JPH11289917A - Preservation of organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preserving an organism, intended for effective utilization for preserving the biological tissue(s) (organ (s)) of an organism by subjecting an organism in a dried state to high-pressure treatment in an inert medium. SOLUTION: An organism is preserved by subjecting the organism in a dried state such as biological tissue(s) being in a tan-state (standing still with the body contracted) or barrel state (humpbacked state) to high-pressure treatment such as under a high hydrostatic pressure of 600 MPa in an inert medium such as fluorocarbon medium. In this case, the organism is pref. a multicellular organism such as Adorybiotas coronifer belonging to the genus Eutardigrada.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for preserving an organism. More specifically, the present invention relates to a method for preserving organisms subjected to high-pressure treatment in a medium.

[0002]

2. Description of the Related Art Normally, when an organism is exposed to a high hydrostatic pressure environment, it affects biological membranes, proteins, DNA molecules, etc. of the organism. At about several tens MPa, the growth and metabolic function of microorganisms are suppressed, and pressure is reduced. It has been found that when the bacteria are further exposed to a high pressure of about 300 to 600 MPa, they almost die.

[0003] In addition, in animal cells other than bacteria, there are no cell walls possessed by bacteria or spores possessed by yeast, and they are made of a thin plasma membrane having a three-layer structure. It is thought that it cannot escape from leakage of cell contents, denaturation of proteins, etc., and therefore, it is used for high-pressure insecticide.

[0004] A similar tendency was observed in the multicellular organism, the viper (formed of about 4,000 cells). In other words, despite the fact that the viper is living at atmospheric pressure (invaded by water) and is considered to have pressure resistance, when subjected to high-pressure treatment using water as the pressurized medium, 100M
Although it can withstand a pressure of about Pa, if it is treated at a higher pressure, the survival rate after 1 hour (SR = Survival rate) becomes 0,
The same result was obtained when fluorocarbon was used as the pressurized medium and a similar high pressure treatment was applied to the active viper.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preserving an organism which can survive exposure to a high hydrostatic pressure of 600 MPa, which is impossible for metazoans to survive. To provide.

[0006]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
This is achieved by a method for preserving multicellular organisms in which the dried organism is subjected to high pressure treatment in an inert medium.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Generally, multicellular organisms are used as organisms. A multicellular organism is an organism having two or more cells, and includes rotifers and the like in addition to the above-mentioned caterpillars.
These multicellular organisms are dried in a dry state, preferably not in a rapidly dry state, but dried in a slow state, for example, in a tan state (a state in which the body is shrunk and stationary) or a barrel state (in a state in which the body is round) Used after The formation of such a tan state or a barrel state is specifically performed by drying on a piece of paper under room temperature conditions.

[0008] The dried organism, preferably a multicellular organism, is immersed in an inert medium, preferably a fluorocarbon liquid, and subjected to high pressure treatment by exposure to a high hydrostatic pressure environment. When water is used in place of the fluorocarbon liquid and high-pressure processing is performed on an active substance, it can withstand a pressure of about 100 MPa, but with higher pressure processing, all individuals stretch and die. It can be said that this shows a result similar to that of general high-pressure insecticidal. Even when a fluorocarbon liquid is used, if the active substance is subjected to high-pressure treatment as it is, the same result as that of water can be obtained.

[0009] From these results, high-pressure treatment of organisms,
It can be seen that it must be dried and then exposed in a fluorocarbon liquid under a high hydrostatic environment. Preferred fluorocarbon liquids as inert media include fluorinated hydrocarbons, typically linear or cyclic saturated or unsaturated perfluorocarbons,
In practice, commercially available products are used as they are.

The high-pressure treatment is performed by exposing to a high hydrostatic pressure environment, and the degree of pressurization is about 100 to 800 MPa,
Preferably it is about 200 to 600 MPa. Where, for example, 600MP
At the pressure of a, it is a region where metazoan animals were not considered viable conventionally.

After the high-pressure treatment, immersion in water or the like is performed to remove the attached fluorocarbon liquid.

[0012]

According to the present invention, an organism that can survive in an anhydrous state such as a beetle is exposed to an arid environment, and all water, that is, protein,
Non-reducing sugars are used as chemicals to replace the bound water in order to maintain the structural integrity of the tissue if the bound water required to maintain biomacromolecules such as cell membrane lipids and nucleic acids is lost. It has been reported that some trehalose is required. The hydroxyl group of trehalose forms a hydrogen bond with biomolecules to form DP of proteins and cell membrane lipids.
Stabilizes PC (connective tissue).

[0013] The Aphid Ador, which belongs to the true aphid
In the case of ybiotus coronifer, it has been confirmed that the weight of trehaas relative to its dry weight increases as the processing time increases at a slow drying rate.

In order to maintain the systematic integrity of biomolecules such as cell membrane phospholipids and proteins in a high-pressure environment, it is the same as the stabilization that trehalose gives to biomolecules of an organism so that it can survive in an anhydrous state. It is considered that stabilization, that is, hydrogen bonding with biomolecules is necessary.However, despite the fact that fluorocarbon which is an inert liquid is used in the present invention, it is not a rapidly dry state, but about 1 to 24 hours. Or if you do a gentle drying process over it,
The amount of trehalose based on the dry weight has also been increased, allowing nearly 100% survival under high-pressure treatment conditions of 600 MPa.

As described above, in the method of the present invention, a relatively high pressurizing speed and a high hydrostatic pressure (600 MPa is a depth of 100
It withstands the extreme physical load of a dwelling state at a pressure of six times the depth of the sea floor (00 m) and decompression at a relatively high speed, enabling the survival of organisms, preferably multicellular organisms. This indicates that it can be effectively used for preserving living tissue of an organism (organ preservation).

[0016]

Next, the present invention will be described by way of examples.

Example 1 A beetle belonging to Echiniscidae, which belonged to the moss under the guard in front of JR Sakuragicho Station in front of JR Sakuragicho Station, was collected on a piece of paper for a day after being collected in order to make it into a barrel state before applying pressure. And let it dry thoroughly. Fluorocarbon liquid is used as a medium for applying pressure while maintaining the dried state of the viper
(Sumitomo 3M product Fluorinert FC77; C ₁₀ F ₇ ) was used.

6 ml of this fluorocarbon solution
And sealed, and then housed in a high-pressure test capsule (R-7K-3-10 manufactured by Yamamoto Hydraulic Industry Co., Ltd.). Exposure to high hydrostatic pressure was carried out in an environment with an outside temperature of 21 ° C and a capsule temperature of 25 ° C. First, pressurize at a pressure of 40MPa / sec, hold at 600MPa for 20 minutes, then about 100MPa
The pressure was reduced at a pressure reduction rate of / sec. After returning to atmospheric pressure, the beetle to which the fluorocarbon liquid was attached was immersed in water and removed. A few minutes later, it was confirmed by an optical microscope (× 40) that 23 of the 30 insects were active extending the body from the barrel state, and the survival rate indicated by the survival rate (SR value) of the active individual was , 76.7%.

Example 2 In Example 1, instead of the viper beetle belonging to the family Aphididae, instead of the viper beetle belonging to the Euridigrada family, the viper beetle Ma belongs to the family Euridigrada.
Using crobiotus occidentalis, it was placed on paper before applying pressure, dried sufficiently in a room (approximately 60-70% RH) to a tan state, and pressurized at various pressures.

After taking out the capsule from the high-pressure test capsule and immersing the stink bug with the fluorocarbon solution in water to remove the stuck solution, the sting bug that shifted from the tan state to the active state after several minutes to one hour was confirmed.

Active population at 1 hour and 10 days later
When the (number of activities) and the survival rate (SR) were calculated, the results shown in the following table were obtained. Table Pressure 1 hour before and 10 days after pressurization (MPa) Number of individuals Activity SR (%) Number of individuals activity SR (%) 100 20 20 100 9 7 78 200 20 18 90 11 4 36 300 20 19 95 18 6 33 400 19 17 89 5 1 20 500 20 16 84 12 4 33 600 20 19 95 8 1 13 Note) Number of individuals after 10 days: Number of individuals living 10 days after decompression

Example 3 In Example 1, when the beetle Echicus japanicus belonging to Heterotardigrada was used in a tan state, the survival rate SR at 600 MPa was 80% (16/20).

COMPARATIVE EXAMPLE 1 In Example 2, the viviparous beetles belonging to the family Aphididae were not put into a tan state, but were subjected to high pressure treatment in an active state. But 200,300,
At 400, 500 or 600 MPa, the values were all 0% (0/20).

COMPARATIVE EXAMPLE 2 In Example 2, a viridifer belonging to the family Aphididae is not put into a tan state, and when high pressure treatment is performed using water instead of a fluorocarbon solution, the survival rate SR is 91% at a pressure of 100 MPa.
(20/22), but 0% (0/20) at 200, 300, 400, 500 or 600 MPa.

[Procedure amendment]

[Submission date] July 7, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 7

[Correction method] Change

[Correction contents]

Claims (7)

[Claims] 1. A method for preserving an organism, which comprises subjecting a dried organism to a high-pressure treatment in an inert medium. 2. The method according to claim 1, wherein the dried state is a tan state. 3. The method according to claim 1, wherein the dried state is a barrel state. 4. The method according to claim 1, wherein the organism is a multicellular organism. 5. The method according to claim 1, wherein the inert medium is a fluorocarbon medium. 6. The method according to claim 1, wherein the high-pressure treatment is performed by high hydrostatic pressure. 7. The method for preserving an organism according to claim 1, wherein the organism in a dry normal state is a living tissue of the organism.