JP3461556B2 - Microorganism testing method using hollow fibers - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to relates to the inspection how to confirm the presence or absence of microorganisms in the sample.

[0002]

2. Description of the Related Art In the fields of various beverages and foods, pharmaceuticals and medical fields, biotechnology, electronics, etc., inspection methods for confirming the presence of microorganisms are extremely important, and various methods have been proposed in the past. ,It has been implemented. The most commonly used method among them is the sterility test method listed in the Japanese Pharmacopoeia. In this method, a part of the sample is directly added to the medium and cultured, and the direct method of checking the presence of microorganisms in the sample and the sample is filtered using a membrane filter, and then the filter is put into the medium and cultured, There is a membrane filter method that confirms the presence or absence of microorganisms inside.

A biologic negative test method is used to test for the presence or absence of mycoplasma, which is one of the microorganisms. In this method as well, the direct smear culture method or the sterility test method is used. There are enrichment culture method and membrane filter method.

On the other hand, it is well known that various filtrations are carried out using hollow fibers, and various techniques for obtaining a desired product by supporting microorganisms on the hollow fibers are also known. However, no attempt has been made to test the presence or absence of microorganisms in a sample using a hollow fiber as in the present invention.

[0005]

Each of the above-mentioned conventional inspection methods has a problem that needs improvement. In particular, since it is qualitative in practice, the above conventional method cannot be said to be a suitable method when it is desired to simply and quickly confirm the presence or absence of microscopic substances in a sample. That is, in the direct method of the sterility test method, since the sample amount is usually as small as 1 to 5 ml, there is a risk of misjudgment when the number of existing microorganisms is small, and usually 10 or more samples are not tested per sample. However, there was a problem in terms of simplicity. And this point was the same in the case of the culture method in the biological drug negative test method.

Further, in the case of the membrane filter method in the sterility test method and the biological drug negativity test method, since the diameter of the membrane filter used is 20 to 50 mm, the diameter usually used for culture and determination is about 16-2
Since it is difficult to put it in a 0 mm test tube as it is, it was necessary to cut it into small pieces with scissors or fold it and then put it in the test tube and culture it. Therefore, there is a problem that it takes time and labor and the risk of contamination by various bacteria increases. Even when the culture solution was placed in a Petri dish instead of the test tube and cultured, there was another problem that the culture solution was easily spilled, and it was difficult to observe changes in turbidity and color tone, and determination was difficult.

Also, in the method of adding the medium to the filtration device, the membrane filter has a small filtration area per unit volume of about 1/5 to 1/10 of that of the hollow fiber. Since it requires a volume of 5 to 10 times that of the hollow fiber, it requires a larger space for testing, and there is a problem in storage at the site where it is necessary to inspect many times.

Therefore, none of the conventional test methods for confirming the presence or absence of microorganisms in a sample is satisfactory in terms of a simple, rapid, and accurate test method aimed at by the present invention. In view of such a current situation, the present invention provides a test method for easily and swiftly and accurately confirming the presence or absence of microorganisms in a sample that is desired in the field such as the above-mentioned field of food and the like, in the laboratory. The purpose is to do.

[0009]

Means for Solving the Problems As a result of intensive research to solve the above problems, the present inventor has devised a compact filter using a hollow fiber as a filter, and after using this filter to filter and wash a sample. , placed in a culture medium of the hollow fiber filtration device each in a test tube, thereby achieving the present invention that examined by looking at the change in turbidity or color tone of the culture of that.

That is, according to the present invention, after the sample is filtered with a hollow fiber filter, washed, and then the microorganisms trapped in the hollow fibers of the hollow fiber filter are brought into contact with a liquid medium to culture the microorganisms. is intended to provide an inspection how microorganisms and judging the presence or absence of microorganisms in a sample by looking at the change in the liquid medium.

The present invention will be described in detail below. Used in the present invention
A hollow fiber filter that can be used is a hollow fiber that is folded into a loop or sealed at one end to a hollow fiber length of 4 to 10 cm.
00, preferably 5 to 50 are bundled to have an outer diameter of 0.5 to 5
The filter is inserted into a cylindrical housing having a length of 5 cm and a length of 5 to 15 cm, and the open end is fixed in the housing with an adhesive or the like. The example shown in FIG.

FIG. 1A shows a hollow fiber filter which can be used in the present invention of the type in which the hollow fibers are packed in the housing at a high density. This type of filter is used by immersing the filter together with the culture solution in a test tube as shown in FIG. 1B. Since a test tube having a diameter of 16 to 30 mm is generally used, the outer diameter of the housing of the filter is preferably 25 mm or less.

In the above drawings , 1 is a hollow fiber, 2 is a housing, 3 is a hollow fiber fixing part, 4 is a sample inlet , and 5 is a sample filtrate outlet.
It is a mouth . The larger the number of hollow fibers 1 in the housing 2 is, the larger the filtration area becomes. However, if the number is too large, the circulation of the culture solution deteriorates, the growth of microorganisms is delayed, and the determination takes time. It is desirable to keep. Usually, it is about 1 to 100, but preferably 5 to 50.

Although not shown in the figure, the hollow fiber is cut into a length of 4 to 10 cm and the upper part of the hollow fiber is melted and sealed by heat without bending the hollow fiber into a loop, and the lower part is bonded. The one fixed in the housing with an agent or the like can be similarly used.

The flow of the sample in the filter is such that the sample liquid injected from the inlet 4 of the filter is outside the hollow fiber, the microorganisms in the sample are trapped, and the filtrate passing through the hollow fiber wall is hollow. The flow enters the inside of the yarn and is then discharged from the filter outlet 5.

As the hollow fibers used in the filter used in the present invention , polysulfone-based, polyvinylidene fluoride-based, polyacrylonitrile-based, polymethacrylate-based, polyamide-based, and cellulose acetate-based hollow fibers are used. The same material can be used.

The outer diameter of the hollow fiber is 50 to 200 μm, preferably 300 to 1000 μm, and the film thickness is 10 to 50.
0 μm, preferably 50 to 300 μm, the pore size is 0.01 to 3.0 μm, preferably 0.01 to 0.2 μm when targeting mycoplasma, and 0.4 to 2. Those having a thickness of 0 μm are used.

As the material of the housing, polycarbonate, polysulfone, polypropylene, acrylic resin, polyethylene, ABS resin, modified PPE resin or the like is used. The sample targeted by the present invention is a sample in which whether or not microorganisms are present is a problem, and is a solid which can be made into a liquid or a solution or a suspension. If the sample is a liquid among these,
Alternatively, dilute with sterilized physiological saline, pure water, buffer solution, etc. before use. In the case of a solid sample, it can be tested by dissolving or suspending it in the above liquid and removing the precipitate by centrifugation or the like, if necessary.

Specific examples of the sample of the present invention which requires determination of the presence or absence of microorganisms include (1) alcoholic beverages such as beer, sake, shochu, and wine, carbonated beverages such as cola and cider, and canned coffee. , Oolong tea, Japanese tea, black tea, sports drinks, mineral water, beverages such as various drinks, (2) injections, pharmaceuticals such as antibiotic preparations, (3) pure water used in the electronics industry, etc.
Examples include washing water, (4) serum, a culture solution for cell culture containing serum, (5) urine and tan that are targets of medical examination. Of these, the target microorganisms of the samples of (1) to (3) are mainly bacteria and / or yeasts except mycoplasma,
(4) and (5) are mainly mycoplasma.

The samples in the above-mentioned fields, especially the sera of (1) to (3) and (4), generally have a small number of microorganisms (eg, 10 ² / ml or less) in many cases, and thus the rapidity in such fields is high. Although it has been difficult in the past to test for the presence of microorganisms, the method of the present invention has made it possible to perform testing in an extremely simple, rapid, and accurate manner. The amount of the sample required for the inspection may be any amount that can be filtered, but generally 10 to 500 ml is sufficient.

Next, the inspection method of the present invention will be described according to the operating procedure. (1) Preparation of sample When the sample can be used as it is as a liquid, it may be used as it is, but in the case where the concentration is too high or when the sample amount is small such as tan, physiological saline, pure water, as described above, Dilute with a buffer etc. to make a sample. In the case of a solid, it is dissolved or suspended in the above liquid as described above, and if necessary, the precipitate is removed by centrifugation to obtain a sample.

(2) The prepared sample is filtered using a hollow fiber filter. The prepared sample is injected from the normal 10~500ml about, the inlet 4 of the middle hollow fiber filter Ri by such press-fitting apparatus <br/> through the sterile syringe or a sterile tube, complement microorganisms on the outside of the hollow fiber After being captured, the filtrate flowing into the hollow fiber is discharged from the outlet 5.

(3) The hollow fiber filter that has filtered the sample is washed with a washing liquid. As the washing liquid, a liquid such as sterilized buffer solution, physiological saline, and pure water that does not dissipate the physiological activity of microorganisms is used, and the injection and washing are performed in the same manner as in the above (2).

(4) The washed hollow fiber filter is brought into contact with the culture solution. As a mode of conflict, as shown in FIG. 1B, a culture solution is placed in a test tube and the hollow fiber filter is immersed in the culture solution.
Then do. In these methods, it is natural to sterilize all the filters, stoppers and other instruments.

As the culture medium, a liquid medium suitable for the microorganism expected to be present in the sample is used. Usually, in the case of bacteria and fungi in pharmaceuticals, thioglycolic acid mediums I and II for sterility test and glucose / peptone medium described in the Pharmacopoeia are used, and in the case of food and water, for example, ordinary broth medium, SCD A medium, a TGE medium or the like may be used, and in the case of Mycoplasma, a liquid medium for Mycoplasma negative test or the medium shown in Examples may be used.

(5) Culture Generally, the culture is carried out at a temperature of 30 to 40 ° C. for 1 day to 2 weeks according to a conventional method. In the case of products for hot benders, the culture may be performed at a temperature of around 55 ° C. Although stationary culture is generally used, shaking culture may be suitable in some cases.

(6) Judgment The change in the liquid medium is observed with the naked eye, and the presence or absence of the growth of microorganisms is confirmed to judge. Specifically, in the case of microorganisms other than mycoplasma, it is generally judged by the turbidity of the culture solution, but depending on the strain, it may be judged by the pellicle on the surface of the medium. Bromthmol Blu for acid-producing microorganisms
e, Crystal Violet, Phenol R
It is also possible to add a pH indicator such as ed to the medium and judge by its color change.

In the case of mycoplasma, it cannot be judged by the turbidity of the culture broth. Therefore, generally, a pH indicator such as Phenol Red is added to judge by the discoloration. In the case of a glucose-utilizing bacterium, the color changes from red-orange to yellow, and in the case of arginine- or urea-utilizing bacterium, the color changes from red-orange to red.

[0029]

EXAMPLES The present invention will be described below with reference to examples.

Example 1 Using the medium shown in Table 1, the cells were cultured at 37 ° C. and then -8.
Mycoplasma arginini [Mycoplasma arginini] [bacteria number about 10 ⁹ cells / ml] thawed was thawed and diluted 10 ⁸ times with a phosphate buffer of pH 7.2 to prepare a sample solution ( About 10 bacteria / m
l). This sample liquid was filtered using the following hollow fiber filter shown in FIG.

(Hollow fiber filter) Housing material: Polycarbonate dimensions (in FIG. 1) a ₁ = 70 mm, b ₁ = 10 mm, c ₁ = 50 mm, d ₁ = 10 m
m, e ₁ = 7 mm, f ₁ = 10 mm, g ₁ = 4 mm ・Hollow fiber Material: Polysulfone Pore diameter: 0.04 μm Outer diameter: 380 μm Film thickness: 100 μm Number: 20

Filtration was carried out by injecting 10 ml of a mixed solution consisting of 1 ml of the sample solution and 9 ml of phosphate buffer into the filter through the inlet 4 of the hollow fiber filter shown in FIG. Then, it was washed with 10 ml of pH 7.2 phosphate buffer, and the filter was immersed in a test tube having a diameter of 18 mm containing 13 ml of the culture solution shown in Table 1 and cultured at 37 ° C. for 48 hours in an incubator. As a control, 1 ml of the above-mentioned 10 ⁸ -fold diluted bacterial solution was placed in a test tube having a diameter of 18 mm containing 12 ml of the culture solution shown in Table 1 and cultured under the same conditions and methods as in the above-mentioned examples. After culturing, when the above-mentioned examples and the target culture solution were observed, both were discolored from red-orange to red, and it was confirmed that Mycoplasma arginiii was present in the sample.

[0033]

[Table 1]

Example 2 A 10% arginine hydrochloride solution in liquid medium of Table 1 (20
ml), and a liquid medium having the same composition (blending amount) as in Table 1 except that a 10% glucose solution (20 ml) was used,
Acoreplasma raidla cultured for 20 to 24 hours at 37 ° C.
wii) [the number of bacteria is about 10 ⁸ / ml] is diluted 10 ⁶ times with a phosphate buffer of pH 7.2, and further diluted 10 times with horse serum to obtain a serum sample of about 10 cells / ml. A sample was prepared. 1 ml of this sample was mixed with 9 ml of phosphate buffer, and the same hollow fiber filter as that used in Example 1 was used, except that the number of hollow fibers was reduced to 5, and filtration and washing were carried out in the same manner as in Example 1. did.

Next, the above glucose-containing liquid medium 13
The filter was immersed in a test tube having a diameter of 18 mm containing ml, and cultured at 37 ° C. for 3 days in an incubator. As a control, 1 ml of the above sample was directly placed in a test tube having a diameter of 18 mm containing 12 ml of the above liquid medium without passing through a hollow fiber filter, and cultured at 37 ° C. for 3 days in an incubator. After culturing,
When the culture solution (medium) was observed, both of them changed from red-orange to yellow, indicating that the microorganisms in the sample, Achole plasmalaid.
It was confirmed that L. awii) existed.

Example 3 Bacillus subtilis cultured in SCD medium (liquid) for 16 hours was diluted 10 ⁵ times and 10 ⁶ times with sterilized physiological saline, and 10 ml of each diluted solution was used in Example 1. Filtered by the hollow fiber filter used in 1.
After washing with 0 ml physiological saline, 15 ml SCD medium (liquid)
It was immersed in a test tube having a diameter of 18 mm. In each case, within 12 hours, the medium became turbid, and Bacillus subtilis sediment was observed at the bottom of the test tube, and turbidity was more clearly observed when shaken.

Example 4 10 ml of a sample solution obtained by diluting a commercially available mineral water (imported product) by 10 ² times was filtered using the same hollow fiber filter as used in Example 1, and then 10 ml of physiological saline solution was used. Washed with. Next, add this to TGE medium (liquid) (standard medium
A medium (liquid) diluted 0 times) was used to soak 13 ml in a test tube having a diameter of 18 mm and cultured at 30 ° C for 4 days. As a result, a precipitate of bacterial cells was observed at the bottom of the test tube and the medium was shaken. It was observed that the (liquid) became cloudy.

Example 5 Ureaplasma urealyticum cultured at 37 ° C. for 20 to 24 hours using the medium shown in Table 2.
aurealiticum) was diluted 10 ⁴ times with physiological saline to prepare a sample solution. 1 ml of the sample solution and 9 ml of physiological saline were filtered through the same hollow fiber filter as used in Example 1, washed with 10 ml of physiological saline, and immersed in a test tube having a diameter of 18 mm containing 13 ml of the medium shown in Table 2. did. On the other hand, 1 ml of 10 ⁴ -fold diluted solution was added to a test tube containing 12 ml of the medium shown in Table 2 as a target. When both were cultured at 37 ° C. for 2 days, the color of the medium (liquid) changed from yellow to red,
Ureaplasma ureaticum (Urea plasma)
It was confirmed that the plasma urealyticum) was present.

[0039]

[Table 2]

Example 6 Mycoplasma hominis cultured at 37 ° C. for 40 to 48 hours using the medium shown in Table 1
minis) was diluted 10 ⁴ times with human urine to prepare a sample solution. After mixing 1 ml of the sample solution and 9 ml of pH 7.2 phosphate buffer and filtering and washing in the same manner as in Example 1 with the same hollow fiber filter as used in Example 1, 13 ml of the medium shown in Table 1 was added. When immersed in a test tube with a diameter of 18 mm and cultured at 37 ° C for 2 days, it was observed that the color of the culture solution changed from red orange to red, and Mycoplasma hominis was present in the sample. Was confirmed.

[0041]

EFFECT OF THE INVENTION Since the test method of the present invention can confirm the presence or absence of microorganisms in a sample quickly, accurately and simply,
It exerts great effects in process control, quality control, research and development, etc. in the beverage / food field, pharmaceutical / medical field, biotechnology / electronics field, etc.

[Brief description of drawings]

FIG. 1A is a cross-sectional view of one embodiment of a hollow fiber filter that can be used in the present invention, and B is a cross-sectional view showing a usage mode thereof .

[Explanation of symbols]

1 hollow fiber 2 housing 3 Hollow fiber fixing part Entrance with 4 stoppers 5 Filter outlet with stopper 6 test tubes 7 Liquid medium

─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hisashi Namura 8-8-17 Nishikasai, Edogawa-ku, Tokyo (56) References JP-A-5-30997 (JP, A) JP-A-3-123497 (JP , A) JP-A 2-163098 (JP, A) JP-A 60-68006 (JP, A) Actual development Sho-62-160605 (JP, U) (58) Fields investigated (Int.Cl. ⁷ , DB) Name) C12Q 1/00-3/00 B01D 53/022 B01D 61/00-71/82 C02F 1/44 C12M 1/00-3/10

Claims (3)

(57) [Claims] 1. A sample is filtered through a hollow fiber filter and then washed, and then the microorganisms and the liquid medium trapped in the hollow fiber of the hollow fiber filter are filtered and washed in the liquid medium in a test tube. in
After culturing the microorganisms by bringing them into contact by immersing an empty fiber filter, the change of the liquid medium is changed to the turbidity of the liquid medium and the
And / or a method for inspecting a microorganism, which comprises determining the presence or absence of a microorganism in a sample by observing the change in color tone . 2. The test method according to claim 1, wherein the sample is a sample collected from beverages, pharmaceuticals, water used in the electronics industry, culture media, or urine / tans. 3. The inspection method according to claim 1, wherein the washing is performed with a washing solution selected from a sterilized buffer solution, physiological saline, and water.