A preservation solution for porcine digestive tract applied to in vitro simulated surgery of digestive endoscopy

Abstract

The present invention discloses a preservation solution for porcine digestive tract applied to in vitro simulated surgery of digestive endoscopy. The preservation solution of the present invention is composed of the following components: potassium citrate 7.65 - 7.68 g / L, sodium citrate 6.42 - 6.47 g / L, calcium chloride 0.38 - 0.42 g / L, magnesium sulfate 2.43 - 2.48 g / L, chlorhexidine 2 - 20 g / L, hydroxyethyl starch 130 / 0.4 60 g / L, glycerol 300 g / L, ampicillin 90 - 110 g / L, streptomycin 48 - 52 g / L, and the balance being distilled water. The in vitro porcine digestive tract preservation solution of the present invention can maximize the freshness and physical properties of the in vitro porcine digestive tract tissue. In addition, tests by a resistivity meter and surgical practice tests show that the preservation solution of the present invention is superior to the original formulation, and the preparation cost and time are greatly reduced, having great advantages in the actual application process and being able to improve the training efficiency.

Description

Technical Field

[0001] The present invention relates to a preservation solution for porcine digestive tract applied to in vitro simulated surgery of digestive endoscopy, belonging to the technical field of biomedical teaching and training. Background Art

[0002] In recent years, the digestive endoscopy technology has made great progress. For early tumors of the digestive tract mucosa, submucosal tumors of the digestive tract, etc., curative resection can be achieved through minimally invasive endoscopic resection. For patients, it can reduce the pain during and after surgery and preserve the integrity of the digestive tract. However, endoscopic resection is relatively professional, and the incidence of various common complications such as bleeding, perforation, and non-curative resection is relatively high when novices perform the surgery. Therefore, a certain number of simulated surgery trainings are required before the actual operation can be started. In this process, the ex vivo porcine stomach model plays an important role. The operator can comprehensively learn a series of problems such as endoscopic instruments, common surgical techniques, and intraoperative complications by performing common endoscopic minimally invasive surgeries such as endoscopic submucosal dissection on the ex vivo porcine stomach.

[0003] In previous usage experiences, we found that doctors often cannot complete all the practice operations at one time after purchasing porcine digestive tract tissues. Therefore, the remaining porcine digestive tract tissues need to be preserved until the next practice time. The common preservation method is to place the tissues in normal saline or formaldehyde solution and then refrigerate them in a 4°C refrigerator. The interval between the two practice times of the operator often needs to be one week or even two weeks. At this time, without proper storage, conditions such as mucosal layer stiffness, poor electrical conductivity of mucosal tissues, and atrophy of the submucosal layer often occur, resulting in ineffective submucosal injection. At this time, the ex vivo porcine digestive tract cannot perform effective endoscopic resection, which affects the training effect.

[0004] The researcher designed a gastric lesion simulation device in the early stage, including a gastric main body model. A plurality of replaceable gastric mucosal lesion operation blocks arranged at intervals are detachably connected to the gastric main body model. The replaceable gastric mucosal lesion operation block includes a fixed outer shell detachably connected to the gastric main body model. The bottom end of the fixed outer shell is open and hollow inside. An installation ring extending towards the center position of the fixed outer shell is fixed along the circumferential direction at the edge of the bottom of the fixed outer shell. An ex vivo porcine gastric lesion block is bonded to the bottom of the installation ring. The ex vivo porcine gastric lesion block is prepared by using the above-mentioned preservation solution for ex vivo animal stomach. It was found during the use process that the porcine gastric tissues of the replaceable module would show atrophy of the submucosal layer and poor electrical conductivity of the mucosal layer after being preserved for 6 - 7 days. In order to minimize the adverse effects of these conditions on the surgical training of endoscopic doctors, a preservation solution for porcine stomach dedicated to in vitro simulated surgery of digestive endoscopy needs to be developed.

[0005] We have extended the animal's digestive tract preservation time from 2-3 days to 1-2 weeks through our previous patent 202210541465.2 "An in vitro animal stomach preservation solution and a gastric lesion simulation device prepared using the same". However, the above preservation solution still has the following problems:

[0006] (1) High preparation cost: The purchase cost of bovine serum albumin, pepsin inhibitor, etc. is high;

[0007] (2) There are many ingredients and the formula is complicated;

[0008] (3) The antibiotics in the formula do not work well in vitro;

[0009] (4) It tends to protect biological and cellular functions, but has poor protection for the physical properties of the pig digestive tract, such as conductivity.

[0010] In view of the above problems, the original formula needs to be optimized. Summary of the invention

[0011] The purpose of the present invention is to provide a novel porcine digestive tract preservation solution for in vitro simulated digestive endoscopy surgery, which can maximize the long-term preservation of the ex vivo porcine digestive tract, and is matched with a set of high-simulation upper gastrointestinal tract simulation training devices with replaceable operation sites for porcine digestive tract mucosal lesions that we have developed in the past, so as to ensure the freshness of the ex vivo porcine digestive tract mucosa and guarantee the smooth progress of digestive endoscopy surgery training.

[0012] In order to achieve the above-mentioned purpose, the present invention provides a porcine digestive tract preservation solution for in vitro simulated digestive endoscopy surgery. The preservation solution uses distilled water as a solvent and is composed of the following components: 7.65-7.68 g / L potassium citrate, 6.42-6.47 g / L sodium citrate, 0.38-0.42 g / L calcium chloride, 2.43-2.48 g / L magnesium sulfate, 2-20 g / L chlorhexidine, 130 / 0.4 60 g / L hydroxyethyl starch, 300 g / L glycerol, 90-110 g / L ampicillin and 48-52 g / L streptomycin.

[0013] The present invention also provides a method for preparing the above-mentioned porcine digestive tract preservation solution for in vitro simulated digestive endoscopy surgery, comprising: weighing each raw material in sequence according to the concentration ratio; adding double distilled water into a container, and then adding potassium citrate, sodium citrate, calcium chloride, magnesium sulfate, chlorhexidine, ampicillin, streptomycin, hydroxyethyl starch 130 / 0.4 and glycerol in sequence and stirring and mixing to obtain a solution; adding double distilled water to adjust the solution to the required volume, filtering it with a bacterial filter membrane, and refrigerating it for later use.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) Aiming at the problem that the preparation cost of the in vitro animal stomach preservation solution in the original patent is high (the purchase costs of bovine serum albumin, pepsin inhibitor, etc. are high), hydroxyethyl starch 130 / 0.4 is used to replace bovine serum albumin as a colloid. Through experiments, the effects of the two are similar;

[0016] (2) Aiming at the problem that the original formula has more components and is complex, the formula is optimized. Through multiple experiments, it is found that removing components such as disodium hydrogen phosphate, potassium dihydrogen phosphate, adenosine, fructose 1,6-diphosphate, dexamethasone, reduced glutathione, mannitol, and low molecular weight heparin can still achieve the same effect;

[0017] (3) Aiming at the problem that the original formula antibiotics do not work well in the in vitro environment, a sufficient amount of chlorhexidine is added;

[0018] (4) Aiming at the problem that the original formula has poor protection for the physical properties of the porcine digestive tract, through repeated attempts, glycerol is added, which can protect cell membranes, remove excessive fat, and at the same time preserve tissue moisture and physical properties; it only needs to be soaked in a glycerol-free electrolyte solution for 6 hours to remove glycerol before using the porcine stomach;

[0019] (5) After optimizing the formula, the present invention can maximize the freshness and physical properties of the in vitro porcine digestive tract tissue. In addition, tests with a resistivity meter and surgical practice tests show that the preservation solution of the present invention is superior to the original formula, and the preparation cost and time are greatly reduced, having great advantages in actual application and can improve training efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a stained diagram of the porcine digestive tract preservation solution in Example 5 of the present invention for preserving the porcine gastric mucosa for 1 month;

[0021] Figure 2 It is a stained diagram of the porcine gastric mucosa preserved with normal saline in Comparative Example 5 for 1 month. DETAILED DESCRIPTION OF THE INVENTION

[0022] To make the present invention more obvious and understandable, preferred embodiments are provided below in conjunction with the accompanying drawings for detailed description as follows.

[0023] Example 1

[0024] This example provides a porcine digestive tract preservation solution for in vitro simulated surgery of digestive endoscopy and its preparation method:

[0025] 1. Composition of the preservation solution

[0026] Potassium citrate 7.66 g, sodium citrate 6.45 g, calcium chloride 0.4 g, magnesium sulfate 2.45 g, chlorhexidine 5 g, hydroxyethyl starch 130 / 0.4 60 g, glycerol 300 g, ampicillin 90 - 110 g, streptomycin 48 - 52 g.

[0027] 2. Preparation method of the preservation solution:

[0028] (1) Weigh each raw material successively according to the said mass concentration; (2) Add 800 mL of double-distilled water into a 1000 mL container, and then successively add potassium citrate, sodium citrate, calcium chloride, magnesium sulfate, chlorhexidine, ampicillin, streptomycin, hydroxyethyl starch 130 / 0.4, and glycerol and stir to mix evenly; (3) Add double-distilled water to make the solution volume up to 1 L, filter the solution using a bacteria-filtering membrane, and store it at 4 °C for standby.

[0029] 3. Obtaining of excised porcine stomach:

[0030] The experimental pigs were placed in the supine position, and low molecular weight heparin was pre-injected to fully heparinize the pigs' circulation. Under normal circumstances, total gastrectomy can be completed through a midline upper abdominal incision, that is, starting from the xiphoid process and winding down around the umbilicus to 2 cm below the umbilicus. If necessary, resection of the xiphoid process can provide sufficient exposure; the stomach was pulled downward, and the left and right gastric arteries and veins at the root of the lesser omentum were ligated with sutures. Subsequently, the left and right gastroepiploic arteries and veins were also ligated. At the same time, the cardia and pylorus were blocked with thick threads; the stomach was lifted upward, and the transverse colon was pulled downward to make the mesentery between the stomach and the transverse colon tense. The operator used the left hand to pull the greater omentum to expose the avascular area, and an electric knife was used to cut along the upper edge of the transverse colon. Starting from the middle part of the colon, it was cut to the lower pole of the spleen on the left side, and then cut to the right side until the hepatic flexure of the transverse colon was reached; before removing the anterior leaf of the transverse mesocolon, groups 14 and 15 lymph nodes were cleared. The incised greater omentum was lifted, and starting from the right side of the middle colic artery, a sharp knife and a gauze ball dissector were used to perform sharp and blunt dissection between the anterior and posterior leaves of the mesocolon. It was easy to find the loose connective tissue space here, and it was easy to clearly dissect and remove the anterior leaf of the mesocolon and its attached fatty lymphoid tissue along this space. Continuing to dissect upward to the lower edge of the pancreas, the confluence of the middle colic vein and the right gastroepiploic vein could be found, and the peritoneum of the pancreas was continuously dissected along this layer to the upper edge of the pancreas. Continuing to dissect upward and to the right, at the lower edge of the pancreatic head and duodenum, the root of the right gastroepiploic vein was ligated and cut. Subsequently, the right gastroepiploic artery branched from the gastroduodenal artery was also ligated and cut. The stomach and duodenum were pulled downward to expose the porta hepatis area and the lesser omentum, and it was cut along the avascular area under the liver. Dissecting from the porta hepatis to the duodenum direction, the peritoneum and loose tissue in front of the hepatoduodenal ligament were cleared. The proper hepatic artery, common bile duct, and portal trunk were exposed. The right gastric artery and vein were found and ligated and cut at the roots respectively. The duodenum was cut 3 cm distal to the pylorus. The excised pig stomach was rinsed clean with normal saline and divided into 3×3 cm squares to obtain excised pig stomach blocks. The excised pig stomach blocks were placed in the prepared excised animal digestive tract preservation solution, and the preservation solution was made to cover the excised pig stomach blocks and stored at 0-4°C.

[0031] Example 2

[0032] This example provides a pig digestive tract preservation solution for in vitro simulated surgery of digestive endoscopes and its preparation method:

[0033] 1. Composition of the preservation solution

[0034] 7.68 g of potassium citrate, 6.46 g of sodium citrate, 0.42 g of calcium chloride, 2.45 g of magnesium sulfate, 10 g of chlorhexidine, 60 g of hydroxyethyl starch 130 / 0.4, 300 g of glycerol, 90-110 g of ampicillin, and 48-52 g of streptomycin were added to 800 mL of distilled water. Double-distilled water was added to make the volume of the solution up to 1 L, and the solution was filtered with a filter membrane and stored at 4°C for standby.

[0035] The preparation method of the isolated animal stomach preservation solution and the use method of the preservation solution described in this example are the same as those in Example 1 and will not be described in detail here.

[0036] 2. Obtaining isolated pig esophagus

[0037] Choose right side lying position, make a posterolateral incision on the left chest, remove the sixth rib, and cut the posterior segment of the fifth rib at the same time, and enter the chest through the costal bed. Pull the lung forward and inward to expose the posterior mediastinum, lift the diaphragm between the liver and spleen with two tissue forceps, cut it between the two forceps, and then extend it in a radial direction, with the front end reaching the proximal costal arch and the rear end pointing to the esophageal hiatus. In order to reduce bleeding and avoid damaging the organs below the diaphragm, use your fingers to guide and lift it upward under the diaphragm when cutting, and stop bleeding while cutting. There are branches of the subphrenic artery near the hiatus, which should be sutured in an 8-shaped pattern with thick silk thread one by one. Leave a few stitches on both sides of the incision for traction. Then reach into the abdominal cavity to explore the gastric fundus, greater and lesser curvatures of the stomach, liver, splenic hilum, left gastric artery and abdominal aorta, greater omentum, mesentery and pelvic cavity for lymph node metastasis or tumor transplantation. If it is clear that resection or palliative surgery can be performed through chest and abdominal exploration, the diaphragmatic incision can be enlarged and the esophageal hiatus can be opened. When separating, the lungs can be collapsed first, and then pulled forward and downward to expose the top of the chest cavity. Gently pull down the lower part of the esophagus, and you can see that the esophagus above the aortic arch moves outside the pleura behind the left subclavian artery. Cut the mediastinal pleura from bottom to top along the left edge of the left subclavian artery to reach the top of the chest. Then use your fingers to separate the esophagus above the aortic arch and wrap it around a thin soft rubber tube as traction. Then, use the right index finger to move downward from above the aortic arch and the left index finger to move upward from below the aortic arch, and gently and bluntly separate the esophagus in the direction of the aortic arch. After fully exposed in this way, esophageal tissue of different lengths can be cut. Use physiological saline to wash the isolated pig esophagus and cut it into 3x3cm squares to obtain isolated pig esophageal blocks. Put the isolated pig esophageal blocks into the prepared isolated animal digestive tract preservation solution so that the preservation solution covers the isolated esophageal blocks and store them at 0-4℃.

[0038] Example 3

[0039] This embodiment provides a pig digestive tract preservation solution for in vitro simulated digestive endoscopy surgery and a preparation method thereof:

[0040] 1. Preservative solution ingredients

[0041] To 800 mL of distilled water, add 7.68 g / L potassium citrate, 6.47 g / L sodium citrate, 0.42 g / L calcium chloride, 2.48 g / L magnesium sulfate, 2 g chlorhexidine, 130 / 0.4 60 g hydroxyethyl starch, 300 g glycerol, 90-110 g ampicillin, and 48-52 g streptomycin, add double distilled water to make the solution volume to 1 L, filter the solution with a bacteria filter membrane, and store it at 4 °C for later use.

[0042] The preparation method of the in vitro animal gastric preservation solution and the usage method of the preservation solution in this embodiment are the same as those in Embodiment 1, and will not be elaborated here.

[0043] 2. Obtaining the in vitro porcine colon

[0044] Make a midline incision or a left paramedian incision, starting from 3 cm or higher above the umbilicus and reaching the pubis. After laparotomy, first turn the greater omentum upward, ligate and cut the blood vessels connected to it near the transverse colon, and try to preserve the greater omentum; in cases of cancer, the greater omentum should be resected together with the transverse colon without reservation. Free the right half of the colon. At 10 - 15 cm from the ileocecal valve, cut the terminal ileum and its mesentery, incise the retroperitoneum behind the cecum, ascending colon and the lateral part of the hepatic flexure, push the colon to the midline, and cut the mesocolon as close to the intestinal wall as possible to cover the exposed surface of the posterior abdominal wall in the future. For other steps, refer to the right hemicolectomy. Free the transverse colon and the left half of the colon. The transverse colon is also separated from the transverse mesocolon in the same way, cut the splenocolic ligament and ligate the blood vessels in it to free the splenic flexure. Cut the retroperitoneum on the outside of the descending colon and sigmoid colon, and free the colon to the midline as in the left hemicolectomy. Resect all the colon. According to the situation, the left and right ureters can be freed and protected by traction with rubber sheets to prevent damage during tissue dissection in the retroperitoneum and pelvis. If there is no malignant lesion, cut the left mesocolon close to the intestinal wall and ligate the blood vessels in it. If there is a malignant lesion in the rectum or the left colon, the inferior mesenteric artery must be cut and ligated close to the abdominal aorta, and all the mesentery of the left half of the colon should be resected. Place a long right-angle clamp at the lower part of the rectum and also at the proximal end to prevent contamination when the intestinal tube is cut, and finally remove all the colon. Wash the in vitro porcine colon with normal saline and divide it into 3x3 cm squares to obtain in vitro porcine colon blocks. Put the in vitro porcine colon blocks into the prepared in vitro animal digestive tract preservation solution so that the preservation solution covers the in vitro porcine colon blocks, and store them at 0 - 4 °C.

[0045] Comparative Example 1

[0046] Wash the in vitro porcine stomach with normal saline and divide it into 3×3 cm squares to obtain in vitro porcine stomach blocks. Put the in vitro porcine stomach blocks into a formaldehyde solution for preservation.

[0047] Comparative Example 2

[0048] Wash the in vitro porcine stomach with normal saline and divide it into 3×3 cm squares to obtain in vitro porcine stomach blocks. Put the in vitro porcine stomach blocks in a refrigerator at -20 °C for cryopreservation.

[0049] Comparative Example 3

[0050] Wash the in vitro porcine stomach blocks with normal saline and divide them into 3×3 cm squares to obtain in vitro porcine stomach blocks. Put the in vitro porcine stomach blocks into normal saline for preservation.

[0051] The morphological changes of the excised porcine gastric blocks 16 obtained in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were observed separately after 1 day, 3 days, 7 days, 14 days, and 1 month of preservation. The results are shown in the following table:

[0052]

[0053] In summary, when using the porcine digestive tract preservation solution of the present invention, the experimental pigs are placed in a supine position, and low molecular weight heparin is pre-injected to fully heparinize the pig's circulation. Fresh excised porcine stomachs are obtained by total gastrectomy. The excised porcine stomachs are rinsed thoroughly with normal saline and cut into 3x3 cm squares to obtain excised porcine gastric blocks. The excised porcine gastric blocks are placed in the excised animal gastric preservation solution prepared previously, with the preservation solution covering the excised porcine gastric blocks, and stored at 0-4°C for up to 1 month.

[0054] Example 4

[0055] This example provides a porcine digestive tract preservation solution for in vitro simulated endoscopic surgery and its preparation method:

[0056] 7.65 g / L of potassium citrate, 6.42 g / L of sodium citrate, 0.38 g / L of calcium chloride, 2.43 g / L of magnesium sulfate, 15 g of chlorhexidine, 60 g of hydroxyethyl starch 130 / 0.4, 300 g of glycerol, 90-110 g of ampicillin, and 48-52 g of streptomycin are added to 800 mL of distilled water. Double-distilled water is added to make the volume of the solution up to 1 L. After filtering the solution with a bactericidal filter membrane, it is stored at 4°C for standby.

[0057] The preparation method of the excised animal gastric preservation solution, the specific operation method of total gastrectomy, and the usage method of the preservation solution in this example are the same as those in Example 1 and will not be repeated here.

[0058] Comparative Example 4

[0059] Add 800 ml of double-distilled water into a 1000 ml container, add 7.6599 g of potassium citrate and stir evenly; add 6.4518 g of sodium citrate and stir evenly; add 400 mg of calcium chloride and stir evenly; add 2.4647 magnesium sulfate and stir evenly; add 2342.3 mg of disodium hydrogen phosphate and stir evenly; add 224.5485 mg of potassium dihydrogen phosphate and stir evenly; add 600 mg of adenosine and stir evenly; add 80 g of fructose 1,6-diphosphate and stir evenly; add 10 mg of dexamethasone and stir evenly; add 3 g of reduced glutathione and stir evenly; add 30 g of mannitol and stir evenly; add 100 g of BSA powder and stir evenly; add 1 mg of protease inhibitor Pepstantin and stir evenly; add 100 g of ampicillin and stir evenly; add 50 g of streptomycin and stir evenly, add 6 mg of low molecular weight heparin and stir evenly. Finally, add double-distilled water to adjust the total volume to 1000 ml, filter with a bacteria-proof membrane and store at 4°C.

[0060] By comparing the new formulated preservation solution in Example 4 with the preservation solution of the original patent formula, the results are shown in the following table:

[0061]

[0062]

[0063] In the comparison between Example 4 and Comparative Example 4, after 1 month of storage, through resistivity meter testing and surgical practice testing, Example 4 is superior to Comparative Example 4, and the solution preparation cost and time cost are greatly reduced, indicating that Example 4 has great advantages in the application process and can improve training efficiency and economic benefits.

[0064] Example 5

[0065] This example provides a preservation solution for porcine digestive tract applied to in vitro simulated surgery of digestive endoscopy and its preparation method:

[0066] Add potassium citrate 7.67 g / L, sodium citrate 6.43 g / L, calcium chloride 0.39 g / L, magnesium sulfate 2.44 g / L, chlorhexidine 5 g, hydroxyethyl starch 130 / 0.4 60 g, glycerol 300 g, ampicillin 90 - 110 g, streptomycin 48 - 52 g into 800 mL of distilled water. Add double-distilled water to make the volume of the solution up to 1 L, filter the solution with a bacteria-proof membrane, and store at 4°C for standby.

[0067] The preparation method of the in vitro animal gastric preservation solution, the specific operation method of total gastrectomy and the usage method of the preservation solution in this example are the same as those in Example 1, and will not be elaborated here. The staining diagram of the porcine gastric mucosa after 1 month of preservation is as Figure 1 shown.

[0068] Comparative Example 5

[0069] Same as Comparative Example 3, the excised porcine gastric blocks were rinsed clean with normal saline and divided into 3×3 cm squares to obtain excised porcine gastric blocks. The staining diagram of the excised porcine gastric blocks stored in normal saline for 1 month is as Figure 2 shown.

[0070] Comparison Figures 1-2 It can be seen that after 1 month of storage, the porcine gastric mucosa stored in the preservation solution of the present invention still maintains a good anatomical structure, while the gastric tissue structure stored in the traditional preservation solution is disordered, loses its original arrangement, and becomes loose.

[0071] The above embodiments are only the preferred embodiments of the present invention, and do not limit the present invention in any formal or substantial way. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A preservation solution for porcine digestive tract applied to in vitro simulated surgery of digestive endoscopy, characterized in that, the preservation solution uses distilled water as a solvent and is composed of the following components: potassium citrate 7.65 - 7.68 g / L, sodium citrate 6.42 - 6.47 g / L, calcium chloride 0.38 - 0.42 g / L, magnesium sulfate 2.43 - 2.48 g / L, chlorhexidine 2 - 20 g / L, hydroxyethyl starch 130 / 0.4 60 g / L, glycerol 300 g / L, ampicillin 90 - 110 g / L, and streptomycin 48 - 52 g / L; the usage method of the preservation solution is as follows: Put the excised porcine gastric block into the preservation solution, immerse the excised porcine gastric block with the preservation solution, and store it at 0 - 4 °C; Or, put the excised porcine esophageal block into the preservation solution, immerse the excised esophageal block with the preservation solution, and store it at 0 - 4 °C; Or, put the excised porcine colon block into the preservation solution, immerse the excised porcine colon block with the preservation solution, and store it at 0 - 4 °C.

2. The preparation method of the preservation solution for porcine digestive tract applied to in vitro simulated surgery of digestive endoscopy according to claim 1, characterized in that, it includes: Weigh each raw material in sequence according to the concentration ratio; add double-distilled water into the container, then add potassium citrate, sodium citrate, calcium chloride, magnesium sulfate, chlorhexidine, ampicillin, streptomycin, hydroxyethyl starch 130 / 0.4, and glycerol in sequence and stir and mix evenly to obtain a solution; add double-distilled water to make the solution volume up to the required volume, and after filtering with a bacteria-filtering membrane, store it in the refrigerator for later use.

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