight concentration, number concentration, etc.) in advance, various amounts of microorganisms can be obtained based on the above subtracted values. Can be quantified in units of.

Quantification of microorganisms Microorganisms (lactic acid bacteria) were quantified using the above apparatus under the following conditions.

[Combustion oxidation conditions]

Carrier gas flow rate 150ml / min Heating temperature 680 ℃ [Microorganism-containing sample solution] Prepared by mixing approximately 5.0mg of lactic acid bacteria in physiological saline (containing glucose equivalent to TC approximately 100ppm) 1.

However, it is not pyrogen free.

[Ultrafiltration, etc.]

Built-in ultrafiltration membrane consisting of 0.2 μm, φ2.5 mm membrane filter made of cellulose (sterilized).

The overpressure is about 0.1 Kg / cm ² .

The sample injection for TC measurement was 5 μm, and the TC calibration was performed at 640 ppm potassium hydrogen phthalate standard value.

Results are shown in FIG. The TC value A was 603.0 ppm TC equivalent, and the TC value B was 107.7 ppm TC equivalent. Therefore, lactic acid bacteria are 49
It is equivalent to 5.3ppm TC. Further, the TC value B shows a good agreement with the background (physiological saline) of about 100 ppm, indicating that the lactic acid bacterium removal rate is good.

Quantification of Pyrogenic Substance On the other hand, quantification of the exothermic substance was carried out using the quantification device shown in FIG. The sample solution used contained a predetermined amount of endotoxin as a pyrogen, and was prepared by dissolving a standard amount of a pyrogen (USP Reference Endotoxin, EC-5) in a standard solution free of endotoxin.

TC values A and TC values B were measured in the same manner as in the quantification method for microorganisms for sample liquids containing various concentrations of pyrogenic substances, and converted into endotoxin concentrations. The time required for quantification was within 5 minutes.

The results are shown in FIG. 3 in comparison with the values obtained by the conventional rim lath test.

As described above, the linearity of the response for the exothermic substance is excellent, and it can be understood that the quantification can be performed with high accuracy equivalent to that of the limus test. In the figure, Plot- ○ -shows the result by the method of the present invention, and Plot-Δ- shows the result by the rimlas test.

In place of the membrane filter in the ultrafilter, reverse osmosis membrane (cellulose triacetate membrane; outer diameter 90 mm, length
420 mm; permeated water 0.018 m ³ / hr) or polysulfone ultrafiltration membrane (outer diameter 40 mm, length 350 mm; nominal molecular weight cutoff 6000, permeated water 0.035 m ³ / hr) about,
The same result as above was obtained.

(F) Effects of the Invention According to the method and apparatus for quantifying microorganisms and the like of the present invention, the amounts of microorganisms and pyrogenic substances can be quantified quickly and easily. Furthermore, since the TC value is used as the basis for quantification, highly reliable quantification can be performed and it can be easily applied to continuous measurement.

[Brief description of drawings]

FIG. 1 is a structural explanatory view showing an embodiment of an apparatus for carrying out a method for quantifying microorganisms of the present invention, and FIGS. 2 and 3 show an analysis chart and an analysis result obtained in the embodiment of the present invention. It is a graph figure which illustrates. 1 ... Quantitative device for microorganisms, 2 ... Sample liquid supply unit containing microorganisms (or pyrogens), 3 ... Filter removal means, 4 ... Three-way valve, 5 ... Metering flow path, 6 ... High pressure hexagon Switching valve, 7 ... Liquid metering means, 8 ... Drainage tank, 9 ... Introducing pipe, 10 ... Carrier gas supply section, 11 ... Total carbon analyzer, 12 ... Combustion pipe, 13 ... Electric heating Drain, 14 …… Drain separator, 15,17 …… Drain pot, 16 …… Electronic cooler, 18 …… Non-dispersive infrared detector, 19 …… Flowmeter, 20 …… Calculator, 21 …… High purity Air cylinder, 22 ... Flow control valve.

Claims (2)

[Claims] 1. A step of measuring a TC (total carbon) value A by directly introducing a predetermined amount of a sample solution containing a microorganism (or a pyrogen) into a total carbon analyzer, and the same microorganism (or pyrogenicity) as described above. Substance) -containing sample solution is introduced into a total carbon analyzer to obtain a demicrobial (or depyrogenic substance) sample solution, and the TC value B is measured. Based on the value obtained by subtraction, the above microorganism (or pyrogen)
A method for quantifying microorganisms and the like, which comprises quantifying microorganisms (or pyrogens) in a contained sample liquid. 2. A sample liquid supply path I extending from a sample liquid supply unit containing a microorganism (or a heat-generating substance), and a sample liquid supply path I extending from the sample liquid supply unit containing a microorganism (or a heat-generating substance) and a microorganism ( Or a sample liquid supply path II provided with a means for removing a heat-generating substance, and a sample solution supply path I and a sample solution supply path II which are switchably connected to supply the sample solution to the liquid measuring means. An apparatus for quantifying microorganisms and the like, which comprises a measuring flow path capable of performing measurement and a total carbon analyzer connected so as to introduce a predetermined amount of sample liquid from a liquid measuring means.