JPH09191870A - Medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medium capable of culturing microorganisms in specimens for an examination them on a large number of specimens in a narrow culturing surface and enabling strict microorganism controlling by using a polyvinyl alcohol. SOLUTION: This medium is composed of a polyvinyl alcohol such as a cold-water soluble polyvinyl alcohol or a carboxylic group modified polyvinyl alcohol. Further, a specimen liquid is preferably added to the medium, and microorganisms, plant cells, plant tissues or plants in the specimen are cultured. When a medium is prepared by adding a water-absorbing polymer such as a grafted starch or a polyacrylic acid to the polyvinyl alcohol, colonies can be formed even in the medium with a smaller amount of polyvinyl alcohol.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention mainly relates to a medium for culturing microorganisms and a method for culturing microorganisms using the medium. More specifically, it relates to a medium for culturing and detecting microorganisms in foods and the environment, and a culturing method thereof. This medium becomes a medium in which microorganisms and the like can grow by adding water or water containing nutrient components.

[0002]

2. Description of the Related Art Usually, a microorganism test contains a nutrient component necessary for the growth of the microorganism or a compound for detecting the nutrient component and / or a compound for inhibiting the growth of a microorganism other than the microorganism to be detected. This is done by applying the test sample solution to the agar medium. Usually, an agar medium having a surface area of 60 to 65 cm ² is used,
The amount of sample that can be applied to the surface of a dried agar medium is usually 20
It is 0 μL. When the amount of microorganisms to be detected is small in the sample, it is necessary to apply more than 200 μL of the sample solution to the medium. In such a case, a method of preparing several or more agar media for one sample or increasing the concentration of agar is used. However, it is the current situation that it is difficult to add 1 mL of sample solution even if the agar concentration is increased.

[0003]

Conventionally, when a sample contains a small amount of microorganisms to be detected, medium 1 of a normal size is used.
There was no way to perform a culture test for the microorganisms in the sample on a single sheet.
The present inventors have found that it is possible to add a larger amount of water than usual to the medium by using polyvinyl alcohol in the medium instead of agar which is usually used for culturing microorganisms and the like. Was completed. That is, the object of the present invention is to obtain a surface area of 60 to 65 cm, which is a size that is usually used, even for a sample containing few microorganisms for detection.
⁽ EN) It is intended to provide a method for easily inspecting a culture of a microorganism in a sample and a microorganism medium used for the method, by using only one medium of ² or less.

[0004]

[Means for Solving the Problems]

(1) A medium using polyvinyl alcohol. (2) A method of culturing microorganisms, plant cells or plant tissues in the sample solution by adding the sample solution to the medium described in (1) above.

The polyvinyl alcohol used in the present invention is
It is polyvinyl alcohol soluble in cold water or polyvinyl alcohol which is dissolved or partially dissolved when water is added after it is dissolved by heating and dried. In addition, a cold water-soluble polyvinyl alcohol derivative or a polyvinyl alcohol derivative that is dissolved by heating, dried, and then dissolved or partially dissolved when water is added can also be used. As the polyvinyl alcohol derivative, for example, carboxyl group-modified polyvinyl alcohol or the like is used. When such a polyvinyl alcohol is used as a medium, even if 1 mL or more of the sample solution is added to the medium, it can be cultured in a culture area of 60 to 65 cm ² or less, and colonies are also formed in this medium.

The method for producing the medium of the present invention and the method for using the medium will be described below. First, polyvinyl alcohol, a nutrient component or a compound for detecting a nutrient component and a microorganism, and / or a compound that inhibits the growth of microorganisms other than the microorganisms for the purpose of detection are dissolved by heating after adding water, Put it in a container such as a petri dish or layer it on a polyethylene terephthalate film,
dry. When cold water-soluble polyvinyl alcohol is used, the medium can be prepared simply by mixing it with nutrients and the like. However, when water is added to a powdery or granular medium in this manner, the uniformity of the constructed medium is poor, so
It is preferable that the powdery or granular medium is once dissolved to form a film. A culture medium in which microorganisms and the like grow can also be obtained by adding water containing nutrients to a cold water-soluble polyvinyl alcohol or a polyvinyl alcohol film that is dried and then dried to form a film. The culture medium may be prepared by arranging nutrient components or the like on the upper side, the lower side, or both sides of the polyvinyl alcohol film. When the nutritional component contains a heat-labile compound, it is preferable to add the nutritional component after the polyvinyl alcohol is heated and dissolved to form a polyvinyl alcohol film.

In order for the microorganisms grown on the medium of the present invention to form colonies, it is necessary that the medium contains 50 mg or more of polyvinyl alcohol per 1 mL of the sample solution added to the medium. By using a medium in which polyvinyl alcohol is mixed with a water-absorbing polymer such as a graft starch type or a polyacrylic acid type, colony formation is possible even with a medium having a smaller amount of polyvinyl alcohol. The medium prepared by the above method is placed in a container such as a petri dish, and a suspension of food, water for wiping the environment, or a sample solution such as a culture solution of bacterial cells is spread on this medium and cultured. Detect microorganisms. The amount of the sample liquid to be added can be adjusted to the volume of the container used by changing the amount of polyvinyl alcohol.

A polyvinyl alcohol film containing nutrients and the like is placed on a plastic film or sheet, a sample solution is added, and then a plastic film or sheet is covered to remove microorganisms etc. in the sample solution. It can also be cultured. A thin container may be used depending on the amount of the sample solution. Alternatively, the sample solution may be added to a polyvinyl alcohol film placed in a petri dish or the like, and then a film made of plastic or the like suitable for the size of the container may be covered and cultured. It is also possible to culture by putting the medium and the sample solution in a plastic bag or the like. It is also possible to culture by adding a sample solution suspended in an aqueous solution of nutritional components to a polyvinyl alcohol film containing no nutritional components.

In the medium of the present invention, not only microorganisms but also
Plant cells and plant tissues can also be cultured. Cultivate plants with polyvinyl alcohol or polyvinyl alcohol derivatives.
A medium capable of culturing and growing plant cells, plant tissues, and plants can be prepared by adding components necessary for growth or adding an aqueous solution of components necessary for culturing and growth.

The nutritional components contained in or added to the polyvinyl alcohol are not limited as long as they are suitable for the microorganism to be cultured. It is possible to use medium components obtained by removing agar from commonly used liquid medium or agar medium, and use dyes to make the resulting colonies easier to see, or chromogenic or fluorescent enzyme substrates for detecting specific microorganisms, and / or detection. A substance that suppresses growth other than the desired microorganism may be added. Nutrients for general viable bacteria inspection include yeast extract / peptone / glucose mixture, meat extract / peptone mixture, peptone / soybean peptone / glucose mixture, etc., to which potassium monohydrogen phosphate and / or sodium chloride are added. Are commonly used, such as E. coli
Nutrients for coliform test include sodium dezoxycholate, peptone, ammonium iron citrate, sodium chloride, potassium dihydrogen phosphate, lactose, neutral red mixture, peptone, lactose, potassium dihydrogen phosphate, eosin Y. Peptone, lactose, etc. to which a methylene blue mixture or the like or a fluorescent and / or chromogenic substrate for β-glucuronidase and / or β-galactosidase was added.
It is possible to use a mixture of nutrients such as sodium chloride / bile salt mixture / potassium hydrogen phosphate / tryptophan mixture that can grow Escherichia coli / Coliform group.Meat extract / peptone / sodium chloride / Mannitol / phenol red / yolk mixture, peptone / meat extract / yeast extract / sodium pyruvate /
Glycine / lithium chloride / tellurite yolk liquor mixture can be used. As nutritional ingredients for vibrio, yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, sodium cholate, ferric citrate, chloride Sodium, beef bile, bromthymol blue, thymol blue mixture, etc. can be used. In addition, as nutrients for enterococci, bovine brain extract, heart extract, peptone, glucose, potassium monohydrogen phosphate, sodium nitride,
Bromthymol blue / 2,3,5-triphenyltetrazolium chloride mixture can be used. Peptone / dextrose mixture, potato extract / dextrose mixture, etc. can be used as fungal nutritional ingredients. Is peptone, corn starch, sodium chloride, esculin, iron (III) citrate ammonium,
Lithium chloride, cycloheximide, colistin sulfate, acriflavine, cefotetan, fosfomycin mixture,
Peptone, starch, sodium chloride, mannite, ammonium iron citrate, esculin, glucose, lithium chloride, phenol red, polymyxin B sulfate, ceftacidium, acriflavine mixture, etc. can be used. As a nutritional ingredient for Salmonella, meat extract is used.・ Peptone ・ Lactose ・ Sucrose ・ Sodium deoxycholate ・ Sodium thiosulfate ・ Sodium citrate ・ Iron ammonium citrate ・ Neutral red mixture, yeast extract ・ Peptone ・ Heart extract ・ Sodium chloride ・ Mannitol ・ L
-Lysine hydrochloride / sodium thiosulfate / ammonium iron citrate / brilliant green / crystal violet mixture can be used.

[0011]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Polyvinyl alcohol 50 g, peptone 11.5 g, corn starch 0.5 g, sodium chloride 2.5 g, esculin 0.
5 g, iron (III) citrate 0.25 g, and lithium chloride 7.5 g were added, and after heating and dissolution, 10 g each was dispensed and dried in a φ90 mm Petri dish to prepare a basal medium for Listeria separation. Basal medium with 44 mg / mL cycloheximide, colistin sulfate,
Acriflavine, cefotetan, fosfomycin (40
(0: 20: 5: 2: 10 (weight ratio)) After adding 25 μL of the mixed solution, 5 mL of the culture solution obtained by culturing various cheeses in EB medium for 48 hours was added and spread over the entire Petri dish, and then at 37 ° C.
Cultured for ~ 48 hours. Bacteria presumed to be Listeria formed green-brown colonies on the medium, and the periphery of the colonies turned black.

Example 2 In 1 L of water, 50 g of polyvinyl alcohol, 2 g of potato extract,
20 g of glucose was added and dissolved by heating, and 20 g of each was dissolved in a Petri dish of φ90 mm and dried to prepare a fungal separation medium. Surface of washed cutting board, work table, etc. 800 to 5000 cm ²
Wipe the cells with a few cotton swabs and remove the bacteria on the cotton swab with sterile water.
The cells were suspended in mL to obtain a bacterial cell suspension. 10 mL of this cell suspension was added to a fungal separation medium, the medium was spread over the entire Petri dish, and then cultured at 25 ° C for 2 to 7 days. Mold growth was observed in about 20% of the tested samples.

Example 3 To 1 L of water, 90 g of polyvinyl alcohol, 4.5 g of yeast extract,
Peptone 9g, Heart extract 1.8g, Sodium chloride 3.6g,
Mannitol 2.7g, L-lysine hydrochloride 4.5g, sodium thiosulfate 3.6g, iron (III) citrate ammonium 0.9g, brilliant green 11.25mg, crystal violet 9
After adding mg and heating and dissolving, 10 g of the lysate was dispensed into a 50 × 50 × 2 mm polystyrene container and dried to prepare a medium. To the prepared medium, 9 mL each of a culture solution obtained by culturing fish meal, rapeseed meal and the like in an SBG medium for 24 hours was added, covered with a 0.04 mm thick polypropylene film, and cultured at 35 ° C. for 24 hours. A colony presumed to be salmonella with a black center was observed in about 5% of the samples.

Example 4 Polyvinyl alcohol 100 g, peptone 3 g, sodium chloride 5 g, sodium dihydrogen phosphate 2.2 g, sodium monohydrogen phosphate 2.7 g, sodium pyruvate 1 g, tryptophan 1 g, sorbitol 1 g, and tarditol 7 in 1.5 L of water. 0.15g
After heating and dissolving, 100 mg of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide was added and mixed,
A polyester film having a thickness of 1 × 2 m and a thickness of 100 μm was laminated, dried, and cut into 15 cm squares. Add 10 times more water to the oysters,
10 mL of the homogenized product was added to the prepared medium. 0.
It was covered with a 04 mm thick polypropylene film and incubated at 35 ° C. for 24 hours. Bacteria with putative E. coli β-glucuronidase activity formed blue colonies.

[0015]

EFFECTS OF THE INVENTION By using the medium of the present invention, by increasing the amount of sample that can be added per surface area as compared with the conventional medium, a sample solution of more than 1 mL is added to a medium having a surface area of 60 to 65 cm ^{2 which is} usually used. Because you can add
Even if the sample has a small number of microorganisms for the purpose of detection, many samples can be added to the medium, so that the sample can be inspected with one medium. If a large amount of sample liquid can be added to one medium, the detection rate of microorganisms will increase and stricter microorganism control will be possible. Further, depending on the amount of polyvinyl alcohol in the medium, the medium of the present invention can be used to inspect an ordinary sample solution of about 1 mL, so that the inspection can be performed more efficiently than the conventional medium.

Claims (2)

[Claims] 1. A medium using polyvinyl alcohol. 2. A sample solution is added to the medium according to claim 1,
A method for culturing a microorganism, plant cell, plant tissue or plant in a sample solution.