JPH05304995A - Method of counting cell bodies of microorganism - Google Patents

Abstract

PURPOSE:To enable high-sensitive and high-accurate count of cell bodies by adding a saccharide and/or polyalcohol to the solution prepared for counting cell bodies of microorganisms according to the luciferin-luciferase reaction. CONSTITUTION:The sample immediately before ATP extraction is treated with the prepared solution containing a saccharide and/or polyalcohol. The ATP in the cell bodies of the microorganism is extracted by the ATP extraction treatment and the cell bodies are counted according to the luciferin-luciferase reaction.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring the number of microbial cells by the luciferin / luciferase reaction.

[0002]

2. Description of the Related Art Conventionally, it has been extremely important to measure the number of microbial cells in many fields such as food, pharmaceuticals, papermaking, water treatment, and clinical fields. The plate method is generally used as the measuring method, but recently, the frequency of use of the measuring method by the luciferin-luciferase reaction has been increasing.

The method for measuring the number of bacterial cells by the luciferin-luciferase reaction is a conventional method in which a sample of a microorganism for which the number of bacterial cells is to be measured is prepared and the sample is subjected to ATP.
AT in sample treated with (adenosine triphosphate) extractant
P was extracted, and then luciferin and luciferase were allowed to act on the extracted ATP to quantify the amount of light generated by the so-called luciferin-luciferase reaction, and this quantitative value was correlated with the previously determined amount of luminescence and the number of bacterial cells. The number of bacterial cells is measured from the calibration curve shown.

By the way, the sample to be subjected to the above-mentioned ATP extraction treatment usually has a microbial cell number within the measurement range (for example, 1 in yeast).
( ⁴ to 10 ⁶ / ml for lactic acid bacteria, 10 ⁶ to 10 ⁸ / ml for lactic acid bacteria, etc.), prepared by a procedure such as washing after cell collection by membrane filtration, centrifugation, and suspension of the cells. The prepared solutions such as the diluting solution, the washing solution, and the suspension solution at this time often use saline solutions having various concentrations.

However, when saline is used during the preparation of the sample, the salt inhibits the luciferin-luciferase reaction, thus lowering the measurement sensitivity. In addition, for example, soy sauce, miso, salted foods, salt-containing foods such as pickles, microbial cultures containing salt, anti-osmotic microorganisms present in seawater, such as halophilic microorganisms, salt-resistant microorganisms, etc. In this case, it is usually 1 to 20% as a preparation liquid such as a diluting liquid, a washing liquid, and a suspension liquid.
Although (w / v) saline is used, many anti-osmotic microbial cells can be used in order to solve the above-mentioned drawbacks by using, for example, sterilized water containing no salt as the preparation liquid. However, it is not suitable because it ruptures due to osmotic pressure and ATP leaks in an extremely short time, and the ATP is decomposed during sample preparation or removed by a washing operation.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for measuring the number of bacterial cells with high sensitivity and accuracy by overcoming the above-mentioned drawbacks in the method of measuring the number of bacterial cells of a microorganism. It was made as.

[0007]

Means for Solving the Problems As a result of various studies to achieve the above-mentioned object, the present inventors have found that in a method for measuring the number of cells by the luciferin-luciferase reaction, a conventional sample preparation liquid for a microorganism has been used. Based on this finding, it was found that the sugar solution and the polyalcohol solution can be used very suitably in place of the saline solution described above, and that the object can be achieved by subjecting the sample prepared using this solution to the ATP extraction treatment. As a result, the present invention has been completed.

That is, the present invention relates to ATP of microbial cells.
Method for measuring the number of bacterial cells by extracting luciferin and luciferin-luciferase reaction, wherein the sample prepared by using a preparation liquid containing sugar and / or polyalcohol is subjected to ATP extraction treatment, Is. Hereinafter, the present invention will be described in detail.

First, the microorganisms in the present invention are mainly yeasts and bacteria, but are not particularly limited. Above all,
The method of the present invention is particularly preferably used for measuring the number of anti-osmotic microorganisms. The anti-osmotic microorganism is not particularly limited as long as it is a microorganism that can grow even in the presence of a high-concentration solute, and may be any microorganism, for example, a halophilic microorganism, a salt-resistant microorganism, and other high concentrations ( Microorganisms that can grow even in solutes (up to about 60%). As the halophilic or salt-tolerant microorganism, for example, a salt concentration of 2.
Examples include those that can grow at 5% (w / v) or higher,
Examples thereof include soy sauce yeast and soy sauce lactic acid bacteria.

The preparation liquid used in the present invention is a solution used for preparing a sample, and means each or all of the diluting liquid, the washing liquid, the suspension liquid and the other treating liquid used. Next, the preparation of the sample for measuring the number of microbial cells is a conventional method except that the preparation of the sample immediately before being subjected to the ATP extraction treatment is carried out using a preparation liquid containing sugar or polyalcohol. The following method may be mentioned, for example.

When measuring the number of microbial cells in a liquid, for example, a certain amount of the solution is taken, and if necessary diluted with a diluting solution, a certain amount thereof is taken and collected by a filtration membrane such as a microfilter. After sterilizing, wash with a washing solution containing sugar or polyalcohol and use it as a sample (with filtration membrane) as it is, or suspend the cells with a suspending solution containing sugar or polyalcohol to obtain a certain amount. Is used as a sample and is subjected to ATP extraction treatment.

When the number of microbial cells existing in a solid or pasty substance is measured, for example, a certain amount thereof is taken, diluted with a diluting solution, and stirred to take a fixed amount of the solution. After collecting the cells with a filtration membrane such as a microfilter, washing with a washing solution containing sugar or polyalcohol, and then collecting the cells as they are or by suspending them in the same manner as in the case of the liquid, or by centrifugation or the like. Then, after washing with a washing solution containing sugar or polyalcohol, if necessary, a suspending solution containing sugar or polyalcohol is further added to suspend, and a certain amount of this is used as a sample, which is subjected to ATP extraction treatment.

Furthermore, when the presence or absence of microbial cells such as a product in which microbial cells are not present or is extremely low is determined or the number of microbial cells is measured, for example, when microbial cells are present, they grow to the measurement range. Cultivation may be carried out under such conditions in advance, and a sample may be prepared for this culture in the same manner as described above. In the present invention, the sample prepared using the preparation liquid containing sugar or polyalcohol as described above is subjected to ATP extraction treatment, that is, at least A
The sample immediately before being subjected to TP extraction treatment is sugar or polyalcohol.
It needs to be prepared using a preparation liquid containing
Therefore, as a preparation liquid before that, a saline solution or the like can be used as in a conventional method, and, of course, a solution containing sugar or polyalcohol may be used.

In the present invention, the sugar used in the preparation liquid includes, for example, glucose, galactose, fructose, xylose, ribose, arabinose, threose, erythrose, maltose, sucrose, lactose, and the concentration thereof is usually 1 to 60% (w
/ W), preferably 5 to 30% (w / w), and used in the range soluble in water.

Examples of the polyalcohol include glycerol and ethylene glycol, and the concentration thereof is usually 1 to 60% (w / w), preferably 5 to 30.
Used in% (w / w). These sugars and polyalcohols may be used alone or in appropriate combination. Here, the result of studying the inhibition of salt on the luciferin-luciferase reaction is shown as Experimental Example 1.

In the following experimental examples and examples described below, "lucifer-LU plus" (luciferase derived from Genji firefly) (manufactured by Kikkoman) was used as the reagent kit for ATP measurement, and photo As a counter, the Biocounter M2500
(Manufactured by Lumac) was used. Experimental Example 1 Saline solutions of various concentrations shown in Table 1 containing 1 ng of ATP standard reagent were prepared, and 100 μl of each was dispensed into a cuvette (measurement container). Then, for these,
100 μl of the TP extraction reagent was added, and 10 seconds later, 100 μl of the luciferin-luciferase luminescence reagent was added, and the amount of luminescence for 10 seconds was immediately measured by a photocounter. Table 1 shows the relationship between the salt concentration and the relative value of the luminescence amount at this time.
And shown in FIG.

The salt concentration shown in Table 1 is that of the saline solution containing the ATP standard reagent as described above, and the salt concentration in the reaction system is 1/3 of this.

[0018]

[Table 1]

From Table 1 and FIG. 1, it can be seen that the inhibitory effect of sodium chloride on the luciferin-luciferase reaction is extremely large. Next, Experimental Example 2 shows the results of examination on the presence or absence of inhibition of sugar and polyalcohol on the luciferin-luciferase reaction. Experimental Example 2 Glucose, galactose and ethylene glycol aqueous solutions of various concentrations (w / v) containing 1 ng of ATP standard reagent were prepared, and the amount of luminescence was measured in the same manner as in Experimental Example 1 below. FIG. 2 shows the relationship between the concentration of each aqueous solution and the relative value of the amount of light emission at this time. In FIG. 2, ○ indicates glucose, ● indicates galactose, and Δ indicates ethylene glycol.

From FIG. 2, it can be seen that the inhibitory effects of glucose, galactose and ethylene glycol on the luciferin-luciferase reaction are hardly observed.
Further, the rupture of cells in the anti-osmotic microorganism was examined and the results are shown as Experimental Example 3. Experimental Example 3 Raw soy sauce 15.0%, NaCl 9.5%, KH2PO4
0.05%, MgSO4 · 7H2O 0.025%,
CaCl2 ・ 2H2O 0.005%, yeast extract
0.01%, glucose 7.0% (% is w /
v), a liquid medium having a composition of pH 5.2 was dispensed to a 500-ml shake flask in an amount of 50 ml, and after sterilization, the soy sauce yeast Tygosaccharomyces lucii (Zygosacc
Haromyces rouxii) ATCC 13
356 was inoculated and aerobically shake-cultured at 30 ° C. for 42 hours by a conventional method to obtain a culture solution.

Next, the culture broth was diluted 1000 times with an aqueous glucose, galactose and glycerol solution having various concentrations (w / v) shown in Table 2, and allowed to stand in a water bath at 20 ° C. for 30 minutes. Pore size 0.45 μm
Was filtered under reduced pressure using a membrane filter (manufactured by Advantic Toyo Co., Ltd.) to obtain a filtrate. With respect to each of the diluted solutions and the filtrate thus obtained, the luminescence amount was measured in the same manner as in Experimental Example 1, and the ratio (%) of the luminescence amount of the filtrate to the diluted solution was determined.

The ratio represents the ratio of the ruptured cells to all the cells, and the results are shown in Table 2.

[0023]

[Table 2]

From Table 2, it can be seen that the addition of glucose, galactose and glycerol markedly suppressed the rupture of bacterial cells. Next, the sample prepared using the preparation liquid containing sugar or polyalcohol as described above is treated with an ATP extractant by a conventional method to extract ATP in the sample, and luciferin and luciferase are added to this. The amount of light generated by the action can be measured using a light quantity measuring device, and the number of bacterial cells can be measured from a calibration curve that shows the relationship between the amount of luminescence and the number of bacterial cells that has been obtained in advance.

The ATP extractant, luciferin, luciferase and the like are not particularly limited, and commercially available reagent kits for measuring the number of bacterial cells, which apply the luciferin / luciferase reaction, are effectively used. In addition, luciferase
It may be of any origin, but those derived from American firefly and Genji firefly are preferable.

[0026]

The method of the present invention, as a sample preparation liquid,
By using a solution containing a sugar or a polyalcohol that shows almost no inhibitory effect on the luciferin-luciferase reaction, the sensitivity and accuracy are much higher than when using saline as a conventional preparation solution. The number of cells can be measured well. Further, when the method of the present invention is applied to the measurement of the number of anti-osmotic microbial cells, it is possible to prevent the cells from rupturing and to measure the number of cells with extremely high accuracy.

[0027]

EXAMPLES The present invention will be specifically described below with reference to examples. However, the technical scope of the present invention is not limited by these examples. Example 1 The same liquid medium as in Experimental Example 3 was dispensed into a 500-ml shake flask in an amount of 50 ml, and after sterilization, this was inoculated with soy sauce yeast T. saccharomyces lucii ATCC 13356, which was then inoculated at 30 ° C. for 42 hours by a conventional method. The culture was performed with shaking under air, and the number of bacterial cells was measured over time. The sample preparation method at this time is as follows.
5% (w / v) galactose aqueous solution, 10% (w / v)
An ethylene glycol aqueous solution and a 10% (w / v) saline solution were used.

First, 1 ml of the culture solution was collected over time as shown in Table 3, and this was filtered under reduced pressure using a pore size 0.45 μm membrane filter (manufactured by Advantic Toyo Co., Ltd.) to collect the bacteria, and then the membrane filter. After washing the above cells with 2 ml of the above-mentioned preparation, put the membrane filter into a clean test tube with clean tweezers, and inject 10 ml of the same preparation to suspend the cells to prepare a sample. ..

The amount of luminescence of each of these samples was measured in the same manner as in Experimental Example 1, and the number of bacterial cells was determined from the calibration curve prepared in advance (the calibration curve for ethylene glycol is omitted) shown in FIG. The results are shown in Table 3. In addition, in FIG.
O indicates that a 15% galactose aqueous solution was used, and ● indicates that a 10% saline solution was used.

[0030]

[Table 3]

From Table 3, according to the method of the present invention, the number of bacterial cells can be measured with high sensitivity, but in the case of the control, the sensitivity is lowered due to the inhibition of the luciferin / luciferase reaction, and the measurable range is You can see that it is happening. Example 2 Glucose 1%, Polypeptone 1%, Yeast extract
0.4%, KH2PO4 0.4%, sodium thioglycolate 0.1%, raw soy sauce 1%, salt 10% (all are w / v), pH 7.0 liquid medium of 5 l Erlenmeyer flask Dissolve 3.5 liters of the soybeans into a sterilized mixture, and sterilize it. Add to this the soy sauce lactic acid bacterium Pediococcus halofils
cus halophilus) I FERM BP-
35 ml of the seed culture solution of 1302 was inoculated, statically cultured at 30 ° C. for 96 hours, and the number of bacterial cells was measured over time.

The sample was prepared by the following method.
First, 1 ml of the culture solution was collected over time as shown in Table 4, and this was filtered under reduced pressure using a pore size 0.2 μm membrane filter (manufactured by Advantic Toyo Co., Ltd.), and then the cells were washed with clean tweezers. The membrane filter to which was adhered was placed in a clean test tube, and 10 ml of a 15% (w / v) glucose aqueous solution as a sample preparation solution was injected to suspend the bacterial cells to prepare a sample.

The amount of luminescence of each of these samples was measured in the same manner as in Experimental Example 1, and the number of cells was determined from the calibration curve previously prepared as shown in FIG. The results are shown in Table 4.

[0034]

[Table 4]

By using the method of the present invention, the number of soy sauce lactic acid bacteria cells could be measured with high sensitivity and accuracy.

[Brief description of drawings]

FIG. 1 is a graph showing the inhibition of salt on the luciferin-luciferase reaction in Experimental Example 1.

FIG. 2 is a graph showing the presence or absence of inhibition of sugar and polyalcohol on the luciferin-luciferase reaction in Experimental Example 2.

FIG. 3 is a graph of a calibration curve used for measuring the number of bacterial cells in Example 1.

FIG. 4 is a graph of a calibration curve used for measuring the number of bacterial cells in Example 2.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satoshi Kawamura 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (1)

[Claims] 1. A method for extracting ATP of microbial cells and measuring the number of cells by a luciferin-luciferase reaction, which comprises subjecting a sample prepared using a preparation solution containing sugar and / or polyalcohol to ATP extraction treatment. A method for measuring the number of bacterial cells, which is characterized by: