JPH08308592A - Determination of bacterial count in calcium carbonate slurry - Google Patents

Abstract

PURPOSE: To easily and accurately determine viable bacterial count without the need of any particular instrument even in case of small viable bacterial count, by conducting both the culture and staining of a calcium carbonate slurry for e.g. coating pigments under specified conditions. CONSTITUTION: A calcium carbonate slurry is mixed with an agar medium with pH8-10 and the mixture is subjected to plate culture; in this case, a 2,3,5- triphenyltetrazolium chloride solution (and glucose solution) is added to the agar medium, and the number of colonies stained red is counted. By this method, the number of colonies produced can be accurately counted by visual inspection even in case of viable bacterial count of as small as <=1×10<3> cfu/ml.

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 the number of bacteria in a calcium carbonate slurry, particularly a calcium carbonate slurry used in a papermaking process.

[0002]

2. Description of the Prior Art Calcium carbonate slurries are mainly used as coating pigments in the paper industry. Light calcium carbonate is usually sold in the form of a high-concentration slurry containing 60% or more and heavy calcium carbonate containing 75% or more. . Since such a calcium carbonate slurry contains a small amount of a low molecular weight polymer as a dispersant, bacteria are proliferated during storage or transportation, and a disorder due to putrefaction such as generation of off-odor, partial discoloration or slime occurs. There are cases.

In order to prevent such microbial damage, the diluted calcium carbonate slurry is mixed with an agar medium and cultivated, and then the viable cell count is measured by the pour-in method in which the number of colonies produced is counted to control quality. Is going. However, according to the conventional pour-out method, when the dilution of the calcium carbonate slurry is high, it is possible to count all the colonies formed inside the agar plate medium by holding the Petri dish over the light. If the slurry is diluted too low, the calcium carbonate particles will turn the agar plate medium into a white background, making it difficult to count the colonies formed on the agar plate medium, especially the colonies formed inside the agar plate medium. Is.

When the number of viable bacteria in the calcium carbonate slurry is small, for example, 1 × 10 ³ cfu / ml (cfu: co
It is necessary to count at a low dilution in the case of less than the long forming unit), but in this case, it is difficult to measure the viable cell count for the above reason. Fluorescent dyes which specifically react with DNA and emit fluorescence, such as 4 ', 6-
Diamidino-2-phenylindole (DAPI),
A method of staining colonies with 3,6-tetramethyldiaminoacridine (acridine orange) or the like can be considered, but in this case also, the colonies formed inside the agar plate medium cannot be counted.

Incidentally, the calcium carbonate slurry may cause spoilage damage even when the viable cell count is small, for example, at 1 × 10 ³ cfu / ml or less. It is difficult to perform sufficient quality control because it is difficult to measure the number.

[0006]

The object of the present invention is to accurately measure the viable cell count by a simple operation without using a special measuring instrument even when the viable cell count in a sample is large or small. It is to propose a method for measuring the number of bacteria in a calcium carbonate slurry that can be used.

[0007]

According to the present invention, a calcium carbonate slurry as a sample and an agar medium having a pH of 8 to 10 are mixed and plated, and then 2,3,5-triphenyltetrazolium chloride is plated on the agar medium. Solution or 2,
A method for measuring the number of bacteria in a calcium carbonate slurry, which comprises adding a 3,5-triphenyltetrazolium chloride solution and a glucose solution and counting the number of colonies stained red.

The calcium carbonate slurry is alkaline with a pH of 9 to 10. Therefore, most of the bacteria that grow in the calcium carbonate slurry are so-called alkalophilic bacteria. It was found that most of these alkalophilic bacteria have dehydrogenase activity and have the ability to reduce 2,3,5-triphenyltetrazolium chloride to formazan. Therefore, in the present invention, 2, 3, 5
Staining alkalophilic bacteria red with triphenyltetrazolium chloride allows an accurate measurement of viable cell counts even at low dilutions.

As the calcium carbonate slurry used as a sample in the present invention, any slurry can be used as long as calcium carbonate is dispersed in a dispersion medium such as water. For example, a dispersant, a fluorescent dye, etc. can be used in the slurry. May be included. The calcium carbonate may be light or heavy. The method of the present invention is preferably applied to a slurry containing a high concentration of calcium carbonate, for example, one containing 60% by weight or more of light calcium carbonate,
Examples include those containing 75% by weight or more of heavy calcium carbonate.

An example of such a calcium carbonate slurry is a calcium carbonate slurry for paper making process, which is suitable for the method of the present invention. Further, according to the method of the present invention, it is possible to accurately measure the number of bacteria in a calcium carbonate slurry which cannot be accurately measured by the conventional method because the number of bacteria in the calcium carbonate slurry is small, and for example, 1 × 1.
A calcium carbonate slurry with a low bacterial count of 0 ³ cfu / ml or less can also be used as a sample.

As the agar medium used in the present invention, an agar medium conventionally used for culturing bacteria can be used,
For example, a medium obtained by adding agar as a support to a medium containing polypeptone, yeast extract, soluble starch, sodium chloride and the like can be used. The pH of the medium is 8 to 10, preferably 9 to 9.5.

In the method of the present invention, first, a calcium carbonate slurry as a sample and an agar medium having a pH of 8 to 10 are mixed and plated. In this case, it is preferable to add an agar medium to the calcium carbonate slurry or its diluted solution and perform plate culture. The culture forms a colony on the agar medium. In this case, colonies are also formed inside the agar layer, but such colonies can also be stained by the staining method described below and counted. The method of applying the sample on the medium after forming the agar plate medium is not preferable because the water in the sample is taken up by the medium and cannot be applied uniformly.

After the culture, a 2,3,5-triphenyltetrazolium chloride (hereinafter referred to as TTC) solution, or a TTC solution and a glucose solution are added to the agar medium to stain the colonies. The concentration of TTC in this case is 1
0-10,000 mg / l, preferably 500-1,0
00 mg / l, glucose concentration 0 to 10,000 m
It is desirable that the amount is g / l, preferably 100 to 1,000 mg / l. The amount of the solution to be used may be an amount sufficient to spread on the agar plate. For example, in the case of a petri dish with a diameter of 11 cm, it is usually 1-5 ml, and in most cases 2-3 m.
l is sufficient. As the solvent for dissolving TTC and glucose, distilled water or pure water is preferable, but dechlorinated tap water may be used. The solution does not need to be sterilized. Note that T
Instead of adding the TC solution and the glucose solution separately, it is also possible to prepare a mixed solution of TTC and glucose in advance and add this mixed solution.

Dyeing is carried out by adding a TTC solution or a TTC solution and a glucose solution at 25 to 40 ° C., preferably 3
The reaction can be carried out by holding at 0 to 37 ° C. for 15 minutes to 2 hours, preferably 30 minutes to 1 hour, and the colony can be stained red. in this case,
Since the colonies formed inside the agar medium can also be stained red, the number of generated colonies can be accurately counted visually.

When the dilution ratio of the calcium carbonate slurry is small, the medium becomes a white background due to the calcium carbonate particles, so the colonies formed inside the agar cannot be counted even if the Petri dish is covered with light, but as described above. By staining, even when the medium is a white background, colonies inside the agar can be accurately counted by giving a red contrast to the white background. For this reason,
The number of bacteria in the calcium carbonate slurry is 1 × 10 ³ cfu
Even when the number of bacteria is as low as / ml or less, the number of bacteria can be accurately measured.

The reason why colonies are stained red is that TT
This is because C is reduced by the dehydrogenase activity of the bacterium and a red, insoluble formazan is formed. In this case, it is impossible to accurately measure the number of bacteria unless all the bacteria that grow in calcium carbonate have dehydrogenase activity, but as will be seen from the examples described below, so-called alkaliphilic bacteria that grow in calcium carbonate slurry. It was revealed by the present inventor that each of these has dehydrogenase activity. Therefore, by staining as described above, the total number of bacteria in the calcium carbonate slurry can be easily measured.

Since TTC has a slight growth inhibitory effect on alkalophilic bacteria, accurate counting cannot be performed unless staining is carried out after culturing as in the present invention. Therefore, in the present invention, the bacteria and TTC are not contacted with each other during the culture period, and the location of the colony can be identified by utilizing only the enzyme activity immediately before the counting, so that accurate counting is possible.

As described above, in the present invention, 1 × 10 ³ cfu
The number of bacteria can be accurately measured even in the case of a calcium carbonate slurry having a low number of bacteria / ml or less. Therefore, in addition to improving quality control, it is possible to detect the initial stage of spoilage of the slurry by measuring the number of bacteria during storage or transportation of the product. Can be prevented. Further, when a bactericide effective for a highly alkaline liquid such as a calcium carbonate slurry is screened in a laboratory, a more accurate test than before can be performed.

[0019]

As described above, according to the method of the present invention, the calcium carbonate slurry and the agar medium are mixed and plated, and then the TTC solution or the TTC solution and the glucose solution are added to the agar medium for staining, Since the colonies are counted, the viable cell count can be accurately measured by a simple operation without using special measuring instruments, even when the viable cell count in the calcium carbonate slurry is high or low. .

[0020]

EXAMPLES Next, examples of the present invention will be described. Example 1 A light calcium carbonate slurry product (containing 75%) in which bacteria have grown to 1.3 × 10 ⁵ cfu / ml or 4.7 × 10 ⁶ cfu / ml during the storage period. Two kinds of seeds and different collection times were selected and serially diluted 10 times with sterile water. These samples (Samples A and B) were used as starch PY agar medium (1 g of polypeptone, 1 g of yeast extract, 1 g of soluble starch, 5 g of salt, 1000 g of tap water.
ml, agar medium of pH 9.0), and 5 series of platings were performed (30 ° C., 3 days).

One each Petri dish in which 40 to 200 bacterial colonies were formed was selected and the number of colonies was counted without staining. In this case, the high dilution ratio of the sample did not interfere with the white color of the slurry, and all the colonies formed inside the agar plate could be counted by holding the Petri dish over the light. Next, TTC solution (100
mg / l) 3 ml, and glucose solution (0.1%)
1 ml was added on the agar medium, and the reaction was carried out in an incubator at 30 ° C. for 1 hour. After the reaction, the number of bacterial colonies in which TTC was reduced and stained red was counted. The results are shown in Table 1.

[0022]

[Table 1]

From the results shown in Table 1, it can be seen that all the alkalophilic bacteria growing in the calcium carbonate slurry have dehydrogenase activity and TTC reducing ability.

Example 2, Comparative Example 1 The number of bacteria was 4.7 (± 0.5) × 10 ⁵ cf during the storage time.
Two kinds of experiments were conducted using a certain calcium carbonate slurry (containing 75%) that had propagated to u / ml as a sample. In the control system, the calcium carbonate slurry was diluted 10 times with sterilized water as in the conventional method to prepare the starch PY.
After plating on an agar medium at 30 ° C. for 3 days, the number of colonies was counted by exposing the Petri dish to light (Comparative Example 1). In the system of the example, a part of the calcium carbonate slurry was taken and sterilized by an autoclave (121 ° C, 15 minutes). 1 ml of each of the sterilized slurries was aseptically dispensed into a Petri dish, and 1 ml of each dilution step sample and 10 ml of the starch PY agar medium were poured and mixed onto the petri dish.
Plated for 3 days at ° C. That is, in the system of the Example, an undiluted slurry sample was added to each Petri dish, and the conditions were the same as when the undiluted slurry was implanted. Only the equivalent has been planted. After culturing, in the system of Example, the cells were stained with TTC in the same manner as in Example 1 and the number of colonies was counted. Table 2 shows the results.

Comparative Example 2 The plating was carried out in the same manner as in Example 1 except that a small amount of fluorescent dye was added to the calcium carbonate slurry used in Example 2. The Petri dish after culturing was brought into a dark room and irradiated with ultraviolet rays using an ultraviolet lamp. The number of colonies detected by absorbing ultraviolet rays and detected as dark spots was counted. The results are shown in Table 3.

Comparative Example 3 After plating in the same manner as in Example 2, 4 ', 6-diamidino-2-phenylindole (hereinafter referred to as DAPI) solution was added on the agar medium, and UV irradiation was performed in a dark room. went. DAPI is a fluorescent dye that specifically reacts with DNA to emit fluorescence, and colonies can be identified as fluorescent spots. The results are shown in Table 3.

Comparative Example 4 The procedure of Comparative Example 3 was repeated except that DAPI was replaced with 3,6-tetramethyldiaminoacridine (hereinafter referred to as acridine orange). The results are shown in Table 3.
Shown in

[0028]

[Table 2] Note) Figures in parentheses are mean ± standard deviation * 1 No sterilized slurry added, no dyeing * 2 Sterile slurry added

[0029]

[Table 3] Note) Average values ± standard deviation in parentheses * 1 Sterile slurry added, UV irradiation * 2 Sterile slurry added, DAPI staining * 3 Sterile slurry added, Acridine orange staining

In the results of Tables 2 and 3, since the number of bacteria in each sample at each dilution step is the same, it is expected that the same number of colonies will be produced, but compared to Comparative Example 1 (which can be visually counted). The number of colonies in Comparative Examples 2 to 4 showed a clearly low count value. This is because neither method can detect colonies formed inside the agar layer. On the other hand, no significant difference is observed between the results of Example 2 and Comparative Example 1, and it can be seen that in Example 2, colonies formed inside the agar layer can also be counted.

Comparative Example 5 In Example 2, an agar plate medium was formed without adding a dilution step sample. Next, the diluted stage sample was smeared on the surface of the agar plate medium, but it was difficult to uniformly smear it by taking water from the medium to solidify.

Comparative Example 6 100 mg / TC of TTC was added to the components of the starch PY agar medium.
After adding and dissolving so that it became 1, the agar was melted by heating and then sterilized by an autoclave at 121 ° C. for 15 minutes. However, the sterilized medium was reddish before it was subjected to the test and could not be used as the medium. It is considered that this is because TTC was alkaline and heated, so that it was subjected to a reducing action to generate red formazan.

Comparative Example 7 In Example 2, when the sample was planted in the agar medium, the TTC solution obtained by sterilization filtration was added to the medium and plated. However, when the added TTC concentration was 10 mg / l, the coloring was weak, and it was difficult to identify the colonies after growth. When the TTC concentration was 100 mg / l, the growth of bacteria was inhibited and the number of colonies was small in some cases.

Example 3 Even with a soft calcium carbonate slurry (containing 75%) delivered with a bactericidal agent added immediately after production, 1 × 10 ³ cfu / ml was stored at the delivery destination in a short time during storage in a tank. I have come to show the above number of bacteria. As the time it takes for bacteria to be detected is getting shorter and shorter,
There are various possibilities such as whether the added bactericide has become invalid due to the fear of failure, or the bactericide addition amount is insufficient, and appropriate measures should be taken. I couldn't.

This situation is 1 × 10 by the conventional method for measuring the number of bacteria.
^Although the cause could not be analyzed because the number of bacteria of ³ cfu / ml or less could not be measured, when the following samples were tested using the TTC staining method of the present invention, the results shown in Table 4 were obtained. .

[Table 4]

That is, no bacteria were detected in the new slurry at the time of delivery, there was no problem in quality, and bacteria began to grow in the residual slurry in the product receiving tank.
It was found that when the old slurry and the new slurry were mixed in the tank, the new slurry was contaminated with bacteria immediately after receiving it in the tank. Based on the above measurement results, when a method of periodically adding a bactericidal agent to a receiving tank was adopted, the problem of increase in the number of bacteria during short-term storage was eliminated, and spoilage could be prevented.

Claims (1)

[Claims] 1. A calcium carbonate slurry as a sample and an agar medium having a pH of 8 to 10 are mixed and plated, and
A 2,3,5-triphenyltetrazolium chloride solution or a 2,3,5-triphenyltetrazolium chloride solution and a glucose solution are added to the agar medium, and the number of red-stained colonies is counted. A method for measuring the number of bacteria in a calcium carbonate slurry.