CN214620314U

CN214620314U - Preparation facilities of quality control bacterial strain ration bobble

Info

Publication number: CN214620314U
Application number: CN202120496378.0U
Authority: CN
Inventors: 蔡向荣; 许英俊
Original assignee: Qingdao Hope Bio Technology Co ltd
Current assignee: Qingdao Hope Bio Technology Co ltd
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2021-11-05
Anticipated expiration: 2031-03-09

Abstract

The invention relates to the technical field of preparation of microorganism freeze-dried balls, in particular to a preparation device of a quality control strain quantitative small ball, which comprises a heat preservation container, a sample application needle and a freezing porous plate, wherein the sample application needle is arranged above the heat preservation container; freezing perforated plate is equipped with the freezing space of several, and freezing hole all is established to every freezing space bottom, the utility model provides a preparation facilities of matter accuse bacterial strain ration bobble has and can satisfy matter accuse sample operation requirement, and operating method is simple, convenient to use's beneficial effect.

Description

Preparation facilities of quality control bacterial strain ration bobble

Technical Field

The invention relates to the technical field of preparation and use of microorganism freeze-dried balls, in particular to a preparation device of a quality control strain quantitative small ball.

Background

Laboratory data reliability and validity are the life lines of 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 device of quantitative pellets of quality control strains, and solves the technical problems that a method for gradient dilution of a bacterial liquid in the prior art is complex to operate, the workload for preparing the quantitative pellets of the quality control strains is large, and a preparation device which is convenient to use is lacked.

The utility model discloses a realize like this: the device comprises a heat-preservation container, a sample application needle and a freezing porous plate, wherein the sample application needle is arranged above the heat-preservation container, and the freezing porous plate is arranged in the heat-preservation container;

freezing perforated plate is equipped with the freezing space of several, and freezing hole all is established to every freezing space bottom.

As a preferred embodiment, the inner diameter of the freezing hole is smaller than the length of any side of the freezing space.

In a preferred embodiment, the bottom of the freezing space is hollowed to form the freezing hole.

In a preferred embodiment, the perforated freezing plate is provided with a plurality of partition plates, and the partition plates are crossed vertically and horizontally to form a freezing space.

As a preferred embodiment, the spotting needle is connected to a micro peristaltic pump.

In a preferred embodiment, the incubation container contains liquid nitrogen, and the bottom end of the spotting needle is 5-8cm away from the liquid level of the liquid nitrogen.

In a preferred embodiment, the freezing multi-well plate is made of a transparent material.

In a preferred embodiment, the freezing porous plate is square, and the thermal container is cylindrical.

The invention has the beneficial effects that: the heat preservation container is used for containing the liquid nitrogen, so that the boiling and the evaporation of the liquid nitrogen can be effectively slowed down, the liquid nitrogen is exposed in the air, the liquid level can be maintained in a calm state for a period of time, and the subsequent freezing operation is facilitated; the bottom of the freezing space of the freezing perforated plate is provided with the freezing holes, and the perforated plate is placed in liquid nitrogen, so that each space can be quickly filled with the liquid nitrogen without adding the liquid nitrogen one by one; the sample application needle drops the bacteria drops into each freezing space one by one, 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 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. The spotting needle may be controlled by a manual or micro peristaltic pump. The utility model provides a preparation facilities simple structure, easily operation, the quality control bacterial strain ration bobble of gained, the outward appearance is complete, and quantity is controllable, and homogeneous stability is good, and the redissolution is good, need not the revival and can directly use, can realize directly obtaining the quality control bacterial strain bobble that contains appointed bacterial count, and it is more convenient to use, has extensive application prospect.

Drawings

FIG. 1 is a schematic view of a freeze multi-well plate according to an embodiment of the present invention installed in the insulated container;

fig. 2 and 3 are two types of frozen multi-well plates of fig. 1, respectively.

FIG. 4 is a schematic view of the overall structure of the fixing device;

fig. 5 is a schematic structural diagram of a limiting block in a top view state.

In the figure: 1-a heat preservation container; 2-freezing a multi-hole plate; 3-a refrigerated space; 4-freezing the wells; 5-a partition plate; 6-a fixing device; 7-a base; 8-fixing the rod; 9-a connecting rod; 10-a limiting block; 11-a fixed through hole; 12-connecting vias; 13-a spotting needle; 14-a clamping section.

Detailed Description

Referring to the attached

drawings

1, 2 and 3, the device for preparing the quality control strain quantitative pellets comprises a heat preservation container 1, a sample application needle and a freezing porous plate 2, wherein the sample application needle is arranged above the heat preservation container 1, the size of the freezing porous plate 2 is matched with that of the heat preservation container 1, and the freezing porous plate 2 is arranged in the heat preservation container 1. Freezing perforated plate 2 install during in the heat preservation container 1, freezing perforated plate 2's upper edge is less than the upper edge of heat retainer 1, promptly freezing perforated plate 2 body is all located in the heat preservation container 1 has better freezing effect. During use, the positions of the freezing porous plate 2 and the heat preservation container 1 are kept relatively fixed. If the upper edge of the freezing perforated plate 2 is higher than the upper edge of the heat preservation container 1, the phenomenon that liquid drops are attached to the partition wall is easy to occur. The freezing porous plate 2 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. Freezing perforated plate 2 is equipped with several freezing space 3, and freezing hole 4 all is established to every freezing space 3 bottom. The sample application needle 14 can be controlled manually or connected with a micro peristaltic pump in a known connection mode, namely the flow speed and flow of the bacteria liquid are controlled by the micro peristaltic pump or the artificial micro peristaltic pump. In this example, the spotting needle was a 5ml syringe-adapted needle. Liquid nitrogen is contained in the heat preservation container, the distance between the bottom end of the sample application needle 14 and the liquid level of the liquid nitrogen is 5-8cm, and therefore smooth dripping of liquid drops is guaranteed. The print pins 14 are height controlled manually, or by means of a fixture 6. Referring to fig. 4 and 5, the fixing device 6 includes a base 7, a fixing rod 8, a connecting rod 9, a limiting block 10 and a clamping portion 14, the fixing rod 8 is mounted on the base 7, the connecting rod 9 is perpendicularly cross-connected with the fixing rod 8, the limiting block 10 is connected with the connecting rod 9 and the fixing rod 8, and the connecting rod 9 can slide up and down along the fixing rod 8 through the limiting block 10, so that the left and right sliding along the limiting block 10 is realized. Clamping part 14 is installed connecting rod 9 one end, clamping part 14 is used for cliping fixedly point sample needle 13, clamping part 14 includes the centre gripping arc board that two one ends are connected, during two centre gripping arc board clamping state, with point sample needle 13 adaptations. Referring to fig. 5, the limiting block 10 is provided with a fixing through hole 11 and a connecting through hole 12, the fixing through hole 11 is not communicated with the connecting through hole 12, and the fixing through hole 11 is in a vertical state and accommodates the fixing rod 8 to extend through. The connecting through hole 12 is horizontal and receives the connecting rod 9 extending therethrough. Fixing hole 11 with connecting hole 12 all is provided with fastening nut, and two fastening nut install respectively on dead lever 8 and connecting rod 9, install fastening nut on the dead lever 8 is located stopper 10's below is installed fastening nut on the connecting rod 9 is located stopper 10 is right-hand. The fixing through hole and the connecting through hole are internally provided with threads, the internal threads are matched with the fastening nut, and the fastening nut is matched with the internal threads to realize the relative positions of the fixed connecting rod 9 and the fixed rod 8. And dripping bacterial liquid into each freezing space one by one through a sample application needle 13, dripping next wave bacterial liquid after the bacterial liquid of one wave is frozen into small balls in a liquid nitrogen environment, repeating the action, and finally forming the frozen small balls which are sunk to the bottom of the heat-insulating container 1 through the freezing holes of the freezing porous plate.

Referring to fig. 1, a plurality of partition plates 5 are mounted on the perforated freezing plate 2, the partition plates 5 include horizontal partition plates and vertical partition plates, all the partition plates 5 are criss-cross to form a freezing space 3, and a plurality of the freezing spaces 3 are uniformly distributed at the bottom of the perforated freezing plate 2. In the present embodiment, the plurality of freezing spaces 3 are uniform in size. The freezing holes 4 may be provided in two types. Referring to fig. 2, the inner diameter of the freezing hole 4 is smaller than the length of any side of the freezing space 3. Referring to fig. 3, the bottom of the freezing space 3 is hollowed to form the freezing hole 4. The freezing porous plate 2 may be provided with any type of freezing holes 4, and may be provided with a mixed type of freezing holes 4. The freezing hole 4 is used for allowing liquid nitrogen to enter the freezing space 3, so that bacterial liquid dropped by the sample application needle is frozen into balls in the freezing space. When the bottom of the freezing space 3 is in a hollow state, the upper and lower surface areas of the freezing holes 4 are equal to those of the single freezing space 3, and at the moment, the freezing porous plate 2 is not easy to float up when being perforated compared with the freezing holes 4. For the convenience of observation, the freezing porous plate 2 is made of transparent material. In this implementation, freezing perforated plate 2 is square, heat preservation container 1 is the tube-shape, and heat preservation container 1 can select for use the thermos cup, double-deck heat preservation container, or other containers that have heat preservation performance.

Examples of the experiments

(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 shaping agent and the balance of water, wherein each 100ml of freeze-dried strain protective agent comprises 10g of protein, 5g of water-soluble sugar, 10g of freeze-drying shaping 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) taking 10ul of the protective agent bacterial liquid obtained in the step (3), and using a preparation device of a quality control strain quantitative pellet in the liquid nitrogen freezing process, wherein in the embodiment, the heat-insulating container 1 is a stainless steel heat-insulating cup, and the sampling needle is an injector needle. Liquid nitrogen is poured into the stainless steel thermos cup, and no matter how much, the liquid nitrogen flows into each freezing space 3, and the liquid nitrogen does not need to be directly poured into each partition one by one. The use is more convenient. Pouring liquid nitrogen into a thermos cup, then placing a freezing porous plate sterilized in advance, after the sample application needle extracts protective agent bacterial liquid, dripping the bacterial liquid one by one into a freezing space 3 on the freezing porous plate 2 above the heat preservation container 1, dripping the bacterial liquid to the bottom of the heat preservation container 1 through the perforation 4, freezing the bacterial liquid by the liquid nitrogen to form a freezing ball, then pre-freezing the frozen ball at the temperature of-35 ℃ and keeping the temperature 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 lyophilized bacterium balls are fully dissolved and uniformly mixed. Need not further dilution, convenient to use, and effectively avoid the error that the dilution process produced.

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 from example 1 were randomly drawn into 10 flasks, 1ml of sterile physiological saline was added to each flask, the mixture was dissolved and mixed, and the bacterial suspension obtained from each flask was counted by pouring with TSA medium, respectively, 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 minus 20 ℃ and minus 80 ℃ have no obvious change in the number of bacteria within one year of placement, and show better stability.

Claims

1. The preparation device of the quality control strain quantitative pellet is characterized by comprising a heat preservation container, a sample application needle and a freezing porous plate, wherein the sample application needle is arranged above the heat preservation container, and the freezing porous plate is arranged in the heat preservation container;

2. The apparatus for preparing quantitative pellets of quality control strains according to claim 1, wherein the inner diameter of the freezing hole is smaller than the length of any side of the freezing space.

3. The apparatus for preparing quantitative pellets of quality control strains as claimed in claim 1, wherein the bottom of the freezing space is hollowed to form the freezing hole.

4. The apparatus for preparing quantitative pellets of quality control strains according to claim 2 or 3, wherein the freezing multi-well plate is provided with a plurality of partition plates, and the partition plates are crossed in a vertical and horizontal direction to form a freezing space.

5. The apparatus for preparing quantitative pellets of quality control strains according to claim 4, wherein the heat-insulating container contains liquid nitrogen, and the bottom end of the spotting needle is 5-8cm away from the liquid level of the liquid nitrogen.

6. The apparatus for preparing quantitative pellets of quality-controlled strains according to claim 1, wherein the spotting needle is connected to a micro peristaltic pump.

7. The apparatus as claimed in claim 6, wherein the freezing multi-well plate is made of transparent material.

8. The apparatus for preparing quantitative pellets of quality control strains according to claim 7, wherein the freezing multi-well plate is square and the thermal insulation container is cylindrical.