JPH047466B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for testing minute impurities in water suitable for quantifying the amount of microorganisms such as bacteria in a sample of ultrapure water.

[Prior art]

Water used in the semiconductor industry and the like needs to be at a particle-free, ultra-pure level. A method called direct microscopic assay (hereinafter referred to as "direct microscopic method") has traditionally been used to manage the water quality.

The principle of direct microscopy is that in order to measure microparticles such as bacteria in water, sample water is filtered through a special filter, the microparticles to be assayed are collected on the filter, and the microparticles are dyed with an appropriate stain. This is to make it easier to observe.

Figure 1 shows the configuration for carrying out the direct observation method, in which 1 is a nitrogen cylinder for pressurization, 2 is a regulator for pressurization adjustment, 3 is a gas pipe, 4 and 6
is an on-off valve, and 5 is a nitrogen gas pressure gauge. 7 is an air filter device that filters and collects particles in the nitrogen gas flowing into the sample tube 8 from the pressurizing nitrogen cylinder 1 in order to prevent them from being mixed into the sample water in the sample tube 8;
A filter element 9 having many micropores of 0.2μ is
It has a structure held in a container by a ring 10,
This makes it possible to trap all microscopic impurities such as bacteria in the nitrogen gas.

Sample tube 8 filled with sample water 11
has a structure that is sealed by a lid 13 equipped with an air vent valve 12, and the sample water 11 is pressurized by nitrogen gas flowing in from the inflow hole 14 of the lid 13, and the sample water 11 is used for sample collection. It is guided to a filter element 16 in a filter device 15. In addition, the filter element 16 is integrally attached and detached with an O-ring 19 together with a spacer 17 and a plate 18 provided on the back side of the filter element 16 so that the sample water is evenly filtered on the filter element 16. It is installed. Note that 20 is a clamp for connecting the sample collection filter device 15 to the sample tube 8, and 21 is a waste liquid tank for accommodating sample water after sample filtration.

In the above-described configuration, the sample filter making section is composed of the sample tube 8 and the sample collection filter device 15. For sample collection, the sample collection filter element 16 is installed in the sample collection filter device 15 as shown in the figure, and after filling the inside with dust-free water to avoid air bubbles, the clamp 20 By sample tube 8
Set to . Inside the sample tube 8,
A fixed amount of sample water, such as ultrapure water, to be tested is filled, and the lid 13 is closed. The inflow hole 14 of this lid 13 is connected to the nitrogen cylinder via the air filter device 7, and initially the regulator 2,
Both the on-off valves 4 and 6 and the air vent valve 12 are in a closed state.

After confirming the closed system as described above, open the regulator 2 and on-off valve 4 to flow nitrogen gas, read the pressure gauge 5, and adjust the gas pressure with the regulator 2 so that the predetermined pressure is reached. Open the on-off valve 6. The nitrogen gas is turned into a dust-free gas by the air filter device 7, flows into the sample tube 8, and is pressurized. The sample water 11 enters the sample collection filter device 15 due to the applied pressure,
The sample water is filtered by a filter element 16 detachably held by an O-ring 19, and minute impurities 22 such as microorganisms in the sample water are collected on the filter element 16.

After the sample is filtered and collected on the filter element 16 in this manner, the air vent valve 12 is opened to return the pressure inside the sample tube 8 to atmospheric pressure. Thereafter, the clamp 20 is removed, the sample collection filter device 15 is removed, and the filter element 16 installed therein is gently taken out using tweezers or the like.
Move on to the dyeing process.

The staining process is a process of staining minute impurities in the sample water captured on the filter element 16 so that they can be easily measured under a microscope. This process is
It also consists of the following steps: drying, staining, washing, re-drying, and slide glass creation.

FIG. 2 is an explanatory diagram of the dyeing process. As shown in FIG.
A dry filter paper 24 is placed inside, and the sample-collected filter element 16, which has been removed from the sample-collecting filter device 15, is placed thereon with the filtration collection side facing up and dried. After that, similarly, as shown in FIG.
A filter paper 26 impregnated with a dyeing solution is spread, and the dried filter element 16 is placed thereon and left for 5 to 6 minutes, thereby dyeing the collected fine impurities on the filter element 16. After dyeing, the filter element 16 is removed using tweezers as shown in FIG.
8, the side to which the staining solution has adhered is cleaned by a wiping method, and then returned to another shear tray or the like and dried again.

Filter element 16 dyed in this way
, as shown in figure c, immersion oil 2
A drop of immersion oil was placed on the filter element 16, a cover glass 31 was placed on top of the slide glass 30, and the field of view shown in Figure 3 was observed using the microscope 32. The stained micro impurities 22 inside are assayed.

The filter element 16 for sample collection used in such a direct microscopy method is made of transparent polycarbonate.
Because of its thickness, there is no need to use special transparency means, and it can be easily observed with an optical microscope as it is.Furthermore, when immersed in a staining solution, the filter element itself made of the above-mentioned material will not be stained at all. It has the advantage that only the captured bacterial cells are stained.

Although the conventional direct microscopic method is a method that can reliably detect microorganisms by using a sample collection filter element having the above-mentioned advantageous characteristics, it has the following drawbacks.

(1) A troublesome staining process is required after sample collection, and it takes too much time and effort until microscopy.

(2) If staining is performed with minute impurities collected, there is a risk of uneven staining, which reduces the measurement accuracy of minute impurities.

〔the purpose〕

The purpose of the present invention is to provide a method for assaying minute impurities in water that can omit staining after sample collection and that can be easily and accurately measured using a microscopic microscope, in order to overcome the drawbacks of the conventional direct microscopy method as described above. This is what we are trying to provide.

〔overview〕

In the method of the present invention, when testing microorganisms and other micro impurities in water, by adding a staining agent that mainly binds to the microorganisms and other micro impurities that are to be detected to the sample water, the microorganisms and other micro impurities present in the sample water are dyed in advance. The method is characterized by reacting with a fluorescent dye and dyeing it fluorescently, then collecting it by filtration and examining it under a microscope.

To summarize, microscopic impurities such as microorganisms in sample water are dyed in advance and then collected by filtration in the same manner as conventional methods. The samples created in this way have already been dyed, so if there are microorganisms in the sample water, their presence can be detected with high accuracy, and there is no need for the post-collection staining process that was previously required. become.

〔Example〕

FIG. 3 is an external view showing an example of the configuration of a sample tube 33 for carrying out the present invention. In this embodiment, in place of the sample tube 8 shown in FIG. 1 in the conventional configuration shown in FIG.
The sample tube 33 shown in the figure is used.

Further, in this example, a case will be described in which a fluorescent substance is used as the staining agent.

The sample tube 33 shown in FIG.
Although it has almost the same configuration as the sample tube 8 used in the conventional method described in the drawings, it differs from the sample tube 8 in FIG. 1 in that a chemical solution inlet 35 is provided in the lid 34. Note that 36 is an air vent hole, and 37 is a lid for a chemical solution inlet.

To carry out the test, fill the sample tube 33 with an appropriate amount of the sample water 11 to be tested, close the lid 34, and then inject a fluorescent substance, such as O-phthalaldehyde, into the sample tube 33 using the syringe 38 or the like. A minute amount of -2 mercaptoethanol solution 39 is dropped from the chemical solution inlet 35, and then the lid 37 is closed.
Other operations may be exactly the same as the conventional method described above. That is, nitrogen gas is supplied from the pressurizing port 40 through the air filter device 7 shown in FIG.
is filtered and collected by a filter element 16 in a sample collection filter device 15 provided as shown in FIG. Thereafter, as in the conventional method, the sample is taken out from the sample collecting filter device 15 and placed on a drying filter paper placed in a suitable container such as a shear dish, with the sample collecting side facing up, and dried.

Note that the operations from pressurizing the sample water to taking out the sample-collected filter element 16 may be the same as in the conventional method described above, so the explanation will be omitted.

In the sample prepared in this manner, the microorganisms present in the sample water are collected on the filter element 16 in the form of a fluorescent substance, so microscopic measurements can be carried out immediately without the need for additional staining work. I can do it.

As is well known, O-phthalaldehyde used as the fluorescent substance in this example is It has the structural formula, and is usually used as a reagent for quantifying primary amines in the field of component analysis. In the presence of an amino group and 2-mercaptoethanol, It reacts according to the following reaction formula, and has the property of emitting strong blue fluorescence with a fluorescence wavelength of around 455 nm when exposed to excitation wavelength light of around 340 nm. In this example, the quantitative properties of the amino group of O-phthalaldehyde as described above were applied to the detection of microbial proteins.
It is designed to detect fine particles, mainly microorganisms, in sample water with high sensitivity.

That is, in the above-described embodiment of the present invention, the microorganisms collected on the filter element 16 have O-phthalaldehyde, a fluorescent substance, already bound to the amino acids of the proteins in the cytoplasm of the microorganisms in the sample tube 33. ing. Therefore, the microorganisms collected on the sample element 16 after sample collection are uniformly fluorescently stained, so if they are dried and observed using, for example, a fluorescence microscope, as explained earlier, Not only can the presence of microorganisms be detected extremely easily by their fluorescence, but the accuracy of the assay can also be further improved.

In addition, in the above example, the case where microorganisms in the sample water are mainly assayed is explained, but the present invention is not limited to this, and when detecting other minute impurities, the microorganisms to be detected are Of course, it is possible to use a fluorescent substance that mainly reacts with impurities as a staining agent, drop a small amount of this into the sample water in the sample tube to cause a reaction, and then collect the dye by filtration.

〔Effect of the invention〕

As explained in detail above, according to the method of the present invention, the staining process for minute impurities such as microorganisms after filtration and collection of sample water as explained in FIGS. It gets easier. Moreover, when microorganisms and the like are collected by filtration, all of the microorganisms and other microscopic impurities have already been dyed, so there is no risk of uneven staining, and the measurement accuracy is further improved.

[Brief explanation of drawings]

Fig. 1 is a diagram showing the configuration of an apparatus for preparing a sample using the conventional direct microscopy method. Fig. 2 is an explanatory diagram of the sample staining process using the conventional direct microscopy method. FIG. 3 is an external view showing an example of the configuration of a sample tube. 7...Air filter device, 8,33...Sample tube, 11...Sample water, 13,3
4, 37...Lid, 15...Filter device for sample collection, 16...Filter element for sample collection, 23, 25, 27...Shearet, 24...Drying filter paper, 26...Dyeing solution impregnated filter paper , 28... Water-containing filter paper, 29... Immersion oil, 30...
Slide glass, 31...Cover glass, 32...
...Optical microscope, 35...Medical solution inlet, 36...Air vent, 38...Syringe, 39...Medical solution, 4
0...Nitrogen gas inlet.

Claims (1)

[Claims] 1. When testing minute impurities such as microorganisms in water, by adding a staining agent that primarily binds to the microorganisms and other minute impurities to be detected to the sample water,
1. A method for assaying micro impurities in water, which comprises reacting micro impurities such as microorganisms present in sample water with the staining agent, staining the water, collecting the sample through filtration, and examining it under a microscope.