JP3070780B2 - Method for measuring bacterial count - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the food industry, the pharmaceutical industry,
A trace amount of viable bacteria present in water, raw materials or products used in the field of the brewing industry or the like is adenosine triphosphate (hereinafter referred to as AT).
P) (abbreviated as P).

[0002]

2. Description of the Related Art Conventionally, for the measurement of live bacteria in a sample, the sample is directly diluted or appropriately diluted, or after supplementing the cells in a simple filter form, cultured on a standard agar medium,
A so-called plate method for detecting colonies is generally used. Recent improved methods include “Quantitative method for viable bacteria” (JP-A-2-57197) and “Method for detecting viable bacteria” (JP-A-2-16398). The former is a method in which live bacteria in a sample are captured by a filtration membrane having a specific pore size, and luciferase is acted thereon to measure ATP by a luminescence reaction. On the other hand, the latter method is a method in which live bacteria in a sample are captured by a filtration membrane having a specific pore size, and then the filtration membrane is washed with a liquid containing no ATP and a luminescent substance in a dissociated state, and luciferase is allowed to act on this. .

[0003]

However, the conventional plate method requires 24 to 72 hours to detect bacteria. JP-A-2-57197 and JP-A-2-163098
In the item (1), the detection time of bacteria is advanced, but measurement is impossible when the amount of bacteria is small. Recently, the number of viable bacteria is about 10 ⁴ cells / cc or less for bacteria and about 10 ³ cells / c for yeast.
It is necessary to be able to measure samples having a concentration of c or less, and in the case of extremely small amounts of viable bacteria, a large amount of samples must be collected by filtration. It is difficult to apply due to restrictions on the device.

In view of this situation, the present inventors have conducted intensive studies, filtered the specimen, added a medium to the viable bacteria collected on the filtration membrane, and cultured the cells for a certain period of time. The present inventors have found that if the sample is destroyed by a surfactant or an ATP releasing agent such as an enzyme and then luciferase is acted on, even if the number of living bacteria is small in a sample, it can be measured, and the present invention has been completed. .

[0005]

That is, according to the present invention, live bacteria in a specimen are collected on a filtration membrane surface of a live bacteria catcher ,
After adding the culture medium to the upper surface and cultivating the viable cell count, extract the adenosine triphosphate of the cells, measure the light generated by the luciferin / luciferase reaction, and calculate the light before growth based on the measured value and the growth conditions. Was measured.
Here, the viable bacteria targeted in the present invention are mainly bacteria and yeasts, but are not particularly limited, and any viable bacteria may be used.

The means for carrying out the present invention will be described with reference to the accompanying drawings. In FIG.
It is composed of a tubular member 2 whose top and bottom are open, and a filtration membrane 3 fixed to a lower portion of the member 2. As the filtration membrane 3 , a medium is added to the upper surface of the filtration membrane 3, and the upper surface of the filtration membrane 3 is added.
Cultivation on the same culture medium to grow viable bacteria.
Extract nosin triphosphate, luciferin luciferer
The light generated by the reaction is measured,
Since the number of bacteria before growth is measured based on the conditions,
Alternatively, a material having hydrophilicity, light transmittance, and chemical resistance for washing is preferable. The size of the pores is generally preferably 0.2 to 1.2 μm, though it depends on the type of living bacteria.

Next, a specified amount of the sample is caused to flow into the cylindrical member 2 of the viable cell catcher 1 and viable cells are collected on the filtration membrane 3. At this time, the funnel-shaped auxiliary container 4 as shown in FIG.
If it is detachably installed on the viable cell catcher 2, viable cells can be efficiently collected from a large number of specimens, and the measurement accuracy is improved.

Then, the viable cells thus supplemented are cultured by a conventional method. First, an optimum medium is added to the viable cells, and then the upper and lower parts of the viable cell catcher 1 are moved up and down. Is covered with a cover to prevent invasion of various bacteria. In this state, the cells are cultured at an appropriate temperature for 2 to 8 hours. After the cultivation, an ATP releasing agent such as trichloroacetic acid is added to the culture to destroy the cell wall of the viable cells and release ATP. That is, after the extraction of ATP, luciferin, luciferase, and the like are added, and the generated luminescence is measured with a luminometer.

[0009]

【Example】

Example 1 Pseudomonas diminuta (IFO14213) was diluted with physiological saline to about 4 × 10 ³ cfu / ml, and 0.1 ml thereof was used as a sample. The sample was introduced into a viable cell trap formed with a Teflon filtration membrane (manufactured by Nippon Millipore Co., Ltd., pore size: 0.22 μm) having a diameter of 25 mm and a height of 12 mm, and was filtered. Add 0.9 ml and incubate at 30 ° C for 8 hours. After that, ATP extraction was performed with a 0.2% solution of trichloroacetic acid, and then luminescence was performed with luciferin and luciferase using an enzyme kit (Lucifer-LU, manufactured by Kikkoman). And the amount of light emission was measured. This measurement is converted into a viable cell count and is shown in Table 1 together with the results obtained by the conventional plate method.

[0010]

[Table 1]

Example 2 Pseudomonas diminuta (IF0114213) was prepared in the same manner as in Example 1, 0.1 ml of the mixture was added to 500 ml of physiological saline, and then
The bacteria were collected by filtration using the auxiliary container 4 shown in the figure. After removing the viable cell catcher 1, 1 ml of medium m-TGE Broth was added thereto, and the cells were cultured at 30 ° C. for 8 hours. After completion of the culture, the amount of luminescence was measured by the same operation as in Example 1, and the following results (Table 2) were obtained.

[0012]

[Table 2]

Example 3 Saccharomyces cerevisiae (IFO 0209) was diluted with physiological saline to about 3 × 10 ² cfu / ml, and 0.1 ml thereof and 0.9 ml of glucose peptone medium were added to the viable cell trap 1. 30 ℃
For 8 hours. Next, after extracting ATP with a 0.2% solution of trichloroacetic acid, the luminescence was measured in the same manner as in Example 1 below, and the following results (Table 3) were obtained.

[0014]

[Table 3]

As in Example 3, Saccharomyces cerevisi
ae (IF0209) was prepared, and 0.1 ml of the resultant was added to 500 ml of physiological saline, followed by filtration and collection using the auxiliary container 4 shown in FIG. The viable cell trap 1 was removed, and 1 ml of the culture solution glucose peptone was added thereto, followed by culturing at 30 ° C. for 8 hours. Next, after extracting ATP with a 2.0% solution of trichloroacetic acid, the luminescence was measured by the same operation as in Example 3, and the following results (Table 4) were obtained.

[0016]

[Table 4]

[0017]

According to the present invention, the viable cell catcher is provided on the filtration membrane surface.
Collect the viable bacteria in the sample, add the medium to the top of the filtration membrane, and culture.
After growing the viable cell count, adenosine triphosphate is extracted from the cells.
Luciferin-luciferase reaction
Measured light, and based on this measured value and growth conditions,
As the number of bacteria was measured, the operation became simple,
Loss of operation time was also eliminated, and the number of viable bacteria was at least so large that it could not be measured, the amount of luminescence was measured after the cells had proliferated once, and the number of viable bacteria before growth was calculated based on the measured value and the growth conditions. Therefore, the number of viable bacteria can at least significantly improve the detection accuracy.

[0018]

[Brief description of the drawings]

FIG. 1 is a front sectional view of a viable cell catcher.

FIG. 2 is a front cross-sectional view of a live bacteria catcher provided with a supplementary container.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... viable bacteria catcher, 2 ... cylindrical member, 3 ... filtration membrane, 4 ... auxiliary container.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C12M 1/26 C12M 1/26 1/34 1/34 A (72) Inventor Shigeru Honma 339 Noda, Noda-shi, Chiba Kikkoma (72) Inventor Motoo Nakajima 339 Noda, Noda, Chiba Pref. Kikkoman Corporation (56) References JP-A-2-163098 (JP, A) JP-A-2-57199 (JP, A) JP-A-53-6480 (JP, A) JP-A-53-31194 (JP, A) JP-A-60-180577 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-1/66 G01N 33/02-33/15 C12M 1/26-1/34

Claims (1)

(57) [Claims] 1. A method for collecting viable bacteria in a specimen on a filtration membrane surface of a viable cell trap, adding a culture medium to the upper surface of the filtration membrane and culturing the number of viable bacteria, and then increasing adenosine triphosphate of the cells. Extract and generate light according to the number of viable cells after growth by luciferin / luciferase reaction, and then calculate the number of viable cells before growth based on the measured light intensity or the number of viable cells after growth based on growth conditions A method for measuring the number of bacteria.