JP2000210099A - Mensuration of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rapidly and highly sensitively mensurate the number of microorganisms in a specimen. SOLUTION: This method for mensurating microorganisms comprises mensurating the microorganisms in a liquid or gaseous specimen containing the microorganisms sequentially according to the following processes (a) to (f). (a) The specimens is sucked and filtered on a membrane filter to immobilize the microorganisms on the filler. (b) The membrane filter is placed on a proper nutritive culture medium to proliferate the microorganisms. (c) The proliferated microorganisms are dried and immobilized together with the membrane filter. (d) The membrane filter is colored with a pigment dissolved in an alcoholic aqueous solution. (e) The dyed membrane filter is decolored with an alcoholic aqueous solution and then dried. (f) The membrane filter is observed by the use of a microscope to mensutate the number of the colonies of the microorganisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring microorganisms based on a method for staining microorganisms, and can be used for rapid and highly sensitive measurement of the number of microorganisms in a sample.

[0002]

2. Description of the Related Art There are a wide range of applications, such as the measurement of the number of E. coli in drinking water and discharged sewage, the testing of bacteria in foods in the food field, the measurement of E. coli and lactic acid bacteria in dairy products, and the testing of gas-phase bacteria in medical aseptic rooms and clean rooms. Bacterial counts are being measured in the field. The measurement of the number of bacteria in such a case has been conventionally performed by an agar plate culture method (24 to 48 hours). A membrane filter method has been widely used as an improved method of this method since about 20 years ago. Since the sample is filtered by suction through a membrane filter, it is possible to concentrate relatively low-concentration microorganisms such as clean water and indoor air.
By culturing the membrane filter on various selective media, the type of microorganism can be roughly specified.
However, it takes 24-48 hours to obtain the results, as in the conventional agar plating method. For this reason, there has been a problem that, for example, control of the number of E. coli in sewage discharge water and food inspection cannot be performed promptly and appropriately.

[0003] As a method for rapidly detecting bacteria, there is a method in which a sample is previously stained with a dye such as fuchsin and then placed on a slide glass and observed with a microscope, or collected with a membrane filter and observed with a microscope. . Since this method does not require culturing, the number of bacteria can be measured quickly (about 1 hour), but has the following disadvantages. (I) A high magnification phase contrast microscope of 1000 times or more is required, (ii) a large number of bacteria needs to be present, (iii) it is difficult to distinguish from trash, and (iv) the type of microorganism is specified. Difficult to do, (v)
Discolors over time and does not preserve the sample.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, and not only allows a novel microorganism staining method to measure microorganisms in a sample quickly, with high sensitivity and simply, but also provides a method for staining microorganisms after staining microorganisms. An object of the present invention is to provide a method for measuring microorganisms in which the degree of coloring hardly changes and which can be stored for a long time.

[0005]

According to a first aspect of the present invention, there is provided a method for measuring microorganisms, comprising sequentially performing the following steps (a) to (f) on a liquid or gas phase sample containing microorganisms. It measures the number of microorganisms in a sample.

(A) A sample is suction-filtered on a membrane filter to immobilize microorganisms on the filter.

(B) Place the membrane filter on a nutrient medium and allow the microorganisms to grow.

(C) The grown microorganisms are dried and solidified together with the membrane filter.

(D) The membrane filter is dyed with a dye dissolved in an aqueous alcoholic solution.

(E) Decolorize and dry the membrane filter with an alcoholic aqueous solution.

(F) Observe the membrane filter with a microscope and count the number of colonies of microorganisms.

Further, according to the method for measuring microorganisms according to claim 2 of the present invention, the pore size of 0.1 to 50 μm is used for the membrane filter in the method for measuring microorganisms according to claim 1.

Further, a method for measuring microorganisms according to claim 3 of the present invention is the method for measuring microorganisms according to claim 1, wherein amide black is used as a dye.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The method for measuring microorganisms according to the present invention is applicable to the fields of environment such as water and sewage and rivers, the field of food such as beverages, the field of cosmetics, the field of medicine such as feces and blood, the indoor and refrigerators, and the clean room. It can be used in a wide range of fields, such as bacterial testing and factory microbiological control testing.

As a result of intensive studies to solve the above problems, the inventor has dried the microbial colonies grown on the membrane filter together with the membrane filter, and then stained and decolored with an alcoholic dye solution. It has been found that the above problem is solved. In particular,
Ethanol or methanol having a concentration of 10 to 80% is used as an aqueous alcoholic solution, and 0.01 to 2% is used as a pigment.
When the concentration of amide black was used, it was found that the microorganisms were retained on the membrane filter, and the number of bacteria (the number of colonies) on the membrane filter could be measured after culturing for 4 to 6 hours. Furthermore, it is possible to stain even microorganisms dried and fixed on a membrane filter several months ago.
It was also found that long-term storage was possible after staining. The present invention has been completed based on such findings. Hereinafter, the method for measuring microorganisms of the present invention will be described.

The membrane filter used in the present invention is the most common and inexpensive cellulose ester type membrane filter, and a sufficient effect can be obtained. Although it may be hydrophilic or hydrophobic, in the case of a polycarbonate membrane filter, microbial colonies on the filter may fall off. In this regard, a cellulose ester-based membrane filter is desirable. The pore size of the filter is selected in the range of 0.1 to 50 μm depending on the target microorganism. In the case of E. coli, 0.45 μm was used. The color of the membrane filter was white.

A liquid or gas phase sample containing microorganisms is collected on a membrane filter by appropriately diluting or changing the suction speed. The membrane filter on which the microorganisms are collected is placed on an agar medium without drying and cultured for a certain period of time. Instead of an agar medium, it may be placed on a filter paper soaked with a culture solution. After culturing for a certain period of time, the membrane filter is dried, and the cultured microbial colonies are fixed on the membrane filter. This drying requires 10 to 30 minutes, but the drying time can be further reduced by using a thermostat at 30 to 40 ° C. After drying, the membrane filter may become round, but it can be restored by soaking it in the staining solution.

In the conventional method, dyeing is performed without drying, but in this method, colonies of microorganisms are easily peeled off from the membrane filter, and minute colonies may be dissolved in the staining solution. In the present invention, microbial colonies grown on a membrane filter before staining are first dried. However, some microorganisms, such as yeast, are easily peeled off from the filter when dried. On the other hand, Escherichia coli, Bacillus subtilis, mold and the like do not peel off and are suitable for the drying method. Optimal colony size is about 1m
m or less.

As the medium for the microorganism, a medium in which the target microorganism grows well or a selective medium in which only a specific microorganism grows is used. If a selective medium of Escherichia coli or yeast is used, only the target microorganism in the sample can be measured. In the present invention, as a medium for Escherichia coli, yeast and Bacillus subtilis, SCDB (Soybean-Ca) of the Japanese Pharmacopoeia sterility test method which is a medium for general bacteria is used.
sein Digest Broth) was used. As a selective medium for Escherichia coli, a deoxycholate agar medium of Ministry of Health and Welfare No. 297 was used. In this agar medium, colonies of Escherichia coli are stained in light red to red, but the measurement is not easy due to weak coloring, and by staining more strongly by the method of the present invention,
It can be easily measured. The culture time is SCDB for E. coli.
4 to 6 hours or more. Thereafter, the membrane filter is dried, and the microorganisms are immobilized on the membrane filter.

Dyes for staining microorganisms and proteins include:
Candidates include methylene blue, fuchsin, malachite green, Victoria blue, Coomassie brilliant blue, eosin, amide black and the like. Among them, methylene blue, malachite green, Victoria blue, and fuchsin are often used for detecting microorganisms and can be used in the present invention. Although eosin has poor staining properties, it was possible to detect microorganisms with fluorescence and thus to perform highly sensitive detection. However, this requires a fluorescence microscope, and when the colonies are very small, it is difficult to distinguish them from dust. On the other hand, amide black is hardly used for microbial staining in the conventional method, but was most suitable for detecting microbial colonies immobilized on the membrane filter in the present invention. The membrane filter was stained with amide black in the same manner as other dyes. However, the background was not increased because the color was easily decolorized, and the dried bacteria (mainly Escherichia coli) were quickly stained and the contrast was high.

As a solution for dissolving the dye, water 100
Diluted alcohol from% to 100% alcohol can be used. However, when the amount of water is large, it takes time for drying, and when the alcohol concentration is increased, the colonies fixed by drying are easily peeled off, and it is difficult to control the decolorization. Therefore, a 50% alcohol aqueous solution was used as an example. The pH of the solution may be neutral as long as it is pH 4 to
No particular problem was observed in the dyeability between No. 9 and No. 9.

Ethanol and methanol were used as alcohols, and both gave similar results. Other solvents and mixed solvents can be used as long as they dissolve the dye well and are soluble in water and do not dissolve the membrane filter or affect the shrinkage or the like.

The concentration of amide black can be used to stain the microorganisms on the membrane filter from 0.01% to several%, but the staining time is from 1 to 30 seconds and from 1 to 30 minutes under the conditions of decolorization within 1 minute. % Was optimal. The dyeing can be repeated as many times as it is light. Microorganisms (preserved microorganisms) fixed on a membrane filter for a long time can also be stained in the same manner. From this, it is possible to observe and measure the number of microorganisms by sampling the microorganisms from the sample, performing only culturing and drying and fixing on the membrane filter, and staining when the need for measurement occurs.

The time required for staining the microorganism immobilized on the membrane filter is several seconds to several tens of seconds. In the case of 1% amide black, the microbial colonies are selectively stained (especially in the case of Escherichia coli) by immersing in a staining solution for about 5 seconds.
On the other hand, since the microorganism colonies are not visible to the naked eye in the culture within 6 hours, the staining is carried out every 10 seconds. For decolorization, an alcohol solution having the same concentration as that of the staining solution is used. Attention must be paid to the fact that if the concentration is different, the decolorizing solution does not permeate the membrane filter and may not be decolored well. If the decolorizing solution for rinsing is separated, 50 to 50 ml of the decolorizing solution is used.
About 0 membrane filters can be decolorized. The bleaching time is completed within one minute. In the case of amide black, the background looks quite high in appearance, but there is no problem since the color becomes lighter when dried. Microscopic observation is possible in a drying time of about 30 minutes at a room temperature of 25 ° C. and 10 to 15 minutes in a thermostat at 37 ° C.

Observation and measurement of the number of bacteria can be performed with the naked eye if the microbial colony is 0.5 mm or more. The 47 mm diameter membrane filter can easily measure microbial colonies after staining even in colonies of 0.5 mm or less by enlarged copy. On the other hand, the microorganism colonies are not visible to the naked eye (50 μm or less) in culture within 6 hours, so a stereoscopic microscope or a biological microscope is used. Since it is difficult to observe with transmitted light, observe with incident light (reflected light) from above. Since a magnification of about 10 to 50 times is sufficient, a stereomicroscope is the easiest to use.

As a result of comparing the number of microorganisms measured by the agar plate method with that of the present staining method, the correlation coefficient is 0.986, and a proportional relationship is recognized. Therefore, no calibration curve is required.

[0027]

Next, the present invention will be described in detail with reference to examples.

As a bacterium, Escherichia coli, I
FO3301), Bacillus subtilis (IFO13719), yeast, and mildew. As a medium, SCDB (Soybean-Casein Digest Broth) liquid and 1.5% agar medium of the Japanese Pharmacopoeia sterility test method, and as a selective medium for Escherichia coli, a deoxycholate agar medium (Nissei) of the Ministry of Health, Labor and Welfare No. 297.
Was used. The actual operation procedure is shown below.

(1) The Escherichia coli culture is diluted with physiological saline so that the number of bacteria becomes 10 to 300 cells / ml.

(2) Put 4 in the holder of the membrane filter.
A 7 mm-diameter membrane filter is aseptically attached, and an E. coli diluent (about 10 ml) is poured onto the membrane filter.

(3) The Escherichia coli dilution is suction-filtered by an aspirator, and Escherichia coli is immobilized on the filter (in the case of a gas phase, contaminated air is sucked as it is).

(4) The membrane filter is aseptically placed on an agar medium and cultured at 37 ° C. for 4 to 24 hours.

(5) After a predetermined time, the membrane filter is dried in a thermostat at 37 ° C. for 15 minutes.

(6) The membrane filter is stained with a dye (1% amide black) dissolved in a 50% aqueous ethanol solution for several seconds to 10 seconds.

(7) Decolorize the membrane filter with a 50% aqueous ethanol solution for several tens of seconds.

(8) Dry the membrane filter in a thermostat at 37 ° C. for 15 minutes.

(9) Observe with a microscope and count the number of microbial colonies.

In this example, 1.0% amide black was dissolved in a 50% aqueous ethanol solution to obtain 0.45 μm
Was filtered as a membrane filter staining solution, and a 50% aqueous ethanol solution was used as the decolorizing solution. The membrane filter has a diameter of 47 mm and a hole diameter of 0.4
5 μm, sterilized cellulose acetate ester (Advantech, Toyo Roshi A080H047W) was used. For microscopic observation, Nikon biological microscope (OPTIP
HOT).

Escherichia coli was inoculated into the SCDB culture solution, and
With shaking for 24 hours (15 hours). OD (optical de
nsity) A culture solution having a wavelength of 600 nm around 1.0 was used as a standard test E. coli suspension. This suspension was diluted with sterile physiological saline to prepare a sample solution of 10 to 300 E. coli / ml. About 10 ml of the diluted E. coli solution was filtered through a membrane filter, quickly placed on an SCDB agar medium or a deoxycholate agar medium, and incubated at 37 ° C. for 4 to 24 hours. Similarly, this E. coli diluted solution was inoculated on each agar medium, and the number of bacteria after 15 to 25 hours was measured and compared with the membrane filter method.

For the detection of suspended microorganisms in the gas phase, a suction pump equipped with the above-mentioned membrane filter is used to detect 10 to 10 microorganisms.
Aspirate 100 liters of contaminated air (3-5 liters /
Min) Then put the membrane filter on an agar medium for 4-24
The cells were cultured at 37 ° C. for a period of time.

The microbial colonies grown on the membrane filter were dried in a thermostat at 37 ° C. for 15 minutes, immersed in 1% amide black staining solution for 5 to 10 seconds, stained, and immediately backgrounded with a decolorizing solution (50% ethanol). Was bleached until it became thin. The bleaching time may be within 1-2 minutes. Then, it was dried in a thermostat at 37 ° C. for 15 minutes and observed with a microscope.

FIGS. 1 to 3 show photographs of membrane filters after culturing on a deoxycholate agar medium (Nissei) for 6, 9, and 15 hours, respectively (no staining). The black line in the figure is a scale line printed on the membrane filter. The arrow A in FIG. 2 shows the colonies of Escherichia coli after culturing for 9 hours, and the arrow B in FIG. 3 shows the colonies after culturing for 15 hours.
In the 6-hour culture in FIG. 1, no detection was possible.

FIGS. 4 to 6 show the SCDB culture medium,
A photograph of a membrane filter stained with amide black after culturing for 9, 15 hours is shown. Arrow C in FIG. 4 is after culture for 6 hours, arrow D in FIG. 5 is culture after 9 hours, and arrow E in FIG.
1 shows colonies of Escherichia coli after culturing for an hour.

Even when using a representative E. coli selective medium and a deoxycholate agar medium in which E. coli colonies develop a red color, it was difficult to observe the colonies under a microscope after culturing for 6 to 15 hours (FIG. 1). ~ 3). However, FIG.
Even when the cells were grown on a general SCDB agar medium (Escherichia coli does not develop color) as described in No. 6, the colonies could be easily measured by staining with amide black.
In particular, E. coli was detected only by this staining method at 6 hours.

According to the measurement method of the present invention, a diluted sample of E. coli was filtered through a membrane filter, cultured on an agar medium for 6 hours, and then stained with amide black.
FIG. 7 shows a comparison between the number of E. coli colonies counted and the value of E. coli colonies that appeared by culturing the E. coli diluted sample as it was on an SCDB agar medium for 24 hours.
Shown in As described above, the correlation coefficient was 0.986, which was almost the same as that of the standard agar plate method. This indicates that the use of the measurement method of the present invention enables the detection of E. coli, which was conventionally required for 24 hours, to be performed within 6 hours with the same accuracy as the agar plate method.

Not only Escherichia coli cultured on the membrane filter but also Bacillus subtilis, mold and the like could be similarly stained with amide black with high sensitivity. 8 to 10 respectively.
The results of amide black staining of Bacillus subtilis and white mold colonies on a membrane filter are shown. The white mold case of FIG. 10 is photographed at a higher magnification than the other cases of FIGS. 8 and 9, and the mycelium is visible because the sample is white mold, but the detection is easy as in the other cases.

FIG. 11 shows a photograph of the membrane filter stained by this staining method two months later. Thus, after dyeing, it can be stored for a long time. FIG. 12 shows the results of culturing Escherichia coli on a membrane filter, drying, storing for 2 months, and then staining by this method. Thus, E. coli colonies could be stained even with the membrane filter stored for a long time.

[0048]

According to the staining method using the membrane filter and amide black of the present invention, the presence or absence and the number of microorganisms, particularly Escherichia coli, in a sample can be measured quickly (within 6 hours), with high sensitivity and easily. Also, the degree of coloring does not change with time,
The sample stained by the method of the present invention has excellent long-term storage stability. Further, those obtained by culturing microorganisms on a membrane filter and then drying can be stained even after long-term storage.
By using such a staining method of the present invention, it can be used for a viable cell count test of raw materials and products, a process microorganism control test, and a microorganism test in the research and development field.

[Brief description of the drawings]

Fig. 1 Deoxycholate agar medium (Nissei)
It is a figure which shows the colony on a membrane filter after culture | cultivating Escherichia coli on a membrane filter for 6 hours.

FIG. 2 Deoxycholate agar medium (Nissei)
It is a figure which shows the colony on a membrane filter after culture | cultivating Escherichia coli on a membrane filter for 9 hours.

FIG. 3. Deoxycholate agar medium (Nissei)
It is a figure which shows the colony on a membrane filter after culture | cultivating Escherichia coli on a membrane filter for 15 hours.

FIG. 4 is a diagram showing colonies on a membrane filter stained with amide black after culturing Escherichia coli on a membrane filter in an SCDB medium for 6 hours.

FIG. 5 is a diagram showing colonies on a membrane filter stained with amide black after culturing E. coli on a membrane filter for 9 hours in an SCDB medium.

FIG. 6 is a diagram showing colonies on a membrane filter stained with amide black after culturing Escherichia coli on a membrane filter for 15 hours in an SCDB medium.

FIG. 7 is a diagram showing a comparison of the number of E. coli measured by the method of the present invention and the agar plate method and a correlation coefficient.

FIG. 8 shows the results of amide black staining of a colony of E. coli on a membrane filter by the method of the present invention.

FIG. 9 shows the results of amide black staining of the Bacillus subtilis colony on a membrane filter by the method of the present invention.

FIG. 10 is a diagram showing the results of amide black staining of a mildew colony on a membrane filter by the method of the present invention.

FIG. 11 is a diagram showing a state of a membrane filter stained by the staining method of the present invention two months later.

FIG. 12. After culturing E. coli on a membrane filter,
FIG. 2 shows the result of staining two months later by the method of the present invention.

Claims (3)

[Claims] 1. A method for measuring the number of microorganisms in a liquid or gas phase sample containing the microorganisms by sequentially performing the following steps (a) to (f) to count the number of microorganisms in the sample. (A) A sample is suction-filtered on a membrane filter to immobilize microorganisms on the filter. (B) Place the membrane filter on a suitable nutrient medium and allow the microorganisms to grow. (C) The grown microorganisms are dried and solidified together with the membrane filter. (D) Dye the membrane filter with a dye dissolved in an aqueous alcoholic solution. (E) Decolorize and dry the membrane filter with an alcoholic aqueous solution. (F) Observe the membrane filter with a microscope and count the number of colonies of microorganisms. 2. The membrane filter has a thickness of 0.1 to 50 μm.
The method for measuring microorganisms according to claim 1, wherein the pore diameter of the microorganism is used. 3. The method according to claim 1, wherein amide black is used as the dye.
The method for measuring microorganisms according to the above.