A liquid nitrogen cryopreservation technology suitable for on-site conditions

  This paper describes a liquid nitrogen cryopreservation technique that is applied to enhance the development of microfilariae of the genus Brucella.

  Peritoneal lavage method was used to obtain the microfilariae of B. elegans and Pahang worm from the abdominal cavity of infected gerbils, and washed in TC199 medium, using modified HAM method (1981, the first step, put microfilaria into 10 %v(/v) Ethylene (ED) 37OC incubation for a minute, the second step, transfer to crushed ice, incubate in 40% ED for 45 seconds, then drop 20 microfilament suspension The cover slips were placed in liquid nitrogen (19o6C) for cryopreservation. During the resuscitation, each cover slip was quickly transferred to 3700 tubes containing at least 1 ml of TC199 solution (containing 20 final heat-inactivated fresh fetal bovine serum) and gently shaken to two Methods The survival level of microfilaria after resuscitation was evaluated.

  One is to observe the activity of microfilariae under a microscope at 37 ° C, to show that the percentage of normal active microfilaments accounted for the total number of microfilaments in 20 flashes, and the specimens of each group were repeated 3 times;

  The other is to observe the ability of cryopreserved and untreated microfilariae to fully develop in the body of Aedes aegypti (eBlsNewtSarin) to evaluate its viability, that is, to mix 9000 microfilaments per milliliter with mouse blood. At 37 °C, the mosquitoes were fed through the pig's intestinal membrane according to the Ruuedge method, kept at room temperature at 28 °C, and fed with 4 (w/v) sucrose solution. At the same time, the mice were fed with blood-free sucrose and sucrose-free control. To evaluate the mosquito mortality caused by microfilariae, 10 days later, 45 mosquitoes were randomly selected from each infection group and placed in TC199 solution. Individual anatomy was performed on the head, chest and abdomen, and the young silkworms were examined, and the mosquitoes of each group were recorded. mortality rate.

  RESULTS: The survival rate of the microfilaments, which were incubated with the second step or the second step without cryopreservation, decreased. There was no significant difference in the results of the variance analysis. The two-step method was used to incubate the liquid nitrogen. The survival of microfilaments increased to 90% within 45 seconds after resuscitation. If the initial 3700 incubation was not given, the survival rate was only 26.6% within 30 seconds after cryopreservation and resuscitation. After resuscitation, microfilariae and control group microfilariae activity, after 5 repeated tests, the former showed that the normal activity of Malay microfilaria was 91% of the latter; the experimental results also confirmed that cryopreservation or no treatment The microfilariae were mixed with rat blood, and the rats were fed with sucrose or sucrose alone to feed Aedes aegypti. After 10 days, the survival rate of the four groups of mosquitoes was 75.1-80.8%, indicating that microfilament had no effect on mosquito viability. Aedes aegypti was mixed with mouse blood with cryopreserved Paphia sinensis and rat blood. After 10 days, the larvae were dissected. On average, 4.04 nl larvae were detected per mosquito (the second stage larvae increased slightly, but no stage I larvae were found). In the cold control group, an average of 5.16 per mosquito was detected. In the 111th larva, the infection level of the former is about 78 of the latter.

  The author believes that the above-mentioned improved method is different from other reported methods, does not require precision equipment, only requires some ice and liquid nitrogen, and is suitable for refrigerating and field application of sheathed microfilaments.

  The back of the hind limbs were perforated, and a drop of physiological saline containing 50 infectious larvae was added dropwise, and then the monkeys were housed in cages. 1~21 months after infection, 50~100% of the labeled sulfur colloid was injected into the metatarsal toe of each hind limb, and the radioisotope content of the abdominal pelvic lymph nodes from the injection site through the lymphatic vessels was detected by radiography. It is measured every tens of minutes for a total of 3 hours. During the test, the hind limbs of the control group showed a flexed shape with 9 limbs, and the straight limbs had 6 limbs to observe the influence of hind limb flexion on lymph fluid flow. A few days after the isotope test, the lymphatic system was stained by injecting sky blue dye into the hind limbs. Ten minutes later, the monkeys were anesthetized and laparotomy, and the lymphatic system was examined in situ with a dissecting microscope.

  In the control group, the limbs were flexed or straightened, and the labeled colloid appeared in the axillary lymph nodes 10 minutes after the injection, which was 10~30 CPM. The 30-120 minutes could be measured in the abdomen-pelvic lymph nodes of most hind limbs. However, there were 4 gelatinous gels that had not reached the abdomen-pelvic lymph nodes within 3 hours. In the experimental group, 8 limbs were 1 to 9 months after infection, and the labeled colloid was first measured in the abdomen-pelvic lymph node 10~7OOPM 30 to 120 minutes after injection, and 4 limbs appeared in the abdomen and pelvic lymph nodes for 10 to 40 minutes. After the lymph node was measured, the other 4 limbs did not appear in the axillary lymph nodes. After n~21 months after infection, the colloid first measured IOCPM in the axillary lymph node, and then measured in the abdomen-pelvic lymph node. The reflux method was similar to that of the control group, and only the reflux rate was lower than that of the control group. Lymphatic staining showed that the dye in the hind limbs of the control group reached the axillary lymph nodes through two tiny lymphatic vessels, which were stained dark blue. The inguinal lymph nodes are light blue. In the hind limbs infected with filars, at 1-9 months, it was found that 2 of the 4 lymphatic vessels were dilated and curved, showing a dark blue color, and there were also pocket-shaped lymphatic vessels that expanded to 3 to 4 long. There are 5 filaments in the varicose lymphatics between the ankle and the axillary lymph nodes, and the 1s/inguinal lymph nodes are dark blue. The structure and coloration of the lymphatic system more than n months after infection were similar to those of the control group.

  The above test showed that after the infection of the Patas monkey by the silkworm, the axillary lymph nodes of the hind limbs were partially obstructed, the lymphatic vessels were varicose, and the limbs were formed. The obstruction disappeared 11 months after the infection.

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