CN111363671A - Intestinal anaerobic microorganism culture bottle and preparation method thereof - Google Patents

Abstract

The invention discloses an intestinal anaerobic microorganism culture bottle and a preparation method thereof, wherein the culture bottle comprises a culture bottle which is sealed and packaged and a culture solution which is filled in the culture bottle; sequentially filling a basic culture solution obtained by mixing a basic culture solution, an L-cysteine saline solution and a resazurin aqueous solution in proportion into a culture bottle, and sealing and packaging; autoclaving the sealed culture flask; finally, mixing the 1640 culture medium solution, the BI serum, the vitamin K and the hemin in proportion to obtain an active additive mixed solution, injecting the active additive mixed solution into a culture bottle which is sterilized under high pressure and cooled to room temperature to complete preparation; the anaerobic microorganism culture bottle adopts the culture bottle body and the packaging part, and all components for preparing the culture solution are commercially available products, so that the raw materials are easy to obtain, the threshold and the cost of anaerobic culture are reduced, the operations of anaerobic culture, storage and transportation are simpler, the preparation method is simple and easy to implement, the requirements of large-scale production are met, and the use requirements of hospitals and scientific research institutions are met.

Description

Intestinal anaerobic microorganism culture bottle and preparation method thereof

Technical Field

The invention relates to the technical field of microbial culture, in particular to an intestinal anaerobic microbial culture bottle and a preparation method thereof.

Background

At present, microorganisms existing in most environments cannot be cultured, and the culturable microorganisms account for about 1% of the total amount, so the culturable microorganisms cannot be cultured, and besides low abundance, the culture conditions are not optimized enough, including temperature, pH, oxygen utilization rate, nutrition and the like. Technically, the temperature and the pH can be adjusted, and the anaerobic treatment and nutrition are generally difficult; therefore, the emphasis of the in vitro culture process of anaerobic microorganisms is on the simulation of the anaerobic environment and its nutrient environment.

At present, in the anaerobic culture technology commonly used in research, an anaerobic box, an anaerobic bag and an anaerobic tank are used, the operation is complex, the cost is higher, a solid culture medium is coated with a plate, and can be cultured in the anaerobic box, the anaerobic bag and the anaerobic tank, but when the liquid culture medium is used for proliferation, the open space is inconvenient to operate, the problem of anaerobic environment is solved by researchers before, but the nutrition in an anaerobic bottle is insufficient, and the growth of microorganisms is not good. The anaerobic culture bottle developed by the invention is added with active nutrient substances on the basis of oxygen removal, ensures the growth of microorganisms, is convenient to operate, has low cost and is suitable for the specific implementation of broad technicians for microorganism research.

In addition, the preservation of anaerobic bacteria has been a big problem, if it is sure to contact with oxygen according to the normal bacteria freezing method, unless it is operated in the anaerobic chamber, but the anaerobic chamber is troublesome to prepare anaerobic environment, inconvenient to operate with rubber gloves, and additionally needs to prepare anaerobic freezing solution. In addition, the transportation of anaerobic bacteria is realized by preparing bacteria into dry powder under specific conditions, then filling the dry powder into an ampoule bottle for oxygen removal and sealing, and reactivating the dry powder after the dry powder reaches the destination. For long-distance transportation, the dry powder is suitable in treatment mode, but for short-distance transportation, the operation cost performance is not high, and from one laboratory to another, the transportation of anaerobic bacteria only depends on the transfer of anaerobic bags and anaerobic tanks, so that the operation is not convenient.

Disclosure of Invention

The invention aims to provide an anaerobic microorganism culture bottle which is convenient for culturing, storing and transporting intestinal anaerobic microorganisms.

The invention also aims to provide a preparation method of the intestinal anaerobic microorganism culture bottle

Therefore, the technical scheme of the invention is as follows:

an intestinal anaerobic microorganism culture bottle comprises a culture bottle which is sealed and packaged and a culture solution filled in the culture bottle; the culture solution is prepared from 100 parts by volume of a basic culture medium aqueous solution, 4 parts of 2.5 wt.% L-cysteine aqueous solution and 0.1 part of 0.1 wt.% resazurin aqueous solution which are mixed together, and an active additive mixed solution of which the volume parts are 4% of that of the basic culture solution; the active additive mixed solution is prepared by mixing 10 parts by volume of 1640 culture solution, 1 part by volume of BI serum, 1 part by volume of vitamin K and 1 part by volume of hemin.

The intestinal anaerobic microorganism culture bottle is sealed, completely meets the anaerobic environment, and the culture solution is used for providing a proper nutrient environment for anaerobic microorganisms; the L-cysteine in the culture solution has strong reducibility, and is subjected to oxidation reaction with oxygen in a sealed culture bottle at high temperature to generate L-cystine, and amino acid meeting the requirement of microbial culture is further provided while oxygen is removed; the resazurin is an oxygen indicator which is used for indicating the oxygen content in the bottle, and pink or light blue when oxygen exists, so that whether the internal state of the culture bottle meets the requirement of an anaerobic culture environment or not can be observed conveniently; the active additive mixed liquor is used for supplementing nutrition to the basic culture solution, and experiments prove that the active additive can meet the growth of most anaerobic microorganisms.

Preferably, the filling volume of the culture solution is 90-95% of the volume of the culture bottle.

Preferably, the basic culture medium is a powdered RAM basic culture medium, a CDC basic culture medium, an RCM basic culture medium or an MRS basic culture medium.

Preferably, the culture bottle comprises a penicillin bottle, a rubber plug inserted at the mouth of the penicillin bottle, a hollow aluminum cover packaged at the end parts of the rubber plug and the mouth of the penicillin bottle, and a top cover covered on the hollow aluminum cover. The penicillin bottle is made of borosilicate glass, has a rigid structure, resists high temperature and high pressure, is used for storing liquid culture medium, and is suitable for sterilization operation and observation of the growth state of bacteria; the penicillin bottle is sealed by the rubber plug, so that the culture medium in the penicillin bottle can be effectively prevented from flowing out, the airtightness is good, the penicillin bottle is convenient to insert by using an injector for carrying out operations such as passage, culture and the like of anaerobic bacteria, and the anaerobic environment in the penicillin bottle is ensured; the hollow aluminum cover plays a role in reinforcing the rubber plug on one hand, and prevents the rubber plug from popping out under high temperature and high pressure, and the hollow structure can facilitate the culture operation of anaerobic bacteria later.

Preferably, the penicillin bottle is a borosilicate glass bottle, and the total amount of microorganisms in the bottle can be measured by measuring the turbidity of liquid in the bottle without opening a cover in the process of culturing anaerobic microorganisms; the top cap adopts the polytetrafluoroethylene top cap, non-deformable under the high temperature high pressure, effectively prevents that the culture flask from storing and the bottleneck pollutes during the transportation.

A preparation method of an anaerobic microorganism culture bottle comprises the following specific steps:

s1, dissolving the powdery basic culture medium in sterile water according to a proportion to prepare a basic culture medium solution; dissolving powdered resazurin in sterile water to prepare a 0.1 wt.% resazurin aqueous solution; dissolving powdered L-cysteine salt in sterile water to obtain 2.5 wt.% of L-cysteine salt aqueous solution;

s2, respectively filling basic culture solution obtained by mixing the basic culture solution, the L-cysteine aqueous solution and the resazurin aqueous solution according to the volume ratio of 100:4:0.1 into a plurality of culture bottles, and sealing and packaging the culture bottles after filling; wherein the filling volume is 90-95% of the volume of the culture bottle;

s3, autoclaving the culture bottle filled and sealed in the step S2;

s4, mixing 1640 culture medium liquid, BI serum, vitamin K and hemin according to a volume ratio of 10:1:1:1 to prepare an active additive mixed solution, and after the temperature of the culture bottle subjected to the high-pressure sterilization in the step S4 is reduced to room temperature, injecting the active additive mixed solution into the culture bottle through an injector in a sterile operation table; the volume ratio of the injection amount of the active additive mixed solution to the basic culture solution is 1: 25.

Compared with the prior art, the anaerobic microorganism culture bottle is assembled by commercial products by adopting the culture bottle, is convenient to purchase and use, the culture solution filled in the culture bottle is prepared by the commercial products, and the preparation and preparation method is simple and easy; this application has realized reducing anaerobic culture's threshold and cost through assembling the anaerobic microorganism blake bottle that obtains with the two, lets the anaerobic culture, stores and the operation of transportation all more simple, and accords with large-scale production's requirement, can satisfy hospital clinical laboratory and relevant scientific research unit's user demand, has greatly made things convenient for scientific research personnel's work, has fine commercial popularization prospect.

Drawings

FIG. 1 is a schematic view of a flask structure of an intestinal anaerobic microorganism flask according to the present invention;

fig. 2 is a difference statistical chart prepared from MCF values of the test results of example 2 according to the present invention.

Detailed Description

The invention will be further described with reference to the following figures and specific examples, which are not intended to limit the invention in any way.

Example 1

The method for preparing the plurality of intestinal anaerobic microorganism culture bottles comprises the following specific steps:

a preparation method of an intestinal anaerobic microorganism culture bottle comprises the following specific steps:

s1, dissolving a powdered RAM basal medium (Haibo) in sterile water according to a ratio of 71:1000 to prepare a basal medium solution; dissolving powdered resazurin in sterile water to prepare a 0.1 wt.% resazurin aqueous solution; dissolving powdered L-cysteine salt in sterile water to obtain 2.5 wt.% of L-cysteine salt aqueous solution;

s2, respectively filling mixed liquor obtained by mixing 100mL of basic culture medium solution, 4mL of L-cysteine saline solution and 0.1mL of resazurin aqueous solution into 10 penicillin bottles with the specification of 10mL, and hermetically packaging the penicillin bottles by inserting rubber plugs into the mouths of the penicillin bottles, packaging hollow aluminum covers at the ends of the rubber plugs and the mouths of the penicillin bottles and covering polytetrafluoroethylene top covers on the hollow aluminum covers; wherein the filling volume of the mixed solution is 10mL, and the bottle neck part of the culture bottle is left empty at the moment;

s3, placing the 10 culture bottles which are filled, sealed and packaged in the step S2 into an autoclave with the model of ZealWay, setting the temperature to 121 ℃, and carrying out autoclaving on the 10 culture bottles for 20 min;

s4, mixing 10mL of 1640 culture solution, 1mL of BI serum, 1mL of vitamin K and 1mL of hemin to prepare active additive mixed solution; after the temperature of the culture bottles sterilized by the high pressure in the step S4 is reduced to room temperature, 0.4mL of active additive mixed solution is injected into each culture bottle through a 1mL syringe in an aseptic operation platform;

s5, repeating the steps S1-S4 until a sufficient number of anaerobic microorganism culture bottles are prepared.

Comparative example 1

Several anaerobic microorganism culture flasks to which the active additive mixture solution was not added were prepared in the same manner as in example 1, i.e., by repeating steps S1 to S3 in example 1.

Example 2

The 40 strains in the following table 1 were anaerobically cultured using two intestinal anaerobic microorganism flasks prepared in example 1 and comparative example 1. The specific culture method comprises the following steps: the freeze-dried powders of 40 kinds of intestinal anaerobic microorganisms in table 1 are respectively and rapidly dissolved in 1mL of PBS according to the serial number, 500 microliters of dissolved bacteria liquid is sucked by a 1mL syringe, and the dissolved bacteria liquid is injected into anaerobic microorganism culture bottles marked with the same serial number and prepared in example 1 and comparative example 1; culturing all culture bottles in a 37 ℃ incubator for 24h, and respectively measuring the turbidity of the cultured microorganisms, wherein the turbidity unit is MCF, 1MCF is 3 x 10 < Lambda > 8CFU/mL, and CFU is the unit of the strain; all flasks had a turbidity of 0MCF before culture.

Table 1:

from the test results in table 1 above, it can be seen that the culture flask manufactured by the method of comparative example 1 can culture the strain, but the culture flask manufactured by the method of example 1 has a significantly better culture effect on the strain. FIG. 2 is a graph showing a difference statistic chart based on the MCF values of the test results in Table 1. As can be seen from FIG. 2, the culture flask manufactured by the method of example 1 has a remarkable effect of culturing the bacterial species, compared with the culture flask manufactured by the method of comparative example 1, and the p value between the two is 0.0154 and less than 0.05, which is statistically significant.

Example 3

Taking out 40 culture bottles prepared in example 1 from the samples cultured in example 2, placing the culture bottles in a refrigerator at 4 ℃, standing for 30 days, taking out, uniformly mixing, standing for 1 hour, and recovering to room temperature; 500. mu.l of the liquid was aspirated from 40 cultures by syringes, respectively, and injected into 40 new anaerobic microorganism culture flasks prepared using example 1, and the 40 new culture flasks were placed in a 37 ℃ incubator for 24 hours.

After the culture was completed, the turbidity of the cultured microorganisms in 40 new culture flasks was measured. The test result shows that: except for bacteria PAC002443, four strains of bacteria including Lachnospiraceae PAC001085_ s, Lachnospiraceae ePAC001459_ s and Lachnospiraceae PAC001772_ s are not grown, other 36 bacteria are activated, the activation rate reaches 90%, and the anaerobic microorganism culture bottle can meet the requirement of short-term storage of anaerobic microorganisms.

In addition, because the liquid is in under the environment of sealed encapsulation all the time in the bottle of the anaerobic microorganism blake bottle of intestinal of this application, does not receive external environment's influence basically, and small in size, consequently also can satisfy the requirement of portable carrying or transportation, can directly be carried in the pocket like other scientific research units need certain anaerobic strain, or arrange closely under the cold-stored condition and transport scientific research institute or hospital.

In conclusion, the intestinal anaerobic microorganism culture bottle disclosed by the application realizes the purpose that the operations of anaerobic culture, storage and transportation are simpler, meets the requirement of large-scale production, and has good market popularization prospect.

Claims (6)

1. An intestinal anaerobic microorganism culture bottle is characterized by comprising a culture bottle which is sealed and packaged and a culture solution filled in the culture bottle; the culture solution is prepared from 100 parts by volume of a basic culture medium aqueous solution, 4 parts of 2.5 wt.% L-cysteine aqueous solution and 0.1 part of 0.1 wt.% resazurin aqueous solution which are mixed together, and an active additive mixed solution of which the volume parts are 4% of that of the basic culture solution; the active additive mixed solution is prepared by mixing 10 parts by volume of 1640 culture solution, 1 part by volume of BI serum, 1 part by volume of vitamin K and 1 part by volume of hemin.

2. The intestinal anaerobic microorganism culture bottle according to claim 1, wherein the filling volume of the culture solution is 90-95% of the volume of the culture bottle.

3. The intestinal anaerobic microorganism culture flask according to claim 1, wherein the basal medium is a powdered RAM basal medium, a CDC basal medium, an RCM basal medium, or an MRS basal medium.

4. The intestinal anaerobic microorganism culture bottle according to claim 1, which comprises a penicillin bottle, a rubber plug inserted into the mouth of the penicillin bottle, a hollow aluminum cap sealed at the end of the rubber plug and the mouth of the penicillin bottle, and a top cap covering the hollow aluminum cap.

5. The intestinal anaerobic microorganism culture bottle according to claim 1, wherein the penicillin bottle is a borosilicate glass bottle; the top cover adopts a polytetrafluoroethylene top cover.

6. A preparation method of an intestinal anaerobic microorganism culture bottle is characterized by comprising the following steps:

s1, dissolving the powdery basic culture medium in sterile water according to a proportion to prepare a basic culture medium solution; dissolving powdered resazurin in sterile water to prepare a 0.1 wt.% resazurin aqueous solution; dissolving powdered L-cysteine salt in sterile water to obtain 2.5 wt.% of L-cysteine salt aqueous solution;

s2, mixing the basic culture medium solution, the L-cysteine aqueous solution and the resazurin aqueous solution according to the volume ratio of 1000:40:1 to obtain a basic culture solution, filling the basic culture solution into a plurality of culture bottles respectively, and sealing and packaging the culture bottles after filling; wherein the filling volume is 90-95% of the volume of the culture bottle;

s3, autoclaving the culture bottle filled and sealed in the step S2;

s4, mixing 1640 culture medium liquid, BI serum, vitamin K and hemin according to a volume ratio of 10:1:1:1 to prepare an active additive mixed solution, and after the temperature of the culture bottle subjected to the high-pressure sterilization in the step S4 is reduced to room temperature, injecting the active additive mixed solution into the culture bottle through an injector in a sterile operation table; the volume ratio of the injection amount of the active additive mixed solution to the basic culture solution is 1: 25.

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