CN109370908A - A kind of culture of chicken coccidial oocyst and save liquid and its application - Google Patents
A kind of culture of chicken coccidial oocyst and save liquid and its application Download PDFInfo
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- CN109370908A CN109370908A CN201811365560.1A CN201811365560A CN109370908A CN 109370908 A CN109370908 A CN 109370908A CN 201811365560 A CN201811365560 A CN 201811365560A CN 109370908 A CN109370908 A CN 109370908A
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- culture
- chicken
- potassium sorbate
- oocyst
- liquid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/10—Protozoa; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The invention discloses a kind of culture of chicken coccidial oocyst and liquid and its application are saved, belongs to veterinary parasitology field.The present invention as culture and saves liquid using potassium sorbate aqueous solution.Test confirms, does not have an impact to egg capsule Sporulated rate, and has obvious bacteriostasis, and can significantly improve the chicken coccidial oocyst vigor saved for a long time at 4 DEG C.The result shows that culture solution and preservation liquid of 1 ~ 3% alternative potassium bichromate solution of potassium sorbate aqueous solution as chicken coccidial oocyst.
Description
Technical field
The invention belongs to veterinary parasitology fields, culture and preservation liquid more particularly to a kind of chicken coccidial oocyst.
Background technique
Eimeria species are enteron aisle parasitic protozoa, and chicken coccidial oocyst is a large amount of with containing simultaneously in excrement discharge animal body, in excrement
Enteric bacteria and microorganism, and the chicken coccidial oocyst of fresh discharge is unsporulated oocysts, does not have infectivity, it need to be in suitable temperature
Sporulated reproduction is completed through certain time under degree, oxygen, damp condition, forms Sporulated Oocysts.Therefore, chicken coccidial oocyst culture
With liquid first choice is saved there is the substance suppression of disinfection bacteriostasis to kill other microorganisms, while Sporulated and work to chicken coccidial oocyst
Power does not have an impact.
Potassium bichromate solution has stronger bactericidal effect, is domestic and international all the time most common chicken coccidial oocyst culture
With save liquid, be generally 2-2.5%(mass volume ratio using concentration), but potassium bichromate has strong and stimulating, to people and animal
Health, environmental sanitation etc. will cause certain influence.The culture used in chicken coccidiasis egg capsule vaccine living at present and save liquid also
There is chloramines substance, such as chloramine B and toluene-sodium-sulfonchloramide.Chloramines is a kind of chlorine-ammonia mixture, has the function of aoxidizing and sterilize, but not
Stablize, disinfecting power is weaker, and bactericidal effect is not as good as free chlorine.Stablize it would therefore be highly desirable to find one kind new safe and efficient, property
Coccidian oocyst culture save liquid and substitute existing coccidian oocyst culture and save liquid, to reduce coccidian oocyst culture and save liquid pair
The influence of human health and environmental sanitation, while the coccidian oocyst time-to-live can also be improved.
Summary of the invention
The object of the present invention is to provide a kind of chicken coccidial oocyst culture and liquid is saved, is deposited with solving existing culture and preservation liquid
The problem of health, the environmental sanitation to people and animal have some impact on.
It is a further object to provide its a kind of applications in chicken coccidial oocyst culture and preservation.
Described in order to achieve the purpose that, the invention is realized by the following technical scheme: a kind of culture of chicken coccidial oocyst and
Liquid is saved, is 0.1~10% potassium sorbate aqueous solution for mass concentration.
It is 1 ~ 3% potassium sorbate aqueous solution for mass concentration as further improvement of the present invention.
Application of the culture and preservation liquid of a kind of above-mentioned chicken coccidial oocyst in chicken coccidial oocyst culture and preservation.
Chicken coccidial oocyst culture and preservation liquid in the present invention are potassium sorbate.Potassium sorbate is colourless to white flakes shape
Crystallization or crystalline powder, odorless or slightly stink, English name: Potassium sorbate, molecular formula C6H7KO2, molecule
Amount is 150.22.Potassium sorbate is made using potassium carbonate or potassium hydroxide and sorbic acid as raw material, unstable in air, can be by oxygen
Change coloring, there is hygroscopicity, soluble easily in water, ethyl alcohol.
Benefit of the invention is that: potassium sorbate is as a kind of safer, efficient preservative, soluble easily in water, use
It is convenient, it can effectively inhibit bacterial growth, egg capsule Sporulated rate is not had an impact, and be remarkably improved the chicken coccidial oocyst of preservation
Vigor is such as applied in chicken coccidiasis live vaccine, will extend vaccine storage life, production of vaccine cost is greatly reduced.
The present invention using potassium sorbate solution as coccidian oocyst culture and save liquid.Test result shows that potassium sorbate is molten
Liquid has obvious bacteriostasis and does not have an impact to egg capsule Sporulated rate, and chicken coccidial oocyst is in 1-3% potassium sorbate solution (matter
Amount volume ratio) in be stored in 4 DEG C, when saving 3,6,9,12 months, the egg capsule vigor of maintenance is significantly higher than in 2.5% potassium bichromate
The egg capsule vigor of middle preservation.Test result shows that potassium sorbate solution can significantly improve the egg capsule vigor saved for a long time, such as answers
With to chicken coccidiasis live vaccine, vaccine storage life will be extended.
Product of the present invention is safe and efficient, property is stable coccidian oocyst culture save liquid substitute existing coccidian oocyst culture and
Liquid is saved, to reduce coccidian oocyst culture and save influence of the liquid to human health and environmental sanitation, while coccidia can also be improved
The egg capsule time-to-live.
Detailed description of the invention
Fig. 1 is Sporulated rate figure of the chicken coccidial oocyst in various concentration potassium sorbate solution.
Specific embodiment
The following examples can further illustrate the present invention, but do not limit the invention in any way.
In the present invention, chicken coccidial oocyst, potassium sorbate aqueous solution dosage do not provide and be particularly limited to, can be according to specific
Application demand is adjusted.
Embodiment 1
Fresh E. pubescens Msxim is stored in the aqueous solution of potassium sorbate, wherein the mass body of potassium sorbate solution
Product specific concentration is 0.1%.
Embodiment 2
Fresh E. pubescens Msxim is stored in the aqueous solution of potassium sorbate, wherein the mass body of potassium sorbate solution
Product specific concentration is 0.5%.
Embodiment 3
Fresh E. pubescens Msxim is stored in the aqueous solution of potassium sorbate, wherein the mass body of potassium sorbate solution
Product specific concentration is 1%.
Embodiment 4
Fresh E. pubescens Msxim is stored in the aqueous solution of potassium sorbate, wherein the mass body of potassium sorbate solution
Product specific concentration is 3%.
Embodiment 5
Fresh E. pubescens Msxim is stored in the aqueous solution of potassium sorbate, wherein the mass body of potassium sorbate solution
Product specific concentration is 5%.
Embodiment 6
Fresh E. pubescens Msxim is stored in the aqueous solution of potassium sorbate, wherein the mass body of potassium sorbate solution
Product specific concentration is 10%.
Chicken coccidial oocyst
YL plants of Eimeria tenella, in the chicken manure of Guangxi Yulin.
Experimental animal and feed
1 age in days yellow-feather broiler, is purchased from Guangdong Wiz Agricultural Science & Technology Co., Ltd., and raising pollutes cage in no Eimeria species
It is interior, it is raised by broiler feeding standard (fowl raising standard NY/T 33-2004).
Feed is to support power feed corporation,Ltd purchased from Lanzhou without any coccidiostat meat chicken complete diet pellet.
Main agents
Pure water；
Potassium sorbate (the pure CP of chemistry, be purchased from Beijing Suo Laibao Science and Technology Ltd)；
Potassium bichromate (the pure CP of chemistry, be purchased from Sinopharm Chemical Reagent Co., Ltd.).
Potassium sorbate solution is prepared: weighing 0.1,0.5,1,3,5,10 g potassium sorbates respectively, pure water is added to be settled to 100
ML dissolves (period can repeatedly stir or dissolve overnight) under room temperature.
Potassium bichromate solution is prepared: being weighed 2.5 g potassium bichromates, is added pure water to be settled to 100 mL, dissolve (period under room temperature
It can repeatedly stir or dissolve overnight).
" influence of the various concentration potassium sorbate solution to chicken coccidial oocyst Sporulated "
5 14 age in days non-ball worms infect yellow plumage chick, and every chicken distinguishes YL plants of Sporulated ovum of per oral inoculation Eimeria tenella
Capsule 5 × 104It is a.168 h after inoculation is cutd open and is killed all chickens, collect bilateral caecum, longitudinally cut off, scraping intestinal contents and
Mucous layer, scraping object are added into 200 mL15% liquor natrii hypochloritis (v/v), are stored at room temperature 10 min after mixing is sufficiently stirred
Afterwards, it is successively filtered through 100 mesh and 200 mesh stainless steels are sieved, collection filtrate, containing fresh E. pubescens Msxim in filtrate.
Prepare 8 50 clean mL centrifuge tubes, number is respectively 1-8, and each pipe is separately added into 20 mL egg capsule filtrates, is centrifuged
(3000 rpm, 10 min) abandon supernatant.The egg capsule precipitating of No. 1 centrifuge tube is resuspended with 40 mL pure water, the egg capsule of No. 2 centrifuge tubes
Precipitating is resuspended with 40 mL, 2.5% potassium bichromate solution, and 3-8 pipe uses 40 mL's 0.1%, 0.5%, 1%, 3%, 5%, 10% respectively
Potassium sorbate solution is resuspended.Egg capsule liquid after each pipe is resuspended moves into 250 mL conical flasks of another cleaning, masking foil sealing
Afterwards in 28 DEG C of constant incubator oscillation (120 rpm) cultures.
After cultivating 48 h, with blood cell counting plate to untreated fish group and each test group egg capsule carry out unsporulated oocysts amount and
Sporulated Oocysts meter number, and calculate Sporulated rate.
Sporulated rate calculation formula are as follows:
Sporulated Oocysts total amount/(Sporulated Oocysts total amount+unsporulated oocysts total amount) × 100%.
After Sporulated, appropriate egg capsule culture solution coating nutrient broth agar plate is respectively taken, in 37 DEG C of constant incubators
Middle culture 24-72 h observes bacterial growth situation.
Each group Sporulated rate calculated result shows, each group Sporulated rate more than 80%, and difference it is not significant ((p> 0.05,
Fig. 1).Wherein, the Sporulated rate of E. pubescens Msxim is respectively 83.18 ± 0.6% Hes in pure water and 2.5% potassium bichromate
84.49±0.6%；0.1%, in 0.5%, 1%, 3%, 5%, 10% potassium sorbate solution E. pubescens Msxim Sporulated
Rate is respectively 82.27 ± 1.9%, 83.65 ± 0.8%, 86.01 ± 0.7%, 85.83 ± 0.4%, 84.87 ± 1.4%, 84.57 ±
0.6%。
Bacterium observation is the results show that in addition to bacterium colony occur in pure water control group and 0.1% potassium sorbate test group, remaining each group
It is showed no bacterium colony to be formed, illustrates that 0.5%-10% potassium sorbate solution can effectively inhibit the life of bacterium in chicken coccidial oocyst culture solution
It is long.
" influence of the long-term preservation to chicken coccidial oocyst vigor "
The Eimeria tenella Yulin strain Sporulated ovum saved with 0.1%, 0.5%, 1%, 3%, 5%, 10% potassium sorbate solution
Capsule saves under the conditions of 4 DEG C, and when saving 3,6,9,12 months, each concentration group is with 3 × 104A Sporulated Oocysts
Infective dose per oral inoculation infects 10 14 age in days non-ball worms and infects chicken, while with same dose in 2.5% potassium bichromate solution
The middle Sporulated Oocysts for saving same time are infection control group.Each group chicken clinical symptoms and bloody stool situation are observed after inoculation,
The 7th day after inoculation, slaughters each group survival chicken and collect the caecum of each group chicken, after longitudinally cutting off caecum, scrape caecal content
Object and mucous epithelium tissue, each group scraping object are added separately to be sufficiently stirred mixed in 200 mL15% liquor natrii hypochloritis (v/v)
After being stored at room temperature 10 min after even, each group egg capsule amount is counted with blood cell counting plate.
For character observation the results show that under the conditions of 4 DEG C, pure water saves chicken coccidial oocyst for a long time, occurs in egg capsule liquid a large amount of
The agglomerating flocculent deposit of black, show it is seriously polluted, it is thus impossible to directly with pure water for a long time save chicken coccidial oocyst.It is adding
After various concentration potassium sorbate or potassium bichromate, each group egg capsule liquid is more uniform, and substrate has a small amount of precipitating (being precipitated as egg capsule).Ovum
Capsule vitality test is the results show that low concentration potassium sorbate solution is maintaining egg capsule vigor ability to be lower than 2.5% potassium bichromate solution；
Chicken coccidial oocyst saves in 1%-3% potassium sorbate solution, and the egg capsule vigor of holding, which is significantly higher than, is stored in 2.5% potassium bichromate
Egg capsule in solution；With the raising of concentration, chicken coccidial oocyst vigor ability is maintained to reduce (result such as table 1: various concentration sorb
The egg capsule vitality test result that sour potassium solution saves for a long time).Test result shows that 1%-3% potassium sorbate solution can be mentioned significantly
High chicken coccidial oocyst vigor, culture and preservation liquid of the alternative potassium bichromate as chicken coccidial oocyst, is such as applied to chicken coccidiasis
Live vaccine will significantly improve vaccine storage life, reduce production cost.
Claims (3)
1. culture and the preservation liquid of a kind of chicken coccidial oocyst, it is characterised in that: it is the sorbic acid that mass concentration is 0.1~10%
Aqueous solutions of potassium.
2. culture and the preservation liquid of a kind of chicken coccidial oocyst according to claim 1, it is characterised in that: it is mass concentration
It is the potassium sorbate aqueous solution of 1-3%.
3. a kind of culture of chicken coccidial oocyst of any of claims 1 or 2 and preservation liquid are in chicken coccidial oocyst culture and saving
Application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111670898A (en) * | 2020-07-16 | 2020-09-18 | 松山湖材料实验室 | Coccidian oocyst cryopreservation agent and preparation method and application thereof |
| CN111713488A (en) * | 2020-07-16 | 2020-09-29 | 松山湖材料实验室 | Cryopreservation agent, preparation method thereof and application thereof in coccidian oocysts |
| CN112400865A (en) * | 2020-11-27 | 2021-02-26 | 松山湖材料实验室 | Wall-breaking agent composition, preparation method thereof and application thereof in oocysts |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111670898A (en) * | 2020-07-16 | 2020-09-18 | 松山湖材料实验室 | Coccidian oocyst cryopreservation agent and preparation method and application thereof |
| CN111713488A (en) * | 2020-07-16 | 2020-09-29 | 松山湖材料实验室 | Cryopreservation agent, preparation method thereof and application thereof in coccidian oocysts |
| CN111670898B (en) * | 2020-07-16 | 2022-03-15 | 松山湖材料实验室 | Coccidian oocyst cryopreservation agent and preparation method and application thereof |
| CN112400865A (en) * | 2020-11-27 | 2021-02-26 | 松山湖材料实验室 | Wall-breaking agent composition, preparation method thereof and application thereof in oocysts |
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