JP2000014398A - Pretreatment for bacterium measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pretreatment method in an inexpensive bacterium measurement having excellent operability without requiring a bacterium grinding apparatus of exclusive use and a reagent. SOLUTION: Indicator bacteria of contamination in a food sanitation examination are ground by an alkali solution and mixed with a buffer solution. The concentration of the alkali solution is preferably 0.1-1.0 N and the treatment time with the alkali solution is preferably 5-10 minutes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay,
The present invention also relates to a pretreatment method for measuring bacteria by a DNA probe method, an enzyme activity measuring method, or the like.

[0002]

2. Description of the Related Art In recent years, serious accidents due to food poisoning have occurred, and with the liberalization of trade, the number of imported foods from overseas has been increasing, and there has been an increasing demand for ensuring the safety of foods.

[0003] In the food hygiene inspection, inspection of contamination indicator bacteria plays a very important role. Gram-negative rods that are typically treated as contamination indicator bacteria in this type of test,
It is particularly important to measure Escherichia coli.

[0004] In order to prevent food poisoning beforehand, it is desired in bacterial tests to be able to quickly and easily measure Escherichia coli, which is a pollution indicator bacterium.

Hitherto, in order to detect bacteria, an immunoassay method, a DNA probe method, an enzyme activity measurement method, and the like have been used for the measurement of bacteria, and a number of such measurement methods have been reported.

When a physiologically active substance contained in a bacterium is measured, the bacterium is pulverized and the target physiologically active substance is extracted. There are a number of methods for disrupting bacterial cells to extract this bioactive substance of interest.

[0007] In the ultrasonic treatment method, a vibrator of a vibration generator is vibrated at 10 to 20 kHz, thereby crushing cells. In addition, a homogenizer method in which cells are crushed in the gap between the outer and inner cylinders made of glass, a blender method in which cells are crushed by rotating a blade attached to the tip of a shaft, and cells that are pressed by bacteria and ejected from a narrow gap A pressurized cell crushing method for crushing cells, a crushing method for crushing cells in the presence of glass powder and the like, and an enzyme treatment method using an enzyme that dissolves cell walls and cell membranes are known.

[0008]

However, many methods for crushing bacterial cells described above can extract physiologically active substances in bacterial cells, but require special equipment and reagents, and are expensive and costly. There was a problem that it was very disadvantageous in terms of sex.

Therefore, an object of the present invention is to provide a pretreatment method for measuring bacteria which is inexpensive and excellent in operability without requiring a dedicated bacterium crushing device or reagent.

[0010]

Therefore, the pretreatment method for measuring bacteria according to the present invention is characterized in that a contamination indicator bacterium in a food hygiene inspection is crushed with an alkaline solution, and a buffer is added thereto.

In the present invention, the bacterial cell membrane is denatured and disrupted by an alkaline solution. Bacterial cells are covered by cell membranes and protect the cytoplasm. It is considered that the cell membrane is denatured by alkali, and the membrane structure changes. That is, the present invention aims to extract a physiologically active substance in a bacterial cell by disrupting a cell membrane by a change in the membrane structure.

Next, in establishing the pretreatment method for measuring bacteria according to the present invention, the following experiment was conducted in order to examine the effect of the concentration of the alkaline solution on bacterial disruption when the contamination indicator bacteria were disrupted with an alkaline solution. Was.

(Effect of alkaline solution concentration on bacterial crushing)
A sodium hydroxide solution of 0 to 0.3N is added to a suspension of E. coli K12 strain containing 150 mM sodium chloride (the number of bacteria: 10 ⁸ cells / ml), the E. coli is disrupted by incubation for 5 minutes, and each treatment is performed. The solution is adjusted to pH 7.0 to 7.5 with sodium dihydrogen phosphate solution. Then, the bacterial count of these prepared samples was measured by an immunoassay using a microplate shown below.

(Measurement of Bacteria) 100 μl of the E. coli sample solution was added to a polystyrene 96-well microplate (Maxi Soap manufactured by Nunc) on which an anti-E. Coli antibody was immobilized.
Leave for 1 hour.

[0015] pH7 containing 150 mM sodium chloride.
After thoroughly washing the wells of the microplate with a tetraphosphate buffer (PBS), the well contains 0.1 mg / ml bovine serum albumin (BSA), which becomes 1.0 μg / ml of a biotinylated modified anti-Escherichia coli antibody. 100 μl of PBS solution
Add 1 and leave for 1 hour.

After thoroughly washing the wells of the microplate with a PBS solution, the concentration of avidin-conjugated horseradish peroxidase becomes 1.0 μg / ml, 0.1 mg / ml.
100 μl of a PBS solution containing BSA is added and left for 1 hour.

After thoroughly washing the wells of the microplate with a PBS solution, orthophenylenediamine 10
100 μl of a citrate borate buffer (pH 5.0, which is a 0.03% aqueous hydrogen peroxide solution) is added, and the mixture is reacted for 10 minutes.

Then, 50 μl of 1.0N hydrochloric acid was added to stop the reaction, and the absorbance at 492 nm was measured. FIG. 1 shows the measurement results.

As a result, Escherichia coli could be almost completely crushed with a 0.01 N or more sodium hydroxide solution.

Therefore, it can be said that the concentration of the alkaline solution for crushing the bacteria is not less than 0.01 N. However, in order to completely denature the bacteria and not to denature the physiologically active substance,
About 0.1-1.0N is preferable.

Further, in establishing the pretreatment method for measuring bacteria according to the present invention, the following experiment was conducted in order to examine the effect of the treatment time on the disruption of bacteria when the indicator bacteria were disrupted with an alkaline solution.

(Effect of Alkaline Solution Treatment Time on Bacterial Disruption) A 0.1N sodium hydroxide solution was added to a suspension of E. coli K12 strain containing 150 mM sodium chloride (the number of bacteria: 1).
In addition to the 0 ⁸ / ml), Escherichia coli crushed between 0-240 minutes. 0, 5, 10, 30, 60, 120, 180, 2
The pH of each treatment solution is adjusted to 7.0 to 7.5 with a sodium dihydrogen phosphate solution every 40 minutes. Then, the bacterial counts of these prepared samples were similarly measured by the immunoassay using the microplate. FIG. 2 shows the measurement results.

As a result, Escherichia coli could be almost completely crushed in 0.1 N sodium hydroxide solution within 10 minutes.

Therefore, it is desirable that the treatment time of the alkaline solution for crushing the bacteria is within 10 minutes, preferably about 5 minutes.

Further, in the method for pretreatment of bacterial measurement of the present invention, the alkaline solution is not particularly limited,
A sodium hydroxide solution or a potassium hydroxide solution having a pH of 10 or more, preferably pH 12 or more was used.

In the method for pretreatment of bacterial measurement of the present invention, the buffer is not particularly limited, but a phosphate buffer,
It is preferable to use at least one selected from acetate buffer, Tris buffer and Good buffer.

[0027]

〔Example〕

(Bacterial disruption) The bacterium to be measured was Escherichia coli K12 strain, and an anti-Escherichia coli rabbit antibody was used as an antibody for immunoassay.

Escherichia coli K12 strains having a cell count of 10 ⁶ to 10 ⁸ cells / ml are each suspended in a 150 mM sodium chloride solution, and a sodium hydroxide solution is added to the E. coli suspension to a final concentration of 0.1N. . After standing for 5 minutes, a sodium dihydrogen phosphate solution is added to a final concentration of 100 mM to adjust the pH to 7.0 to 7.5. Then, the bacterial counts of these prepared samples were similarly measured by the immunoassay using the microplate. FIG. 3 shows the measurement results. [Comparative Example] As a comparative example, an example in which bacteria were disrupted by ultrasonic waves is shown.

Escherichia coli K12 strains having a cell count of 10 ⁶ to 10 ⁸ cells / ml were each suspended in a 150 mM sodium chloride solution, and the E. coli suspension was subjected to a sonicator for disrupting bacteria for 5 minutes to disrupt Escherichia coli. A sodium dihydrogen phosphate solution was added to the ultrasonically disrupted Escherichia coli so as to have a final concentration of 100 mM and a pH of 7.0 to 7.5, and the number of bacteria was similarly measured by the immunoassay using the microplate. . FIG. 3 shows the measurement results.

As a result, almost the same measurement results were obtained when the bacteria were crushed by the alkaline solution in the pretreatment method of the present invention and when the bacteria were crushed by ultrasonic waves. When measuring bacteria, ultrasonic waves are generally used to disrupt the bacteria, and the disruption by the ultrasonic waves is almost completely performed. Therefore, even in the case of using the alkaline solution in the pretreatment method of the present invention, the bacteria could be almost completely disrupted.

[0031]

Since the method for pretreatment of bacterial measurement of the present invention is configured as described above, it does not require a dedicated bacterium crushing device or reagent, and is inexpensive and easily crushes bacterial cells. It can be done.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of an investigation on the effect of alkaline solution concentration on bacterial disruption in the present invention.

FIG. 2 is a diagram showing the results of examining the effect of the treatment time of an alkaline solution on bacterial disruption in the present invention.

FIG. 3 is a diagram showing a comparison between the result of crushing bacteria by an alkaline solution and the result of crushing bacteria by conventional ultrasonic waves in the present invention.

Continued on the front page F term (reference) 4B024 AA11 GA19 HA15 4B029 AA07 AA15 BB02 CC01 FA03 4B063 QA01 QA18 QQ06 QR02 QR25 QR48 QR84 QS03 QS33 QX01 4B065 AA26X CA24 CA46

Claims (3)

[Claims] 1. A pretreatment method for measuring bacteria, characterized in that indicator bacteria for contamination in a food hygiene test are crushed with an alkaline solution, and a buffer is added thereto. 2. The method according to claim 1, wherein the concentration of the alkaline solution is 0.1 to
2. The pretreatment method for measuring bacteria according to claim 1, wherein the method is 1.0 N. 3. The treatment time with the alkaline solution is 5 minutes.
2. The pretreatment method for measuring bacteria according to claim 1, wherein the treatment time is 10 minutes to 10 minutes.