JP3937019B2 - Dispersion medium for preservation of microorganism and container for preservation of microorganism - Google Patents

Description

  The present invention relates to a dispersion medium for storing microorganisms and a container for storing microorganisms, and more specifically, a dispersion medium for storing microorganisms and microorganisms that can preserve microorganisms for a long period of time even when they are repeatedly frozen and thawed during storage. It relates to a storage container.

  Microbial preservation methods are roughly classified into freezing methods, drying methods, and subculture methods. Of these, freezing and drying methods are used as long-term stable storage methods, and subculture methods are used for microorganisms to which these techniques are not applicable.

  The freezing method is a method in which a living reaction is stopped by freezing intracellular moisture of a target microorganism, and a long-term survival is achieved in a resting state. Storage containers such as ampoules and vials containing the target microorganisms are stored in a freezer consisting of an ultra-low temperature tank or a liquid nitrogen tank at -50 ° C to -80 ° C. Microorganisms that are resistant to freezing can be stored by freezing.

  In addition, the drying method is a method in which a living reaction is stopped by removing intracellular moisture of a target microorganism, and a long-term survival is achieved in a resting state. The drying method is divided into two depending on the water removal method, and there are a freeze-drying method in which water is removed by sublimation from the solid phase after freezing, and an L-drying method in which water is removed by evaporation from the liquid phase without freezing.

  In the freeze-drying method, a storage container containing a target microorganism is attached to a vacuum freeze-dryer to remove moisture. The dried specimen from which moisture has been removed as described above is sealed in a vacuum, or returned to normal pressure and sealed, and stored in a cool and dark place. The freeze-drying method can be applied only to microorganisms that are resistant to both drying and freezing.

  The L-drying method is a method in which the target microorganism is placed in a storage container and attached to a vacuum freeze dryer, and the water inside and outside the microorganism cell is removed from the liquid phase by evaporation so as not to boil or freeze under reduced pressure. It is. The dried specimen from which moisture has been removed is sealed under vacuum and stored in a cool dark place. Microorganisms resistant to drying can be stored by the L-drying method.

  On the other hand, the subculture method is a method in which microorganisms are periodically transplanted and preserved while maintaining an active state, and can be applied to all culturable microorganisms, but the microbial cells are not in a dormant state, so that the properties of the microbial strain are The risk of changing is higher compared to freezing and drying methods.

  In the case of using a long-term storage microorganism strain, in the freezing method, a cryopreserved specimen is taken out of a freezer, immersed in hot water at room temperature to 37 ° C. and thawed, and then the contents are transplanted into a medium to proliferate the microorganism. To use. In the drying method, a preserved specimen is opened, sterilized distilled water is added to liquefy the contents, and then the contents are transplanted into a medium to grow microorganisms for use. In either method, the preserved specimen after use is discarded.

  The freezing operation in the freezing method or the drying operation in the drying method adversely affects the microorganisms and leads to a decrease in the number of viable bacteria. Therefore, the microorganisms to be stored are substances prepared for the purpose of preserving the microorganisms (hereinafter referred to as the microorganisms). , And then dispersed in a storage container, followed by freezing and drying. As the dispersion medium for the freezing method, glycerin, dimethyl sulfoxide or the like is used, and other methods such as adding fructan (see, for example, Patent Document 1) have been reported. As the dispersion medium of the drying method, skim milk, sucrose, glucose, bouillon, sodium glutamate, cysteine, gelatin are used, and a method of adding a cellulose-based biopolymer (see, for example, Patent Document 2) or an amino acid such as arginine. (For example, refer to Patent Document 3) and the like have been reported.

  However, when a microorganism is used, if it is restored from the preserved specimen by the freezing method or the drying method each time, a large quantity of preserved specimen must be retained for the frequently used microorganism. For this reason, generally, a method is used in which a long-term storage microorganism strain is restored by the above method at an appropriate interval and connected by the subculture method from the previous restoration to the next restoration. However, the method of connecting by the subculture method has the above-mentioned problems even in a short period of time and is laborious.

  Therefore, in a microorganism that can be repeatedly frozen and thawed, a cryopreservation method (hereinafter referred to as a repeated use cryopreservation method) on the premise of repeated use is used instead of the subculture method, and subculture is performed. Efforts are made to reduce labor and other problems by connecting with the law.

  When using preserved microorganisms by the repeated use cryopreservation method, the contents of the storage container by the freeze method or the drying method are transplanted to a culture medium to grow the microorganisms, and the grown microorganisms contain a dispersion medium. Store in a storage container for repeated use cryopreservation.

  When using microorganisms stored by the above-mentioned repeated use cryopreservation method, the storage container for repeated use cryopreservation is kept at room temperature to 37 ° C. to thaw the contents, and one of the microorganisms suspended with the dispersion medium. A part is taken out and transplanted to a medium, and microorganisms are grown and used. The dispersion medium in which the microorganisms remaining in the storage container are suspended is again put in the freezer and frozen, and the stored microorganisms are stored until the next use.

  As a dispersion medium used in the repeated use cryopreservation method, skim milk to which sodium glutamate is added has been generally used. In addition, the storage temperature in the repeated freezing storage method is generally in the range of −20 ° C. to −40 ° C., where there is little risk of freezing injury even if the operator handles it with bare hands to increase work efficiency.

  However, in the above-mentioned repeated use cryopreservation method, as the number of repeated treatments increases, the number of surviving bacteria remarkably decreases, and the types of microorganisms that can be used are limited. Improvement of protection performance was desired.

JP-A-7-99965 Japanese Patent Laid-Open No. 10-243781 JP 2003-219862 A

  The present invention solves the above-described problems, and provides a dispersion medium that can be used for various microorganisms even when a repeated use cryopreservation method is used, and a storage container containing the dispersion medium. Let it be an issue.

  The inventors of the present invention repeatedly applied freezing and thawing treatment to a wide range of microorganisms by adding a disaccharide to a dispersion medium consisting of skim milk and sodium glutamate, which has been conventionally used in the repeated use cryopreservation method. However, it has been found that long-term storage is possible, leading to the present invention. That is, the present invention is as follows.

  A microorganism-preserving dispersion medium comprising sodium glutamate, skim milk, and a disaccharide and capable of being repeatedly used for freezing and thawing during storage.

  The dispersion medium for microbial storage according to the present invention is a dispersion medium for microbial storage in which sodium glutamate is in the range of 0.5 to 3.0 mass%, skim milk is 3 to 20 mass%, and disaccharide is in the range of 3 to 20 mass%.

  In the microorganism storage dispersion medium, the disaccharide is a microorganism storage dispersion medium in which the disaccharide is one or more of lactose, trehalose, maltose, and sucrose.

  Furthermore, the present invention is a microorganism storage container characterized in that sodium glutamate, skim milk, and disaccharide are contained in the container.

  In the microorganism storage container, the microorganism storage container includes 0.5 to 3.0 parts by mass of sodium glutamate, 3 to 20 parts by mass of skim milk, and 3 to 20 parts by mass of disaccharides.

  In the storage container, the disaccharide is a microorganism storage container in which the disaccharide is one or more of lactose, trehalose, maltose, and sucrose.

  The storage container is a sterilized microorganism storage container.

  The dispersion medium for preserving microorganisms of the present invention can be used for various microorganisms even using a repeated use cryopreservation method, and a small number of samples can be obtained by a freezing method for long-term stable storage or a drying method. It enables various experiments of microorganisms to be performed efficiently. Further, the microorganism storage container of the present invention is a container sterilized by previously containing the components of the dispersion medium of the present invention, and by adding sterilized distilled water, it is possible to prepare a microorganism dispersion medium that can be easily frozen and stored.

  The dispersion medium for preserving microorganisms of the present invention is characterized by containing sodium glutamate, skim milk, and a disaccharide. Preferably, the disaccharide is one or more of lactose, trehalose, maltose, and sucrose.

  The sodium glutamate is preferably 0.5 to 3.0% by mass, and more preferably 1.0 to 2.0% by mass of the dispersion medium. The sodium glutamate is not necessarily highly pure, and commercially available edible sodium glutamate can be preferably used.

  The skim milk is preferably 3 to 20% by mass of the dispersion medium, and more preferably 5 to 15% by mass. The skimmed milk is not necessarily highly pure, and commercially available skimmed milk can be preferably used, but skimmed milk for microbial testing is more preferred.

  The disaccharide is preferably one or more of lactose, trehalose, maltose, and sucrose. Among them, lactose, trehalose, and maltose are more preferable, and lactose is particularly preferable from the viewpoint of easy availability. The disaccharide is preferably 3 to 20% by mass of the dispersion medium, and more preferably 5 to 15% by mass.

  The ratio of sodium glutamate, skim milk, and disaccharide is preferably about 20 parts by mass of skim milk and 10-20 parts by mass of disaccharide with respect to 3 parts by mass of sodium glutamate.

  As the solvent of the dispersion medium of the present invention, water and an aqueous glycerin solution are preferable.

  Furthermore, the present invention is a microorganism storage container, wherein 0.5 to 3.0 parts by mass of sodium glutamate, 3 to 20 parts by mass of skim milk, and 3 to 20 parts by mass of a disaccharide are contained in the container.

  The storage container may be made of any material such as glass or plastic, but it is preferably one that does not deform or change in the range of + 125 ° C to -196 ° C.

  The storage container is preferably sterilized. Although the sterilization method is not particularly limited, it is preferably sterilized at 110 ° C. to 115 ° C. when heated. A method of irradiating radiation can also be preferably used, and among them, sterilization by a method of irradiating γ rays is particularly preferable.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. In the following, “part” means mass part, and “%” means mass%.
<Example 1>
10 parts of commercially available skim milk for microorganism testing (trade name: DIFCO 232100, manufactured by BECTONDICKINSON), sodium glutamate (trade name: sodium glutamate monohydrate, Wako reagent special grade, manufactured by Wako Pure Chemical Industries, Ltd.), 1.5 parts, lactose (Product name: lactose monohydrate, JIS reagent special grade, manufactured by Wako Pure Chemical Industries, Ltd.) To 5 parts, distilled water was added so that the total mass was 100 parts, and dissolved to prepare a dispersion medium of Test 1. . Dispense 2 ml of the dispersion medium into a screw cap test tube having an outer diameter of 16.5 mm and a length of 12 cm at 115 ° C. using a high-pressure steam sterilizer (trade name: HICLAVE HV-50, manufactured by Hirayama Seisakusho). And autoclaved for 15 minutes.

  Furthermore, with respect to the test 1, only the lactose content was changed from 5 parts to 10 parts (Test 2), 15 parts (Test 3), and 0 parts (Comparative 1). Similarly, the dispersion media of Test 2, Test 3, and Comparative 1 were prepared.

In the test, a soft rot fungus (Erwinia carotovora subsp. Carotovora MAFF 301393) was used. The MAFF 301393 is an abbreviation for microbial genetic resources of the Ministry of Agriculture, Forestry and Fisheries. The test bacteria dissolved in the vial by the freezing method are grown in a medium, further cultured in a slant medium at room temperature for 1 day, suspended in sterilized distilled water, and a concentration of about 10 ⁷ cfu / ml. Adjusted. 100 μl of this suspension was dispensed into the test tubes into which the dispersion media of Tests 1 to 3 and Comparative 1 were injected, and immediately stored in a freezer at −20 ° C.

  The test tube stored in the −20 ° C. freezer was immersed in tap water for 10 minutes one day later to melt the contents. This was defined as one freezing / thawing treatment. Thereafter, the thawed specimen is immediately returned to a −20 ° C. freezer, and thereafter, the same freezing and thawing process is performed every day. The second day is treated twice and the third day is treated three times. The survey was conducted 8 times, 12 times. The number of surviving bacteria was diluted with sterilized distilled water after thawing the frozen specimen, and 50 μl of the diluted solution was smeared on a 9 cm diameter ordinary agar plate and cultured at room temperature for 4 days. The original bacterial count was calculated by multiplying by the dilution rate. The results are shown in Table 1.

  From the results of Table 1, the dispersion media of Test 1 to Test 3 are all 6 to 8 times when the number of treatments is 8, compared to the dispersion media of Comparative 1 that have been conventionally used and do not contain lactose. When the number of treatments was 12, 22 to 32 times the number of bacteria was obtained.

<Example 2>
In Example 1, instead of lactose in Test 1, trehalose (trade name: trehalose dihydrate, Wako Reagent Special Grade, Wako Pure Chemical Industries, Test 4), maltose (trade name: maltose monohydrate, Wako) Tests 4 to 4 were performed in the same manner as Test 1 except that the reagent grade, Wako Pure Chemical Industries, Test 5), and sucrose (trade name: sucrose, JIS reagent special grade, Wako Pure Chemical Industries, Test 6) were used. A dispersion medium of Test 6 was prepared.

  The method of freezing / thawing was the same as in Test 1, and the number of treatments was investigated once and 12 times. The number of surviving bacteria was measured in the same manner as in Example 1. The results are shown in Table 2.

  From the results in Table 2, it was found that sucrose was slightly inferior, but when trehalose and maltose were added in the same manner as lactose, these additions were effective in alleviating frost damage.

<Example 3>
A dispersion medium of Example 3 was prepared in the same manner as in Example 2 except that Escherichia coli JCM 1649 was used instead of the soft rot test bacteria used in Example 2. The freezing / thawing treatment method and the number of surviving bacteria were measured in the same manner as in Example 1, and the number of treatments was investigated once and 15 times. The JCM 1649 is an abbreviation for RIKEN System Storage Facility. The results are shown in Table 3.

  From the results in Table 3, it can be seen that the dispersion medium of the present invention is also effective in E. coli.

According to the present invention, even if a freeze storage method for repeated use is used, it can be used for various microorganisms such as soft rot fungi and Escherichia coli, which have not been able to be used in the conventional method, and the storage management and utilization of microorganisms are possible. It became easy. Further, by preparing in advance a dispersion medium composition of the present invention in a sterilized container, it becomes possible to easily obtain a storage container containing the dispersion medium.

Claims (4)

In the method for preserving microorganisms , freezing and thawing are repeated by using a dispersion medium for preservation of microorganisms containing 0.5 to 3.0% by mass of sodium glutamate, 3 to 20% by mass of skim milk, and 10 to 15% by mass of disaccharide. A method for preserving microorganisms . The method for preserving microorganisms according to claim 1, wherein the disaccharide is one or more of lactose, trehalose, maltose, and sucrose. The method for preserving microorganisms according to claim 1 or 2, wherein the dispersion medium for preserving microorganisms is placed in a container for preserving microorganisms . The method for preserving microorganisms according to claim 3, wherein the container for preserving microorganisms is sterilized.