CN112980692B - Preparation and use method of quality control strain quantitative pellet - Google Patents
Preparation and use method of quality control strain quantitative pellet Download PDFInfo
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Abstract
The invention relates to the technical field of preparation and use of microbial freeze-dried balls, in particular to a preparation and use method of quality control strain quantitative balls. The preparation method comprises the following steps: (1) selecting single colony of the quality control strain into a liquid culture medium, and culturing to logarithmic phase to obtain a bacterial liquid; (2) taking a quantitative bacterial liquid into a freeze-dried strain protective agent, and diluting with the protective agent to obtain a protective agent bacterial liquid; (3) taking a quantitative protective agent bacterial liquid, freezing the bacterial liquid into balls by using liquid nitrogen, and then pre-freezing the balls; (4) carrying out vacuum freeze drying on the pre-frozen pellets obtained in the step (3) to obtain dried pellets; (5) and (6) subpackaging. The using method comprises the following steps: adding 1ml of sterile normal saline into a penicillin bottle filled with freeze-dried bacteria balls, fully dissolving and uniformly mixing to obtain a bacteria liquid containing a certain bacteria number, and directly using the bacteria liquid in a subsequent experiment. The quantitative pellet prepared by the invention has the beneficial effects of capability of meeting the use requirement of a quality control sample, simple preparation method and convenience in use.
Description
Technical Field
The invention relates to the technical field of preparation and use of microbial freeze-dried balls, in particular to a preparation and use method of quality control strain quantitative balls.
Background
Laboratory data reliability and validity are the life lines for inspection work and scientific research.
The microbial standard strain is more and more emphasized to carry out a verification methodology verification test, and after the microbial verification method applicability test is carried out according to the requirements of Chinese pharmacopoeia, related requirements are also put forward in the health-care food and cosmetic risk monitoring work. The method utilizes standard strains as reference standard substances to control the microbial testing process of medicines, health-care foods and cosmetics, is an important link for ensuring the scientific and accurate testing result, and has clear requirements on Chinese pharmacopoeia, health-care food and cosmetic risk monitoring workbooks and the like.
The applicability test of the microbial detection methodology needs to use a standard strain containing a certain number of bacteria, and the traditional gradient dilution method is complex to operate and has large workload. The microorganism quantitative quality control strain is an important tool for examining the accuracy and reliability of laboratory test results, and the quantitative quality control strain suitable for the microorganism detection of food and medicines is a bottleneck for restricting the microorganism test standardization.
Disclosure of Invention
The invention provides a preparation and application method of a quality control strain quantitative pellet, and solves the technical problems of complicated operation and large workload of a bacterial liquid gradient dilution method in the prior art.
The invention is realized by the following steps: the method comprises the following steps:
(1) selecting single colony of the quality control strain to a liquid culture medium, and culturing to logarithmic phase to obtain a bacterial liquid;
(2) taking a quantitative bacterial liquid into a freeze-dried strain protective agent, and diluting with the protective agent to obtain a protective agent bacterial liquid;
(3) taking a quantitative protective agent bacterial liquid, forming into balls by using liquid nitrogen, and pre-freezing;
(4) carrying out vacuum freeze drying on the pre-frozen pellets obtained in the step (3) to obtain dried pellets;
(5) and (6) subpackaging.
As a preferred embodiment, in the step (2), the lyophilized strain protective agent comprises the following components in parts by mass: 10% of protein, 5% of water-soluble sugar, 10% of freeze-drying shaping agent and the balance of water.
As a preferable embodiment, the lyophilized strain protective agent is prepared by dissolving protein, water-soluble sugar and lyophilized shaping agent, autoclaving at 115 deg.C for 20 min, and cooling.
As a preferred embodiment, the protein is at least one of human serum albumin, calf serum, skimmed milk powder; the water-soluble sugar is at least one of sucrose, glucose, trehalose, lactose and galactose; the freeze-drying shaping agent is at least one of polyethylene glycol 4000, polyethylene glycol 8000, polyethylene glycol 20000, mannitol and polyvinylpyrrolidone.
As a preferred embodiment, in step (3), use liquid nitrogen freezing bobble device in the liquid nitrogen freezing process, liquid nitrogen freezing bobble device includes heat preservation container, application of sample needle and freezing perforated plate, the liquid nitrogen is held in the heat preservation container, freezing perforated plate is arranged in inside the heat preservation container, freezing perforated plate is equipped with the several space bar, the space bar vertically and horizontally alternately forms the freezing space of several, and the freezing hole is established to every freezing space bottom, the application of sample needle is arranged in the heat preservation container top, application of sample needle bottom distance liquid nitrogen liquid level 5-8cm, to freezing hole drip liquid fungus liquid, treat all freezing equal one drop fungus liquid in space, after the fungus liquid freezes into the bobble, repeat again to the operation of freezing space drip fungus liquid.
In a preferred embodiment, in the step (2), 50ul of the bacterial liquid is sucked.
In a preferred embodiment, in the step (2), the bacterial liquid is diluted by 100 times.
As a preferred embodiment, in the step (3), the prefreezing condition is-35 ℃ for 2 hours.
As a preferred embodiment, in the step (4), the vacuum freeze-drying parameter: drying for 6-7h at-35 to-30 ℃; secondary drying for 2-3h at 10-25 ℃.
A method for using quality control strain quantitative pellets comprises the steps of adding 1ml of sterile normal saline into a penicillin bottle filled with freeze-dried pellets, fully dissolving and uniformly mixing to obtain bacterial liquid containing a certain number of bacteria, and directly using the bacterial liquid in subsequent experiments.
The invention has the beneficial effects that: the protein is dissolved in water to form a colloidal solution, the molecular diameter is 1-2nm, the colloidal solution is wrapped on the outer side of the thallus to form a protein membrane, the cell is protected, freeze-drying enzymes are fixed, the leakage of intracellular substances caused by the damage of cell wall protein is prevented, and the damage of microbial cells caused by freeze drying can be reduced. The water soluble sugar, especially trehalose has special hydration, can stabilize cell membrane and protein structure, resist stress, protect thallus, and reduce damage in freeze drying process. The selected frozen plastic agent has no toxicity, no stimulation, good hygroscopicity, lubricity and cohesiveness, and is beneficial to the forming of frozen pellets and the stability of the shape after drying. The thickness of the needle head of the sample application needle and the height of the needle head from liquid nitrogen are controlled to be 5-8cm, the bacterial liquid is stably dripped, and liquid cracking is effectively avoided. Freezing hole is equipped with to freeze the freezing space bottom of perforated plate, and this freezing hole is the perforation or freezing space bottom is the fretwork form, and the point sample needle drips into the fungus drop to every freezing space one by one, keeps apart the bobble that does not solidify completely, avoids liquid nitrogen freezing in-process, and the bobble glues the phenomenon of piling up. The preparation method provided by the invention has the advantages of simple process and low cost, and the obtained quality control strain quantitative pellet has the advantages of complete appearance, controllable quantity, uniformity, good stability and good redissolution property, can be directly used without revival, can directly obtain the quality control strain pellet containing the specified number of bacteria, is more convenient to use and has wide application prospect.
Detailed Description
A preparation method of quantitative pellets of quality control strains comprises the following steps:
(1) selecting the single colony of the quality control strain into a liquid culture medium, and culturing to logarithmic phase to obtain a bacterial liquid;
(2) taking a quantitative bacterial liquid to dilute in a freeze-dried strain protective agent to obtain a protective agent bacterial liquid;
(3) taking a quantitative protective agent bacterial liquid, forming into balls by using liquid nitrogen, and pre-freezing;
(4) carrying out vacuum freeze drying on the pre-frozen pellets obtained in the step (3) to obtain dried pellets;
(5) subpackaging into sterile penicillin bottles, and filling a dry pellet in each sterile penicillin bottle.
In the step (2), each 100ml of the freeze-dried strain protective agent comprises the following components in parts by mass: 10% of protein, 5% of water-soluble sugar, 10% of freeze-drying shaping agent and the balance of water.
The logarithmic growth phase of the single colony culture of the quality control strain is correspondingly adjusted according to different strains, and the logarithmic growth phases of the different strains are different. Escherichia coli ATCC 25922 is used as an example, and cultured for 18-24h under 36 ℃ environment to obtain a bacterial liquid.
Further, the freeze-dried strain protective agent is obtained by fully dissolving all the components, then carrying out high-pressure sterilization at 115 ℃ for 20 minutes, taking out and cooling.
Further, the protein is at least one of human serum albumin, calf serum and skimmed milk powder; the water-soluble sugar is at least one of sucrose, glucose, trehalose, lactose and galactose; the freeze-drying shaping agent is at least one of polyethylene glycol 4000, polyethylene glycol 8000, polyethylene glycol 20000, mannitol, and polyvinylpyrrolidone.
In step (3), use liquid nitrogen freezing bobble device among the liquid nitrogen freezing process, liquid nitrogen freezing bobble device includes the insulated container, point sample needle and freezing perforated plate, the liquid nitrogen is held in the insulated container, freezing perforated plate install in inside the insulated container, the last edge of freezing perforated plate is less than the last edge of heat retainer, freezing perforated plate with the position of insulated container keeps relatively fixed. If the upper edge of the freezing perforated plate is higher than the upper edge of the heat preservation container, the phenomenon that liquid drops are attached to the partition wall is easy to occur. The freezing porous plate can be placed first, and then liquid nitrogen is poured in, so that the problem that the freezing porous plate floats due to too much liquid nitrogen is avoided. The freezing perforated plate is provided with a plurality of partition plates which vertically extend upwards, the partition plates are criss-cross to form a plurality of freezing spaces, the bottom of each freezing space is provided with a freezing hole, the freezing holes are through holes or the bottoms of the freezing spaces are hollow to form a freezing channel, and the freezing holes are used for enabling liquid nitrogen to enter the freezing spaces and enabling the bacterial liquid to be frozen into balls. When freezing space bottom was the fretwork state wherein, the upper and lower surface area in freezing hole equals with the upper and lower surface area in single freezing space, and at this moment, freezing perforated plate is difficult for floating when freezing the hole for perforating. The sample application needle can be a needle head matched with a 5ml syringe. After the sample application needles extract the protective agent bacteria liquid, the bottom ends of the sample application needles are 5-8cm away from the liquid level of liquid nitrogen above the heat preservation container, so that liquid drops are guaranteed to smoothly drip, the bacteria liquid is dripped into each freezing space one by one, after one wave of bacteria liquid is frozen into small balls in a liquid nitrogen environment, the next wave of bacteria liquid is dripped, the actions are repeated, and finally formed frozen small balls sink to the bottom of the heat preservation container through the through holes of the porous plate. The flow speed and the flow of the bacteria liquid can be controlled by a micro peristaltic pump or manually. The heat preservation container can be a vacuum cup, a double-layer heat preservation container or other containers with heat preservation performance.
Further, in the step (2), 50ul of the bacterial liquid is sucked.
Further, in the step (2), the bacterial solution is diluted by 100 times.
Further, in the step (3), the pre-freezing condition is-35 ℃ for 2 h.
Further, in the step (4), the vacuum freeze-drying parameters are as follows: drying for 6-7h at-35 to-30 ℃; secondary drying for 2-3h at 10-25 ℃.
A method for using quality control strain quantitative pellet comprises adding 1ml sterile physiological saline into penicillin bottle containing lyophilized pellet, dissolving completely and mixing. Each penicillin bottle is matched with 1ml of sterile normal saline, further dilution is not needed, the use is convenient, and errors generated in the dilution process are effectively avoided. The penicillin bottle is a storage container for freeze-dried pellets and a dilution container during use, and the dilution container is not required to be additionally arranged, so that the operation is convenient.
Examples
(1) Selecting single colony of Escherichia coli of the quality control strain, and culturing in trypticase soy peptone broth to logarithmic phase, i.e. culturing at 36 ℃ for 18-24h to obtain high-purity bacterial liquid with better activity;
(2) the freeze-dried strain protective agent comprises the following components in parts by mass: 10% of protein, 5% of water-soluble sugar, 10% of freeze-drying plastic agent and the balance of water, wherein each 100ml of freeze-drying strain protective agent comprises 10g of protein, 5g of water-soluble sugar, 10g of freeze-drying plastic agent and the balance of water. After all the components are fully dissolved, sterilizing for 20 minutes at 115 ℃ under high pressure, taking out, and cooling for later use. Wherein, the protein comprises 3.0g of calf serum and 7.0g of skimmed milk powder, and the water-soluble sugar comprises 3.0g of sucrose and 2.0g of trehalose; the freeze-dried plastic forming agent comprises 40003.0 g of polyethylene glycol, 60004.0 g of polyethylene glycol and 80003.0 g of polyethylene glycol.
(3) 50ul of the high-purity bacterial liquid obtained in the step (1) is sucked into the protective agent used by the freeze-dried strain for dilution until the concentration is 10-3The bacterial liquid of (a);
(4) get 10ul of protective agent fungus liquid that step (3) obtained, liquid nitrogen freezing in-process uses liquid nitrogen freezing bobble device, liquid nitrogen freezing bobble device includes the insulated container, point sample needle and freezing perforated plate, hold the liquid nitrogen in the insulated container, freezing perforated plate is arranged in inside the insulated container, freezing perforated plate is equipped with the several space bar, the space bar is criss-cross to form the several freezing space, all is equipped with the perforation or the same through-hole of upper and lower surface area in every freezing space. In this embodiment, the thermos cup is selected for use to the insulated container, and the syringe needle is selected for use to the sampling needle. And pouring liquid nitrogen into the stainless steel thermos cup, wherein no matter how much, the liquid nitrogen flows into each freezing space, and the liquid nitrogen does not need to be poured into each partition one by one directly. The use is more convenient. Pouring liquid nitrogen into a thermos cup, then placing a freezing porous plate sterilized in advance, after the protective agent bacterial liquid is extracted by the sample application needle, dripping the bacterial liquid into a freezing space on the freezing porous plate one by one above the heat preservation container, dripping the bacterial liquid to the bottom of the heat preservation container through the perforation, freezing the bacterial liquid by the liquid nitrogen to form a freezing ball, then pre-freezing at the temperature of-35 ℃ for 2 hours;
(5) and (3) carrying out vacuum freeze drying on the pre-frozen small balls to obtain dried small balls, wherein the vacuum freeze drying parameters are as follows: drying for 6-7h at-35 to-30 ℃; secondary drying at 10-25 ℃ for 2-3 h;
(6) and (5) subpackaging the dried pellets obtained in the step (5) into sterile penicillin bottles, vacuumizing, plugging and capping to form the product. Each sterile vial is filled with a dry pellet.
(7) Is matched with 1ml of sterile normal saline. When in use, the matched physiological saline is directly sucked into a penicillin bottle filled with the freeze-dried bacterium balls, and the penicillin bottle is fully dissolved and uniformly mixed. Further dilution is not needed, the use is convenient, and errors generated in the dilution process are effectively avoided.
Test verification
Counting culture proves that the quality control sample stored at low temperature has better uniformity and stability.
A uniformity verification
Optionally, 5 batches of the product obtained in example 1 were randomly drawn into 10 bottles, 1ml of sterile physiological saline was added to each bottle, the mixture was dissolved and mixed, and the bacterial suspension obtained from each bottle was counted by pouring with TSA medium, and the results are shown in Table 1:
TABLE 1 bacterial suspension count results (unit: CFU)
As can be seen from Table 1, the number of bacteria contained in 5 batches of samples is on average 73, 58, 86, 45 and 66 respectively, and the number of bacteria in 10 bottles of samples taken from each batch is in the range of 10-100 of the required number of bacteria, so that the uniformity of the quantitative globules of the strain meets the requirement.
B detection of stability of bacterial count at different storage temperatures
3 batches of quality control samples obtained in example 1 were stored in refrigerators at 4 ℃, -20 ℃ and-80 ℃ respectively, taken out at regular intervals, and tested for changes in the number of bacteria, the results of which are shown in Table 2.
TABLE 2 number of bacteria contained in the pellets of the quantitative strains stored at different temperatures for different periods of time
As can be seen from Table 2, the bacterial strains preserved at 4 ℃ are quantitatively pellets, and have no obvious change when being stored for 30 days, and the number of contained bacteria is obviously reduced to half in 60 days along with the time extension; the pellets placed at the temperature of-20 ℃ and-80 ℃ have no obvious change of the bacterial count within one year of placement, and show better stability.
The invention has the beneficial effects that: the protein is dissolved in water to form a colloidal solution, the molecular diameter is 1-2nm, the colloidal solution is wrapped on the outer side of the thallus to form a protein membrane, the cell is protected, freeze-drying enzymes are fixed, the leakage of intracellular substances caused by the damage of cell wall protein is prevented, and the damage of microbial cells caused by freeze drying can be reduced. The water soluble sugar, especially trehalose has special hydration, can stabilize cell membrane and protein structure, resist stress, protect thallus and reduce damage in freeze drying process. The selected frozen plastic agent has no toxicity, no stimulation, good hygroscopicity, lubricity and cohesiveness, and is beneficial to the forming of frozen pellets and the stability of the shape after drying. The thickness of control application of sample needle syringe needle and syringe needle are apart from the height of liquid nitrogen, and the fungus liquid is steadily dripped into, effectively avoids liquid to burst apart. By means of the freezing perforated plate, the sample application needle drops the bacteria one by one into each freezing space, small balls which are not completely solidified are isolated, and the phenomenon that the small balls are adhered to form a pile in the liquid nitrogen freezing process is avoided. The preparation method provided by the invention has the advantages of simple process and low cost, the obtained quality control strain quantitative pellets have complete appearance, controllable quantity, good uniformity and stability and good redissolution property, can be directly used without revival, can directly obtain the quality control strain pellets containing the specified number of bacteria, and has more convenient use and wide application prospect.
Claims (6)
1. A preparation method of quantitative pellets of quality control strains is characterized by comprising the following steps:
(1) selecting single colony of the quality control strain into a liquid culture medium, and culturing to logarithmic phase to obtain a bacterial liquid;
(2) taking a quantitative bacterial liquid into a freeze-dried strain protective agent, and diluting with the protective agent to obtain a protective agent bacterial liquid;
(3) taking a quantitative protective agent bacterial liquid, freezing the bacterial liquid into balls by using liquid nitrogen, and then pre-freezing the balls;
(4) carrying out vacuum freeze drying on the pre-frozen pellets obtained in the step (3) to obtain dried pellets;
(5) subpackaging into penicillin bottles;
in the step (2), each 100ml of the freeze-dried strain protective agent comprises the following components in parts by mass: 10% of protein, 5% of water-soluble sugar, 10% of freeze-drying shaping agent and the balance of water; the freeze-dried strain protective agent is obtained by dissolving protein, water-soluble sugar and freeze-dried shaping agent completely, sterilizing at 115 ℃ for 20 minutes under high pressure and cooling;
in step (3), use liquid nitrogen freezing bobble device in the liquid nitrogen freezing process, liquid nitrogen freezing bobble device includes the insulated container, application of sample needle and freezing perforated plate, the liquid nitrogen is held to the insulated container content, freezing perforated plate is arranged in the insulated container, freezing perforated plate is equipped with the several space bar, the space bar vertically and horizontally alternately forms the freezing space of several, and the freezing hole is established to every freezing space bottom, the application of sample needle is arranged in the insulated container top, application of sample needle bottom distance liquid nitrogen liquid level 5-8cm, to freezing interval space dropwise add fungus liquid, treat all freezing space all drips one drop fungus liquid, after the fungus liquid design becomes the bobble, repeat again to the operation of freezing space dropwise add fungus liquid.
2. The method for preparing quantitative beads for quality control strains according to claim 1, wherein the protein is at least one of human serum albumin, calf serum and skimmed milk powder; the water-soluble sugar is at least one of sucrose, glucose, trehalose, lactose and galactose; the freeze-drying shaping agent is at least one of polyethylene glycol 4000, polyethylene glycol 8000, polyethylene glycol 20000, mannitol and polyvinylpyrrolidone.
3. The method for preparing quantitative beads of quality control strains according to claim 1, wherein 50ul of bacterial liquid is aspirated in the step (2).
4. The method for preparing quantitative beads of quality control strains according to claim 3, wherein in the step (2), the bacterial liquid is diluted by 100 times.
5. The method for preparing quantitative beads for quality control strains according to claim 4, wherein the pre-freezing condition in the step (3) is-35 ℃ for 2 h.
6. The method for preparing quantitative beads for quality control strains as claimed in claim 5, wherein in the step (4), the vacuum freeze-drying parameters are as follows: drying for 6-7h at-35 to-30 ℃; secondary drying for 2-3h at 10-25 ℃.
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