JP4895642B2 - Microbial separation method - Google Patents

Description

  The present invention relates to a method for separating microorganisms, and in particular, in order to detect infectious microorganisms such as protozoa, bacteria, and viruses present in water such as environmental water such as rivers and lakes and treated water of each treatment process of water and sewage, The present invention relates to a method for separating microorganisms from collected sample water.

  Since there are a wide variety of chemical substances in the environment, it is thought that environmental waters such as rivers and lakes that are raw water for water supply are also contaminated with various chemical substances. However, in addition to water quality problems in the water environment, the occurrence of water-borne infections caused by protozoa such as Cryptosporidium, bacteria such as enterohemorrhagic Escherichia coli O157 and Legionella, and viruses has become a major social problem.

  For example, in the method for testing protozoa such as Cryptosporidium and Giardia, which are the cause of emerging waterborne infections (for example, Non-Patent Document 1), the Cryptosporidium test procedure is roughly divided into sampling, filtration concentration, and peeling. It consists of suspension, separation and purification, immunofluorescence staining, and microscopic observation.

  However, this Cryptosporidium inspection method requires many steps as described above, and the work is cumbersome and it takes more than half a day to complete the inspection. Can not do it. In particular, in the above test method, when Cryptosporidium is generally captured and recovered from a sample, the disk filter is clogged due to the turbidity of the sample, and the filtration speed decreases in a short time, so the time required for the entire test process In other words, the length of the sample becomes long and the measurement cannot be performed quickly, and a large amount of sample cannot be processed efficiently. Furthermore, the recovery rate of Cryptosporidium in the above-mentioned filtration concentration and exfoliation suspension is as low as around several percent to 80%, and the detection error may be judged to be negative.

  Further, a Cryptosporidium concentration method using calcium carbonate (Non-Patent Document 2) has been reported as a separation and concentration method that does not rely on filtration. In this concentration method, a calcium chloride solution and a sodium hydrogen carbonate solution are added to the sample water, and when the pH is raised to 10 with the sodium hydroxide solution, insoluble calcium carbonate crystals are precipitated. The microorganism to be detected is adsorbed and the precipitate is collected. The principle of this concentration method is considered as follows.

  That is, the surface of Cryptosporidium is negatively charged. When a calcium chloride solution is added, cationic calcium ions gather in the vicinity of Cryptosporidium, and when a sodium hydrogen carbonate solution is added, the anionic carbonate ion is Cryptosporidium. The calcium carbonate crystal is precipitated at the same time as Cryptosporidium is taken into the crystal, and the crystal grows and settles. In order to separate Cryptosporidium from this calcium carbonate crystal, sulfamic acid, which is an acid, is added to the calcium carbonate precipitate, and only the calcium carbonate is dissolved to isolate Cryptosporidium.

  However, in this method, in order to precipitate calcium carbonate adsorbed by Cryptosporidium, it has to be allowed to stand for 4 hours or more or overnight, and the time required for all the test steps becomes long, and there is a problem that rapid measurement cannot be performed. is there.

On the other hand, in Patent Document 1, calcium hydroxide is added to a suspension containing true bacteria to agglomerate the bacteria to a pH of 10 or more, and ferric chloride is further added to grow the aggregates. A method is described in which the grown agglomerates are dehydrated with a centrifuge or the like to separate true bacteria. In this method, the water content of the precipitate forms a large floc of 60 to 80%, and the precipitate can be recovered by a press filter or centrifugal separation. However, there is a problem that the apparatus is complicated and large-scale.
“Cryptosporidium Provisional Countermeasure Guidelines for Water Supply”, Ministry of Health, Labor and Welfare, revised 1998 G. Vesey et al., “A new method for the concentration of Cryptosporidium oocysts from water”, Journal of Applied Bacteriology ), 1993, 75, p. 82-86 Japanese Patent Publication No.55-49826

  In view of the above problems, an object of the present invention is to provide a method for separating a microorganism that can precipitate a microorganism-containing precipitate in a short time and can increase the detection sensitivity of the microorganism. .

  In order to achieve the above object, a method for separating a microorganism according to the present invention includes a step of adding an anion solution and a cation solution to sample water containing microorganisms to precipitate crystals of an inorganic substance that is hardly soluble in water. And a step of further adding an iron-based flocculant to the sample water, aggregating the precipitate to settle as a floc, and dissolving the precipitate with an acid to obtain the microorganism. .

As the anion solution, a solution containing SO ₄ ²⁻ , CO ₃ ²⁻ or PO ₄ ³⁻ is used, and as the cation solution, Mg ²⁺ , Ca ²⁺ , Sr ²⁺ or Ba ²⁺ is used. It is preferred to use a solution. It is preferable to use ferric chloride as the iron-based flocculant. It is preferable to use hydrochloric acid as the acid. The step of dissolving the precipitate with an acid to obtain a microorganism includes adding the acid to the precipitate and then passing a dissolution time for the acid to dissolve the precipitate, and then the solid containing the microorganism and the solid A step of separating into a precipitate solution is preferred.

  In this way, after adding an anion solution and a cation solution, and precipitating a poorly water-soluble inorganic substance incorporating microorganisms, adding an iron-based flocculant causes the precipitate to aggregate and form a floc. The sedimentation speed is greatly improved, and the supernatant and sediment can be separated in a short time. Further, by dissolving the precipitate with an acid, the microorganism can be separated from the precipitate without reducing the detection sensitivity when detecting the microorganism. Therefore, labor saving and automation of the work of separating microorganisms can be achieved. Furthermore, a method of solid-liquid separation with a simple mechanism such as suction removal of the supernatant can be applied.

  As the flocculant for water treatment process, among inorganic flocculants, aluminum flocculants such as polyaluminum chloride (PAC) and aluminum sulfate (sulfuric acid band) and iron such as polyiron sulfate and ferric chloride are used. Examples of organic flocculants made of polymers include polyacrylic acid and alginic acid. However, since the inorganic substance adsorbing microorganisms is dissolved with acid, it is difficult to use an organic flocculant from the viewpoint of solubility in acid. As for the inorganic aluminum flocculant, aluminum is suspected of being a causative substance of Alzheimer's disease and cranial nervous system disease, and therefore, it is in the direction to be subject to emission control in the future, and it is desirable to refrain from using it.

  Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic diagram showing a microorganism detection apparatus suitable for carrying out the microorganism separation method according to the present invention. The following is a description when the detection target microorganism is Cryptosporidium, but the present invention is not limited to this and can be widely applied to microorganisms such as protozoa, bacteria, and viruses.

  As shown in FIG. 1, the microorganism detection apparatus according to the present embodiment includes a concentration tank 13 that aggregates microorganisms in sample water to form a floc, a sample purification unit 16 that separates microorganisms from the floc, and microorganisms. An antibody reaction unit 25 that performs an antigen-antibody reaction by adding a fluorescently labeled antibody, a fluorescence detection unit 37 that detects microorganisms, and a sample storage unit 44 that stores the detected microorganisms are mainly configured.

  First, sample water 1 is supplied into the concentration tank 13 by the sample water supply pump 2. While stirring the sample water 1 with the agitator 14 provided in the concentration tank 13, the calcium chloride solution 3 is supplied to the concentration tank 13 with the cation solution supply pump 4, and the sodium hydrogen carbonate solution 5 is supplied with the anion solution. Supply to the concentration tank 13 by the pump 6. Then, in order to raise the pH to 10 to generate calcium carbonate, the sodium hydroxide solution 7 is supplied to the concentration tank 13 by the pH adjuster supply pump 8. Thereby, calcium carbonate in which Cryptosporidium is taken in is deposited.

  After the calcium carbonate is precipitated, ferric chloride 9 is supplied to the concentration tank 13 by the flocculant supply pump 10. Thereby, calcium carbonate aggregates and forms a floc. Then, the stirrer 14 is stopped and flocs are allowed to settle naturally.

  Next, the precipitate precipitated on the bottom of the concentration tank 13 is supplied to the sample purification unit 16. At the lowest end of the bottom of the inverted conical shape of the concentration tank 13, a pipe connected to the precipitate dissolution tank 19 is provided. By opening the valve 15 provided in this pipe, the precipitate in which Cryptosporidium is taken in Is sent to the precipitate dissolution tank 19. The remaining supernatant is discharged through a pipe (not shown) separately provided at the bottom of the concentration tank 13. After discharging the supernatant, the washing water 11 is supplied to the concentration tank 13 by the washing water supply pump 12 and washed, and then the next sample water is supplied.

  Hydrochloric acid 17 is injected into the precipitate dissolution tank 19 by an acid supply pump 18. Thereby, only calcium carbonate dissolves in the precipitate of calcium carbonate adsorbed by Cryptosporidium, and thus a concentrated sample solution in which Cryptosporidium is concentrated is obtained.

  Since this concentrated sample solution contains salts such as calcium ions and the pH is lowered by hydrochloric acid, it interferes with the binding between Cryptosporidium and the fluorescently labeled antibody that labels Cryptosporidium. Therefore, in order to remove the salts, the valve 20 at the lower end of the precipitate dissolving tank 19 is opened, and the concentrated sample solution in the precipitate dissolving tank 19 is supplied to the desalting and defoaming tank 21.

  Cryptosporidium (diameter 5 μm) can pass through the desalting and defoaming tank 21 and can capture contaminants larger than the detection target microorganism, for example, a contaminant removal filter having a pore size of 10 μm or more, and cryptosporidium, For example, a microorganism capturing filter having a pore size of 1 μm or less that permeates a solution containing a salt is provided in two upper and lower stages. Thereby, the impurities in the concentrated sample solution and the solution containing a high concentration of salts can be removed. The sample containing Cryptosporidium trapped in the microorganism capture filter of the desalting and defoaming tank 21 is washed out with salts with the wash water 22 supplied by the wash water supply pump, and then the valve 24 is opened and the antibody reaction unit 25 is opened. Supply.

  The antibody reaction unit 25 includes an antibody mixing tank 26 for mixing a sample containing Cryptosporidium and a fluorescently labeled antibody for labeling Cryptosporidium, and an antibody reaction tank 27 for performing an antigen-antibody reaction. First, in the antibody mixing tank 26, a fluorescently labeled antibody 28 that specifically recognizes cryptosporidium is injected by a labeled antibody supply pump 29 and mixed with a sample. At this time, when one type of fluorescently labeled antibody is used, there is a possibility that a fluorescently labeled antibody that is not specifically bound to a contaminant may be determined as a false positive. Therefore, a plurality of antibodies with different antigen recognition sites are prepared, and each of them is fluorescent. A method of multiply labeling the detection target microorganisms with fluorescently labeled antibodies combined with fluorescent substances having different wavelengths is more preferable because the detection accuracy of the detection target microorganisms is increased.

  The antigen-antibody reaction with many commercially available antibodies is 20 to 30 minutes at 37 ° C. and 1 hour or more at room temperature, and in order to measure more frequently, after mixing the sample and the fluorescently labeled antibody, It is preferable that the mixed liquid be transferred to another container so that the next sample can be supplied. Furthermore, in order to measure Cryptosporidium at a high frequency, it is preferable to prepare a plurality of antibody reaction tanks 27 and supply a different antibody reaction tank for each sample by switching the valve 34.

  Therefore, as shown in FIG. 1, first, the fluorescently labeled antibody 28 is mixed with the first sample, and then the valve 33 is opened, and this mixed solution is washed away with the washing water 31 from the washing water supply pump 32. To supply. Then, the valve 36 is opened, and the antibody mixing tank 26 is washed with the washing water 31, and then the valve 36 is closed to supply the second sample to the antibody mixing tank 26. This second sample is mixed with the fluorescently labeled antibody 28 in the same manner as the first sample, and this mixed solution is washed away with the washing water 31 and sent to the antibody reaction tank 27. At this time, the second mixed liquid is supplied to the antibody reaction tank 27 different from the first mixed liquid by switching the valve 34.

  In order to advance the antigen-antibody reaction efficiently, the antibody reaction part 25 or the antibody reaction tank 27 is preferably equipped with a thermostat (not shown) for keeping the sample temperature at 37 ° C. The sample for which the antigen-antibody reaction has been completed is supplied to the fluorescence detection unit 37 by the pump 35 by opening the corresponding valve 34.

  The fluorescence detection unit 37 measures the fluorescence intensity of Cryptosporidium bound with the fluorescence labeled antibody with a fluorescence detector 38 such as a fluorescence microscope observation or a flow cytometer, and sends the measurement data to the determination unit 40. When performing multiple staining using a plurality of fluorescently labeled antibodies, it is preferable to use a fluorescence detection unit that can measure a plurality of fluorescence wavelengths simultaneously. In order to further improve the detection accuracy, it is preferable to circulate and supply the sample to the fluorescence detector 38 and integrate the measurement data.

  Therefore, as shown in FIG. 1, a three-pronged sample supply switching valve 41 is provided on the inlet side of the fluorescence detector 38, and a three-pronged sample discharge switching valve 39 is provided on the outlet side. At the same time, by providing a sample circulation pump 42 between the fluorescence detector 38 and the sample discharge switching valve 39, the sample measured by the fluorescence detector 38 is transferred by the sample circulation channel 43 by switching the valves 41 and 39. It can be supplied to the fluorescence detector 38 again. The determination unit 40 can switch the valves 41 and 39 based on the measurement data to control whether the sample is circulated or sent to the sample storage unit 44.

  In the sample storage unit 44, the determination unit 40 determines whether the sample storage is necessary based on the measurement data detected by the fluorescence detector 38. That is, when Cryptosporidium is detected in the sample by the fluorescence detector 38, the channel is switched by the three-way channel switching valve 46, and the sample discharged from the fluorescence detection unit 37 is supplied into the storage container 45. ,save. The stored sample can be provided to a test for determining whether the contaminating microorganism to be detected is alive or infectious. On the other hand, when Cryptosporidium is not detected, it is discarded.

In FIG. 1, the calcium chloride solution 3 is supplied from the cation solution supply pump 4 and the sodium carbonate solution 5 is supplied from the anion solution supply pump 6 to generate calcium carbonate. And a combination of a cation solution and an anion solution that produce a precipitate that dissolves in an acidic solution. For example, a solution containing Mg ²⁺ , Ca ²⁺ , Sr ²⁺ or Ba ²⁺ is used as the cation solution, and SO ₄ ²⁻ , CO ₃ ²⁻ or PO ₄ ³⁻ is used as the anion solution. A solution can be used. Examples of the precipitate generated thereby include magnesium carbonate, calcium sulfate, calcium carbonate, calcium phosphate, strontium sulfate, strontium carbonate, barium carbonate, and barium phosphate.

  In FIG. 1, the sodium hydroxide solution 7 is supplied from the pH adjuster supply pump 8, but potassium hydroxide, aqueous ammonia, or the like can be used as the pH adjuster. Ferric chloride 9 was supplied from the flocculant supply pump 10, but polyiron sulfate, polysilica iron, or the like can be used as the iron-based flocculant. Although hydrochloric acid 17 was supplied from the acid supply pump 18, nitric acid, citric acid, acetic acid, etc. can be used in addition to the acid. An acid having a concentration of 1 mol / L is preferably added at 20 mL to 1 g of the precipitate. In addition, after the acid is added to the precipitate in the precipitation dissolution tank 19 and before the microorganisms are separated in the desalting and defoaming tank 21, the concentrated sample solution to which the acid has been added is allowed to stand for 10 minutes or more to dissolve. Is preferably provided. During the dissolution time, it is preferable to stir and actively promote dissolution.

  Furthermore, although FIG. 1 demonstrated the case where a membrane filter (microfiltration membrane) was used as the desalting and defoaming tank 21, the method of desalting and defoaming is not limited to this, and the size of microorganisms to be separated. Depending on the microorganism, a method using microorganisms, a method using an ultrafiltration membrane, a dialysis using a dialysis membrane, or the like, or a method using a centrifuge may be used.

(Sedimentability test of iron-based flocculant)
Among iron-based flocculants, polyiron sulfate and ferric chloride are generally used, and therefore, those flocculants that are suitable for the method of separating microorganisms were screened. That is, calcium carbonate is produced in pure water, and a flocculant is added here to precipitate calcium carbonate. The suspension is allowed to stand, and the turbidity of the supernatant is measured at regular intervals to agglomerate. The sedimentation properties of the precipitates by the agents were compared. Details will be described below.

  1 L of pure water was placed in a polypropylene beaker, and a 1 mol / L calcium chloride solution and a 1 mol / L sodium hydrogen carbonate solution were first added while stirring with a jar fermenter equipped with a constant temperature water bath at 25 ° C. Next, 1N (1N) sodium hydroxide solution was added to adjust the pH to 10 to produce insoluble calcium carbonate. Then, each flocculant is added at an injection rate of 2.5, 5.0, 7.5, 10.0 mg-Fe / L, and rapid stirring is performed at a speed of 40, 50, 60, 70 cm / s for 1 minute. The mixture was gently stirred at 15 cm / s for 10 minutes and then allowed to stand.

  The supernatant was sampled at regular intervals, the absorbance at 660 nm was measured with an absorptiometer, and the turbidity was calculated. Turbidity was calculated in advance using a turbidity standard solution 100 degrees (polystyrene) (manufactured by Wako Pure Chemical Industries, Ltd.), and the turbidity was calculated from the absorbance of the supernatant. The results are shown in Table 1. ○ in Table 1 indicates that the supernatant turbidity is 0.1 or less after 45 minutes from standing, Δ indicates that the supernatant turbidity is 0.1 or less after 60 minutes, and × indicates 60 minutes. Later, the supernatant turbidity did not become 0.1 or less, and nt represents that the test was not performed.

  As shown in Table 1, ferric chloride was used as a condition for aggregating and precipitating calcium carbonate with an iron-based flocculant, and the injection rate was 2.5 mg-Fe / L. 50 or 70 cm / s has been found suitable. Furthermore, when compared with a rapid stirring intensity of 50 and 70 cm / s under these conditions, the rate of decrease in the supernatant turbidity was faster at 70 cm / s, and the precipitation of calcium carbonate was better.

(Acid microbiological recoverability and labeling test)
Depending on the type of acid used to dissolve the precipitate, the precipitate cannot be completely dissolved, and the remaining precipitate may interfere with microorganism detection, or the antigenic determinant on the cell membrane of the microorganism to be detected may be acid. As a result, the binding property with the labeled antibody was lowered, and as a result, the labeling property of the labeled antibody was lowered. That is, a predetermined solution and an aggregating agent are added to pure water to which Cryptosporidium is added to obtain a precipitate in which Cryptosporidium is incorporated, which is dissolved with various acids, and the Cryptosporidium separated and recovered is fluorescently labeled with a labeled antibody. Then, by detecting with a flow cytometer, the recovery rate and labeling property of microorganisms by acid were compared. Details will be described below.

  While taking 1 L of pure water into a polypropylene beaker and stirring with a jar fermenter equipped with a constant temperature water bath at 25 ° C., about 1,000 Cryptosporidium oocysts (manufactured by Waterborne) were added, and the following solution was added. . First, a 1 mol / L calcium chloride solution and a 1 mol / L sodium hydrogen carbonate solution are added and stirred, then a 1N (1N) sodium hydroxide solution is added, the pH is adjusted to 10, and an insoluble carbonic acid solution is added. Calcium was generated. Then, ferric chloride was added at an injection rate of 2.5 mg-Fe / L, rapid stirring was performed at a speed of 70 cm / s for 1 minute, slow stirring was performed at 15 cm / s for 10 minutes, and then allowed to stand. . After 1 hour, the supernatant was removed by suction with a suction pump, and various acids were added to the remaining precipitate. The acids used for comparative study are sulfamic acid, inorganic acid hydrochloric acid, nitric acid, sulfuric acid, and organic acid citric acid used in the conventional method.

  The acid solution obtained by adding the acid is transferred to a centrifuge bottle, centrifuged at 3000 g for 10 minutes, and then the supernatant is removed, and the precipitate is washed with 1% PET (0.02% sodium pyrophosphate, 0.03% (EDTA-3Na, 0.01% Tween 80) was suspended in 30 mL, and transferred to a 50 mL polypropylene centrifuge tube. Again, centrifuge at 3000 g for 10 minutes, then remove the supernatant and suspend the precipitate in 1 mL of 1% PET (0.02% sodium pyrophosphate, 0.03% EDTA-3Na, 0.01% Tween 80) It was transferred to a 1.5 mL polypropylene microtube. This was centrifuged at 15000 rpm for 2 minutes, the supernatant was removed, and then the precipitate was resuspended in 1 mL of PBS to obtain a Cryptosporidium concentrated sample.

  100 μL of labeled antibody (EasyStain C + G, manufactured by Wako Pure Chemical Industries, Ltd.) conjugated with a fluorescent substance FITC (fluorescein insothiocyanate) was added to this concentrated sample, and an antigen-antibody reaction was allowed to proceed at 37 ° C. for 30 minutes or at room temperature for 1 hour. It was. The forward scattered light intensity and FITC fluorescence intensity of fluorescently labeled Cryptosporidium, and the fluorescence intensity (FL3) at 650 nm were measured with a flow cytometer (FACS Calbur, Becton Dickinson). In the case of FACS Calibur, the light source of the light irradiation unit uses an argon laser (488 nm), FSC (forward scattered light), SSC (side scattered light), FL1 (530 ± 15 nm), FL2 (585 ± 21 nm), FL3 ( 650 nm or more) and a corresponding optical detector detects an optical signal. In this measurement, the measurement results of FSC, FL1, and FL3 were used. Since FL1 represents fluorescence derived from FITC, the FL1 intensity of Cryptosporidium bound to the FITC-labeled antibody is increased. On the other hand, FL3 is a photosynthetic organ-constituting substance derived from dinoflagellate and represents fluorescence of PerCP (peridinin chlorophyll protein), which is a fluorescent substance, and can therefore be used for detecting algal chlorophyll.

  The recovery rate of Cryptosporidium is the ratio between the number of Cryptosporidium detected in the sample labeled with the untreated Cryptosporidium and the number of Cryptosporidium detected by labeling the concentrated sample obtained by the above procedure. Calculated from The results are shown in Table 2.

  As shown in Table 2, when the sulfamic acid was added and immediately after the concentration treatment, a brown precipitate, which was thought to contain the iron component of the flocculant, remained in the concentrated sample. The recovery rate of Cryptosporidium was also as low as less than 10%, and this brown precipitate was considered to be the cause of lowering the labeling property of the labeled antibody. Therefore, in order to prevent the brown precipitate from remaining, the addition amount was increased from 20 mL to 30 mL, and after further adding sulfamic acid, the mixture was stirred for 10 minutes to dissolve the precipitate and then concentrated. As a result, the brown precipitate in the concentrated sample disappeared, but the crypt recovery rate was still as low as about 10%, and the combination of the iron component of the flocculant and sulfamic acid had some adverse effects on the labeling properties of the labeled antibody. It was.

  On the other hand, when hydrochloric acid, nitric acid, or citric acid was used, no brown precipitate remained in the concentrated sample, and calcium carbonate was almost completely dissolved. In addition, when sulfuric acid was used, calcium ions generated by dissolving with acid and sulfate ions were combined to form a white precipitate of poorly water-soluble calcium sulfate, which was not suitable for the method of separating microorganisms.

  When compared with the recovery rate of Cryptosporidium, it was found that the recovery rate was about 80% for hydrochloric acid and about 77% for citric acid, which was relatively good, whereas that for nitric acid was about 41%.

  Next, the evaluation of the labeling property will be described in detail. The forward scattered light (FSC), FITC fluorescence intensity (FL1), and FL3 intensity of fluorescently labeled Cryptosporidium were measured with a flow cytometer and compared.

  Since FSC (forward scattered light) corresponds to the particle size of the particles, the FSC intensity corresponding to the particle size of Cryptosporidium can be grasped by measuring Cryptosporidium in advance. Cryptosporidium was suspended in PBS, and a sample to which a FITC-labeled anti-cryptospodium antibody was added was measured.

  FIG. 2 is a dot plot of FL3 and FL1 in which untreated Cryptosporidium is labeled with a labeled antibody, and FIG. 3 is a dot plot of FSC and FL1. In each dot plot, an area where Cryptosporidium exists (in the solid line frame in the figure) was set. 4 and FIG. 5 show the Cryptosporidium existing region set in both the dot plots of FIG. 2 and FIG. 3 among the particles distributed in the Cryptosporidium existing region set in each dot plot of FIG. 2 and FIG. It is the dot plot which extracted only the particle | grains distributed to. Thus, for example, if particles that do not enter the Cryptosporidium presence region set in the dot plot of FIG. 3 among the particles distributed in the Cryptosporidium presence region set in the dot plot of FIG. As shown in the dot plot, impurities showing false positives can be excluded.

  Similarly, FIG. 6 is a dot plot of FL3 and FL1 of Cryptosporidium labeled with a labeled antibody when hydrochloric acid is used to dissolve calcium carbonate, and FIG. 7 is a dot plot of FSC and FL1. The solid line frame in the dot plot is a region where Cryptosporidium set in FIGS. 2 and 3 is present. FIG. 8 and FIG. 9 show the Cryptosporidium existing region set in both the dot plots of FIG. 6 and FIG. 7 among the particles distributed in the Cryptosporidium existing region set in each dot plot of FIG. 6 and FIG. It is the dot plot which extracted only the particle | grains distributed to.

  When comparing the distribution of untreated Cryptosporidium and the distribution of Cryptosporidium concentrated using hydrochloric acid to dissolve calcium carbonate, the fluorescence intensity of FITC hardly changed. In the sample using hydrochloric acid, the FL3 intensity slightly increased, but the possibility that impurities were mixed in the Cryptosporidium existing region was small. Similar results were obtained with sulfamic acid and nitric acid.

  FIG. 10 is a dot plot of FL3 and FL1 of Cryptosporidium labeled with a labeled antibody when citric acid is used to dissolve calcium carbonate, and FIG. 11 is a dot plot of FSC and FL1. The solid line frame in the dot plot is a region where Cryptosporidium set in FIGS. 2 and 3 is present. As a result of using citric acid, the region where Cryptosporidium labeled with the labeled antibody was present was different from the distribution of untreated Cryptosporidium, so the result using citric acid was Cryptosporidium labeled with the labeled antibody. The area where is present is indicated by a dotted frame.

  12 and FIG. 13 are set in the dot plots of both FIG. 10 and FIG. 11 among the particles distributed in the Cryptosporidium existing region (in the dotted frame) set in each dot plot of FIG. 10 and FIG. It is the dot plot which extracted only the particle | grains distributed in the cryptosporidium presence area | region which carried out.

  Comparing the distribution of untreated Cryptosporidium with the concentration of Cryptosporidium concentrated using citric acid to dissolve calcium carbonate, the fluorescence intensity of FITC decreased and the FL3 intensity increased in the sample using citric acid. It was found that there was a high possibility that impurities were mixed in the area where the putospodium was present.

It is a schematic diagram which shows the detection apparatus of microorganisms suitable for enforcing the separation method of microorganisms concerning this invention. 2 is a two-dimensional dot plot of untreated Cryptosporidium FL3 and FL1. 2 is a two-dimensional dot plot of untreated Cryptosporidium FSC and FL1. FIG. 4 is a two-dimensional dot plot of FL3 and FL1 in which only distributed particles are extracted in both Cryptosporidium existing regions in FIG. 2 and FIG. 3. FIG. 4 is a two-dimensional dot plot of FSC and FL1 in which only distributed particles are extracted in both Cryptosporidium existing regions of FIG. 2 and FIG. It is a two-dimensional dot plot of FL3 and FL1 when hydrochloric acid is used. It is a two-dimensional dot plot of FSC and FL1 when hydrochloric acid is used. FIG. 8 is a two-dimensional dot plot of FL3 and FL1 in which only distributed particles are extracted in both Cryptosporidium existing regions in FIG. 6 and FIG. 7. FIG. 8 is a two-dimensional dot plot of FSC and FL1 in which only distributed particles are extracted in both Cryptosporidium existing regions of FIG. 6 and FIG. 7. It is a two-dimensional dot plot of FL3 and FL1 when citric acid is used. It is a two-dimensional dot plot of FSC and FL1 when citric acid is used. 12 is a two-dimensional dot plot of FL3 and FL1 in which only distributed particles are extracted in both the Cryptosporidium-existing regions of FIG. 10 and FIG. FIG. 12 is a two-dimensional dot plot of FSC and FL1 in which only distributed particles are extracted in both Cryptosporidium existing regions of FIG. 10 and FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample water 2 Sample water supply pump 3 Calcium chloride solution 4 Cation solution supply pump 5 Sodium hydrogen carbonate solution 6 Anion solution supply pump 7 Sodium hydroxide solution 8 pH adjuster supply pump 9 Ferric chloride solution 10 Coagulant supply Pump 11, 22, 31 Wash water 12, 23, 32 Wash water supply pump 13 Concentration tank 14 Stirrer 15, 20, 24, 30, 33, 34, 36 Valve 16 Sample purification section 17 Hydrochloric acid solution 18 Acid supply pump 19 Precipitation dissolution Tank 21 Desalting Defoaming Tank 25 Antibody Reaction Unit 26 Antibody Mixing Tank 27 Antibody Reaction Tank 28 Fluorescent Labeled Antibody 29 Labeled Antibody Supply Pump 35 Pump 37 Fluorescence Detection Unit 38 Fluorescence Detector 39 Sample Discharge Switching Valve 40 Judgment Unit 41 Sample Supply Switching Valve 42 Sample circulation pump 43 Sample circulation channel 44 Sample storage unit 45 Charge storage container 46 the flow path switching valve

Claims (5)

Adding an anion solution and a cation solution to a sample water containing microorganisms, taking in crystals of inorganic substances that are hardly soluble in water, and depositing the microorganisms ;
Then, the sample water further added iron-based coagulant, allowed to settle as flocs by aggregating the precipitates, the steps separated,
A step of dissolving the precipitate with an acid to obtain the microorganism. A solution containing SO ₄ ²⁻ , CO ₃ ²⁻ or PO ₄ ³⁻ is used as the anion solution, and a solution containing Mg ²⁺ , Ca ²⁺ , Sr ²⁺ or Ba ²⁺ is used as the cation solution. The method for separating a microorganism according to claim 1, which is used.   The method for separating microorganisms according to claim 1, wherein ferric chloride is used as the iron-based flocculant.   The method for separating a microorganism according to any one of claims 1 to 3, wherein hydrochloric acid is used as the acid.   The step of dissolving the sediment with an acid to obtain a microorganism comprises adding the acid to the sediment, and after the dissolution time for the acid to dissolve the sediment, the solid containing the microorganism and the precipitation The method for separating a microorganism according to any one of claims 1 to 4, wherein the method is a step of separating the product into a product lysate.