Method for freeze-preservation and recovery of uronema marinum isolated from fugu rubripes

A technology of redfin puffer and cryopreservation, applied in the biological field, can solve the problems of loss of insect strains, weakening of moth-eaten infection ability, cumbersome passage, etc., to avoid bacterial contamination, prevent genetic drift of insect strains, and avoid cumbersome processes Effect

Active Publication Date: 2018-12-21
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing shield ciliates used for research are mainly preserved through in vitro continuous subculture, which has the disadvantages of cumbersome in vitro culture and subculture, loss of worm strains due to failure of subculture, bacterial contamination, weakened ability of moth infection, germplasm mutation and loss of immunogenicity And other issues
At present, the cryopreservation and recovery of scutellum ciliates are still blank, especially there is no relevant report on the cryopreservation of marine C.

Method used

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  • Method for freeze-preservation and recovery of uronema marinum isolated from fugu rubripes
  • Method for freeze-preservation and recovery of uronema marinum isolated from fugu rubripes

Examples

Experimental program
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Effect test

Embodiment 1

[0019] a. Scutellum ciliates were isolated from the festered tissue of the sick redfin puffer in a farm in Dalian City, Liaoning Province, and identified as marine cercia ( Uronema marinum ), inoculate the obtained marine C. filariae in a broth medium for in vitro acclimatization and culture for 15 days to obtain a marine C. cerevisiae liquid. The broth medium is 5 g of red-finned oriental puffer per 100 mL of sterilized seawater The homogenate was boiled for 10 minutes, and then filtered through 200-mesh gauze;

[0020] b. Then inoculate the marine C. filariae solution in the broth medium at a volume ratio of 1:100, and culture it at 20°C for 120 hrs to obtain the marine C. cerevisiae solution in the exponential growth phase;

[0021] c. Pipette 1 mL of marine C. filariae solution in exponential growth phase into a sterilized 1.5 mL centrifuge tube, centrifuge at 2000 rpm for 10 minutes, discard the supernatant and slowly add 1 mL L-15 complete medium to the centrifuge tube ...

Embodiment 2

[0028] The recovery method of the present invention corresponding to Example 1 is as follows: quickly put the cryopreservation tube stored in liquid nitrogen for 24 hours into a water bath, bathe in 37 °C for 2 min, centrifuge at 4000 rpm for 5 min, discard the supernatant, add L-15 to completely The culture medium was resuspended, transferred to a multi-well plate, and cultured for seven days in a constant temperature incubator at 20°C.

[0029] Observe and count under the microscope every day (the value is expressed as the mean value ± standard error), the result: from the first day to the second day, it was in a stagnant period, and no worms with strong vitality were observed; on the third day, vigorous worms were observed , counting its population density was 1586±82 / mL; on the fourth day, counting its population density was 158400±1728 / mL; on the fifth day, counting its population density was 590000±20406 / mL; on the sixth day, counting The population density was 460133±27...

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Abstract

The invention discloses a method for freeze-preservation and recovery of uronema marinum isolated from fugu rubripes. The method comprises the following steps of using an L-15 complete culture mediumto culture the uronema marinum to the exponential growth phase, exponential phase and adult worm phase, and using the L-15 complete culture medium to resuspend until the density is 200000 uronema marinum per 1ml; sucking an adult worm solution of uronema marinum, adding into a freeze-preservation tube, adding 40vol% sterilizing glycerin and 10vol% dimethyl sulfoxide, uniformly mixing, quickly putting the freeze-preservation tube into a programmable gradient cooling cell freeze-preservation box, staying overnight at the temperature of -80 DEG C, and putting into liquid nitrogen, so as to preserve for a long time; fetching the freeze-preservation tube out of the liquid nitrogen, quickly putting into a water bath pot, treating for 2min in a water bath with the temperature of 37 DEG C, centrifuging for 5min at the rotation speed of 4000rpm, discarding a supernatant, adding the L-15 complete culture medium to resuspend, transferring into a porous plate, and culturing for three days in a constant-temperature culture box with temperature of 20 DEG C, so as to complete recovery.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a preservation and recovery method of scutellum ciliates, in particular to a marine cercifilaria ( Uronema marinum ) cryopreservation and recovery methods. Background technique [0002] Marine Cercia ( Uronema marinum ) belongs to the order Scutellaria, ciliates, and is a small individual facultative parasitic ciliate. Marine cercariae are highly adaptable and can live freely in various aquaculture water bodies. Once there is too much residual bait in the aquaculture water, the environment deteriorates and the organic matter content is too high, the marine cercariae will reproduce in large numbers and parasitize the fish in each aquaculture period, especially the fish with body surface damage. The debris is food, and in severe cases, secondary bacterial diseases cause a large number of farmed fish to die. It has been reported that marine cercariasis can cause disease in tur...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0284Y02A40/81
Inventor 黎睿君高延奇叶仕根王伟
Owner DALIAN OCEAN UNIV
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