CN101864387A - Marine crustacean mycoplasma culture medium and separation and purification method thereof - Google Patents
Marine crustacean mycoplasma culture medium and separation and purification method thereof Download PDFInfo
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Abstract
The invention relates to a marine crustacean mycoplasma culture medium and a separation and purification method thereof. The method is characterized in that mycoplasma broth medium, phenol red and redistilled water are sterilized, then calf serum is added and inactivation is performed, then fresh yeast decoction, glucose solution, thallium acetate aqueous solution and penicillin are added to mix evenly and obtain a liquid culture medium; and mycoplasma broth medium, redistilled water and agar powder are sterilized, then calf serum, fresh yeast decoction, thallium acetate aqueous solution and penicillin are added to mix evenly, the mixed solution is poured on a sterilized flat plate to prepare a solid culture medium. The separation and purification method using the culture medium, of marine crustacean mycoplasma is to isolate colonies on the solid culture medium, place the isolated colonies in the liquid culture medium for growth in culture, then place on the solid culture medium for purification and repeat the above steps to obtain the purified mycoplasma. By using the method of the invention, the success rate of isolating crustacean mycoplasma is high.
Description
Technical field
The present invention relates to a kind of mycoplasma culture medium and separation purification method.
Background technology
Existing Mycoplasma is in procaryote, be at present can independent existence, self-reproduction in the non-activity cell culture medium minimum microorganism, acellular wall construction has the polymorphism of height, can see through millipore filtration.Mycoplasma is except causing the human infectious disease, can also cause domestic animal, poultry and crop pest (Razin S.Peculiar properties ofmycoplasmas:the smallest self-replicating prokaryotes.FEMS Microbiol Lett, 1992,79 (1-3): 423-431.).Isolated since the first strain people is mycoplasma from nineteen thirty-seven, mycoplasma species is reported successively surplus in the of existing 120 so far, and be more common in Mammals and Jie Zhi animal Insecta kind (Razin S.Yogev D.Naot Y.Molecular biology and pathogenicity of mycoplasmas.Microbiol Mol Biol Rev.1998,62 (4): 1094-1156.).Yet relevant Crustacea mycoplasma is all seldom reported at present both at home and abroad.(Yang Jifang, Chinese prawn mycoplasmas ultrastructure and host's main ultra micro pathological change, Oceanologia et Limnologia Sinica, 1997, Vol.28, No.2 such as Yang Jifang; Yang Jifang, the popular hepatopancreas of Chinese prawn downright bad syndromes research I virus and class mycoplasmas concurrent infection prawn hepatopancreas, 1993, ocean, the East Sea, Vol 11, NO3, the 34-39.) polyinfection of discovery mycoplasma and bacterium and mycoplasma and white spot syndrome virus from culture Chinese prawn enteron aisle sarcoidosis and white spot syndrome.The no pathogenicity mycoplasma in environment a large amount of exist and in the healthy animal body long-term existence, the animal of carrying mycoplasma can't show any symptom before not being subjected to coercing.Yet, isolating mycoplasma pure growth can make healthy prawn morbidity from the prawn eye of ill death, the gill, brain, or make easier virus and infection (the Ghadersohi A of bacterium of being subjected to of healthy animal, Leigh O.Isolation, charactersation and DNA analysis of Mycoplasma spp.from moribund prawnsPenaeus monodon cultured in Austualia.Dis Aquat Org, 1999,35:53-61).
The crustacean mycoplasma is carried out scientific research, and primarily the bottleneck problem that will solve is the substratum that preparation is fit to the breeding of crustacean mycoplasma growth in vitro.The substratum that is used for the cultivation of the mankind and livestock and poultry mycoplasma at present all can not satisfy the growth demand of crustacean mycoplasma.
Summary of the invention
Technical problem to be solved by this invention is that the present situation at prior art provides a kind of marine crustacean mycoplasma culture medium.
Another technical problem to be solved by this invention is that the present situation at prior art provides a kind of marine crustacean mycoplasma culture medium separation purification method.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: this marine crustacean mycoplasma culture medium, comprise liquid nutrient medium and solid medium, and it is characterized in that:
Will
Mycoplasma broth culture 12.0～15.0g
0.4-1wt% phenolsulfonphthalein 2.35～3.00mL
Redistilled water 470～600mL
At pH is under 7.0-8.0,110 ℃-121 ℃ the condition behind the sterilization 10-20min, add the 5-30mL calf serum at 56 ℃ of deactivation 30min, and then add 10-40v% fresh yeast leach liquor 5-20mL, 5-10wt% glucose solution 0.5-3mL, 5-15wt% thaliium acetate aqueous solution 0.1-0.5mL and 5-20mg/mL penicillin 0.5-2mL and mix and obtain described liquid nutrient medium;
Will
Mycoplasma broth culture 12.0～15.0g
Redistilled water 470～600mL
Agar powder 0.94～1.2wt%
10-20min sterilizes under pH7.0-8.0,110 ℃ of-121 ℃ of conditions, to be cooled after 55～60 ℃, the calf serum 5-30mL, 10-40v% fresh yeast leach liquor 5-20mL, 5-15wt% thaliium acetate aqueous solution 0.1-0.5mL and the 5-15mg/mL penicillin 0.5-2mL that add filtration sterilization after mixing are poured into mixed solution on the sterilization flat board and make solid medium; The consumption of above-mentioned agar powder is that every 100mL solid medium adds 0.94-1.2g.
Wherein, the preparation method of described fresh yeast leach liquor is as follows:
Take by weighing the high reactivity yeast of working, be suspended in the 2-6 distilled water doubly, 20-40 ℃ stir 5-30min after, the 15-60min that at room temperature ferments is heated to boiling point then, cooling obtains fermented liquid; Fermented liquid with the centrifugal 15-30min of 2000-4000rpm, is got supernatant liquor,, then supernatant liquor is placed 90～95 ℃ of water-bath 10-20min with NaOH adjust pH to 7.0～8.0; After the cooling,, get supernatant liquor and promptly obtain fresh yeast leach liquor with the degerming of 0.22-0.45 μ m membrane filtration again with the centrifugal 15-30min of 7000～8000rpm.
Use aforesaid liquid substratum and solid medium to carry out the separation purification method of marine crustacean mycoplasma, it is characterized in that comprising the steps:
1) the sick sample organ of aseptic clip, add the mycoplasma broth culture of 3～6 times of sick sample organ volumes and the quartz sand of the doubly sick sample organ of 0.5-1 volume, the aseptic emulsion that grinds to form, the centrifugal 10-20min of 3000-5000rpm, with supernatant liquor through the sterile filtration of 0.22-0.45 μ m film, remove the impurity elimination bacterium, obtain aseptic supernatant liquor;
2) getting aseptic supernatant liquor is inoculated in 10 times of described liquid nutrient mediums of volume, placing 22-41 ℃, concentration is that the CO2gas incubator of 5%～10v% is cultivated, the 48-72h blind passage carries out colony growth with blind passage for being inoculated on the described solid medium, obtain the feature bacterium colony;
3) described feature bacterium colony is inoculated incubation growth on the described liquid nutrient medium, the culture that obtains is inoculated into carries out colony growth on the described solid medium; Repeat this step, until the feature bacterium colony that obtains required mycoplasma;
4) with sterile razor blade picking described feature colony inoculation in described liquid nutrient medium, shake cultivations in 22-41 ℃, 50-100rpm, to liquid nutrient medium color taking-up culture during by the red stain Huang; This culture and described liquid nutrient medium are carried out 10 times of dilution methods, be diluted to desired concn, obtain diluent; Get the above-mentioned diluent of 0.1-1mL and be inoculated in solid medium continuation cultivation, isolate mycoplasma feature germ;
5) repeating step obtains the ocean crustaceans mycoplasma of purifying more than 4 three times.
The separation purification method of another kind of marine crustacean mycoplasma is characterized in that comprising the steps:
1) the sick sample organ of aseptic clip, add the mycoplasma broth culture of 3～6 times of sick sample organ volumes and the quartz sand of the doubly sick sample organ of 0.5-1 volume, the aseptic emulsion that grinds to form, the centrifugal 10-20min of 3000-5000rpm, supernatant liquor is filtered through 0.22-0.45 μ m sterile filters, remove the impurity elimination bacterium, obtain aseptic supernatant liquor;
2) get the aseptic supernatant liquor of 0.2-1mL and coat on the described solid medium, place the wet environment of CO2gas incubator to cultivate CO under 22-41 ℃
2Concentration range is 5%～10v%, cultivates 3～5 days, obtains mycoplasma feature bacterium colony; Choose described feature colony inoculation in liquid nutrient medium with sterile razor blade, concussion is cultivated under 22-41 ℃, 50-100rpm condition, obtains culture during to its colour changed into yellow of liquid nutrient medium; In this culture, add described liquid nutrient medium and carry out 10 times of dilution methods, obtain the diluent of desired concn; This diluent is seeded to described solid medium continues to cultivate, isolate mycoplasma feature bacterium colony;
3) the feature bacterium colony repeating step that obtains more than 2 three times, is obtained the ocean crustaceans mycoplasma of purifying.
The separation purification method of another marine crustacean mycoplasma is characterized in that comprising the steps:
1) gets 3-5mm with aseptic the picking of transfering loop
3The sick sample tissue block of size is directly streak inoculation on described solid medium, places the CO2gas incubator of wet environment, 22-41 ℃ to cultivate CO
2Concentration range is 5%～10v%, cultivates 3～14 days, grows bacterium colony;
2) choose required colony inoculation in liquid nutrient medium with sterile razor blade, put 22-41 ℃, 50-100rpm and shake cultivation, obtain the mycoplasma culture during to the liquid nutrient medium its colour changed into yellow; In this culture, add described liquid nutrient medium and carry out 10 times of dilution methods, obtain the diluent of desired concn; This diluent is seeded to described solid medium continues to cultivate, isolate mycoplasma feature bacterium colony;
3) the feature bacterium colony repeating step that obtains more than 2 three times, is obtained the ocean crustaceans mycoplasma of purifying.
The separation purification method of another marine crustacean mycoplasma is characterized in that comprising the steps:
1) gets the sick sample of live body, the sick sample of aseptic condition down cut, effusive tissue juice is inoculated in 10 times of described liquid nutrient mediums of volume, placing 22-41 ℃, concentration is that the CO2gas incubator of 5%～10v% is cultivated, the 48-72h blind passage, blind passage is carried out colony growth for being inoculated on the described solid medium, obtain the feature bacterium colony;
2) described feature bacterium colony is inoculated incubation growth on the described liquid nutrient medium, the culture that obtains is inoculated into carries out colony growth on the described solid medium; Repeat this step, until the feature bacterium colony that obtains required mycoplasma;
3) with sterile razor blade picking described feature colony inoculation in described liquid nutrient medium, shake cultivations in 22-41 ℃, 50-100rpm, to liquid nutrient medium color taking-up culture during by the red stain Huang; This culture and described liquid nutrient medium are carried out 10 times of dilution methods, be diluted to desired concn, obtain diluent; Get the above-mentioned diluent of 0.1-1mL and be inoculated in solid medium continuation cultivation, isolate mycoplasma feature germ;
4) repeating step obtains the ocean crustaceans mycoplasma of purifying more than 4 three times.
Compared with prior art, the invention provides and a kind ofly be used for the substratum of crustacean, especially mud crab mycoplasma vitro culture and use the separation purification method of this substratum the crustacean mycoplasma.This liquid and solid medium can effectively promote the mycoplasma growth in vitro, and it is higher that our bright separation method is isolated in the Crustacean body success ratio of mycoplasma.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1
Medium preparation:
1, the preparation of liquid nutrient medium: take by weighing mycoplasma broth culture 2.04g and be dissolved in the 80mL redistilled water, add the 0.4mL0.4% phenolsulfonphthalein, transfer pH to 7.8,121 ℃ of sterilization 15min with 1M NaOH; The calf serum 20mL of filtration sterilization will be added after 55 ℃ of the liquid nutrient medium that the obtain coolings, 25v% fresh yeast leach liquor 10mL, 10wt% glucose solution 1mL, 10wt% thaliium acetate aqueous solution 0.2mL, 10mg/mL penicillin 1mL mixes the back and uses the packing of 5mL test tube standby.
2, the preparation of solid medium: take by weighing mycoplasma broth culture 2.04g and be dissolved in the 80mL redistilled water, transfer pH to 7.8 with 1MNaOH, adding final concentration is the agar powder of 1.02wt%, 121 ℃ of sterilization 15min, to be cooled to about 57 ℃, the calf serum 20mL, 25v% fresh yeast leach liquor 10mL, 10wt% thaliium acetate aqueous solution 0.2mL and the 10mg/mL penicillin 1mL that add filtration sterilization are poured into after mixing that to make solid medium on the 10mL sterilization flat board standby.
The preparation method of above-mentioned fresh yeast leach liquor is: take by weighing the 250g high reactivity yeast of working, be suspended in the 1000mL distilled water, 37 ℃ stir 20min after, the 40min that at room temperature ferments is heated to boiling point then, cooling obtains fermented liquid; Fermented liquid with the centrifugal 20min of 3000rpm, is got supernatant liquor,, then supernatant liquor is placed 90～95 ℃ of water-bath 15min with 1M NaOH adjust pH to 7.8; After the cooling,, get supernatant liquor and promptly obtain fresh yeast leach liquor with 0.25 μ m membrane filtration degerming again with the centrifugal 20min of 7500rpm.
The substratum for preparing in the present embodiment uses for embodiment 2-6.
Embodiment 2
1) under the aseptic condition, take the about 5.7g of mud crab cheek tissue to add 15mL mycoplasma broth culture lapping liquid and 3mL quartz sand, the aseptic emulsion that grinds to form, the centrifugal 15min of 4000rpm; With removing the impurity elimination bacterium through 0.45 μ m membrane filtration in the supernatant liquor inhalation syringe, obtain aseptic supernatant liquor.
2) get the 0.5ml supernatant liquor and be inoculated in the 5mL liquid nutrient medium, place 37 ℃, 5v%CO
2CO2gas incubator in cultivate.A 72h blind passage generation is inoculated into liquid nutrient medium and solid medium with blind passage respectively for being divided into two.Liquid nutrient medium is used for preserving and this blind subculture of growth; On the solid medium at 37 ℃, 5v%CO
2Cultivate the growing state of observing bacterium colony on the solid medium after 3 days at microscopically every day under the condition.The solid medium of selecting colony growth to be evenly distributed, the typical single mycoplasma feature bacterium colony of mark under low-power microscope.If can not obtain the feature bacterium colony, can carry out many for blind passage, until obtaining the feature bacterium colony through generation blind passage.Generally, through promptly isolating the feature bacterium colony behind three generations's blind passage,, may not contain mycoplasma in this culture if also do not find the feature bacterium colony behind three generations's blind passage.
Among the embodiment 2 to embodiment 5 on the solid medium grown cultures in separation, purification condition and the bacterium colony liquid medium within of feature bacterium colony all be in 22-41 ℃, concentration are the CO2gas incubator of 5%～10v%, to carry out.
3) the feature colony inoculation that obtains with the sterile razor blade picking is in liquid nutrient medium, shakes cultivation at 37 ℃, 80rpm, to the liquid nutrient medium color by the red stain Huang; Get culture and be diluted to 10 for 10 times
-6After, obtain diluent; This diluent is inoculated on the solid medium, under wet environment, 37 ℃, 5%CO
2Continue under the condition to cultivate, observe behind the 5d and occur equally distributed, big or small bacterium colony on the solid medium as tip-like, under low-power microscope, observe, these bacterium colonies are typically " fried egg " sample, bacterium colony is rounded, all less than 100 μ m, middle portion is thicker for diameter, for being absorbed in the growth part of agar inside; Neat in edge, periphery is the very thin clear area of one deck, the growth part that this scatters at agar surface for bacterium colony.Bacterium colony is less to be colourless often, becomes faint yellow or pale brown look after outmoded, the densely distributed pearl shape that is of part bacterium colony, and circular surface is mellow and full smooth, gos deep in the agar, about the about 50 μ m of size.The single bacterium colony of picking is inoculated in liquid nutrient medium under aseptic condition, 80rpm concussion is cultivated under 37 ℃ of wet environments, to the liquid nutrient medium its colour changed into yellow again 10 times be diluted to 10
-6, coat on the solid medium and cultivate, obtain mycoplasma feature bacterium colony.
4) the single mycoplasma feature of picking bacterium colony, repeating step can obtain the marine crustacean mycoplasma of purifying more than 3 three times successively, can preserve in-80 ℃ of very low temperature glycerine.
Embodiment 3
1) the sick sample organ of aseptic clip, can be the organs such as the cheek, stomach, intestines, hepatopancreas or heart of marine crustacean, the mycoplasma broth culture and the 0.5～1g quartz sand that add 4 times of volumes, regulating pH with NaOH is 8, the aseptic emulsion that grinds to form, 3000rpm is centrifugal, and 20min gets supernatant liquor, in the inhalation syringe through 0.45 μ m film sterile filtration, remove the impurity elimination bacterium, obtain aseptic supernatant liquor.
2) get the aseptic supernatant liquor of 0.5mL and be inoculated in the 5mL liquid nutrient medium, place 37 ℃ CO2gas incubator to cultivate CO
2Concentration range is 7%, blind passage behind the 72h, also inoculate solid medium when blind passage is seeded in liquid nutrient medium, the culture that is seeded on the liquid nutrient medium is used for keeping and this blind passage bacterial classification of growing, and the culture that is seeded on the solid medium is used for separation, the needed mycoplasma bacterium colony of purifying.
3) be seeded in culture on the solid medium at 37 ℃, 5v%CO
2Cultivate (culture condition after 3 days under the condition?) observe the colony growth situation every day at microscopically.The solid medium of selecting colony growth to be evenly distributed, the typical single bacterium colony of mark under low-power microscope, with sterile razor blade picking mark position colony inoculation in liquid nutrient medium, put 37 ℃, 80rpm and shake cultivation,, get 10 times of methods of culture and be diluted to 10 when yellow by red stain to the liquid nutrient medium color
-4, obtain dilution.Get this diluent of 0.2mL and be seeded to solid medium continuation cultivation.
Repeating step 3 at least three times obtains the ocean jacket class animal mycoplasma of purifying, can preserve standby in-80 ℃ of very low temperature glycerine.
Embodiment 4
1) is inoculated in solid medium with the aseptic disease crab ascites that picks of transfering loop, gets about 3mm
3The tissue block of size is directly streak inoculation on solid medium, places wet environment, 37 ℃ CO2gas incubator to cultivate CO
2Concentration is 6v%, cultivates behind the 3d at microscopically and observes the colony growth situation every day.
2) solid medium of selecting colony growth to be evenly distributed, under low-power microscope behind the typical single bacterium colony of mark, choose mark position colony inoculation in liquid nutrient medium with sterile razor blade, put 37 ℃, 80rpm and shake cultivation, get culture 10 times dilution methods by red stain when yellow to the liquid nutrient medium color and be diluted to 10
-4, obtain diluent; Get the 0.2mL diluent and be seeded to solid medium continuation cultivation, obtain the mycoplasma feature bacterium colony of purifying.
3) picking mycoplasma feature bacterium colony, repeating step obtain the ocean jacket class animal mycoplasma of purifying more than 2 three times, can preserve standby in-80 ℃ of very low temperature glycerine.
Embodiment 5
1) get the sick sample of live body, outside with the alcohol swab sterilization, the sick sample of aseptic condition down cut splashes into the effusive tissue juice of sick sample and carries out grown cultures in the liquid nutrient medium.Simultaneously with the sample tissue drop on solid medium, coating is placed in wet environment, 37 ℃ the CO2gas incubator and cultivates CO
2Concentration range is 5v%.Blind passage after 72 hours, blind passage is cultivated for being inoculated on the described solid medium flat board, examine under a microscope the growing state of bacterium colony on the solid medium every day after 3 days, the solid medium of selecting colony growth to be evenly distributed, the typical single mycoplasma feature bacterium colony of mark under low-power microscope.If can not obtain the feature bacterium colony, can carry out many for blind passage, until obtaining the feature bacterium colony through generation blind passage.Generally, through promptly isolating the feature bacterium colony behind three generations's blind passage,, may not contain mycoplasma in this culture if also do not find the feature bacterium colony behind three generations's blind passage.
2) solid medium of selecting colony growth to be evenly distributed, under low-power microscope behind the typical single bacterium colony of mark, choose mark position colony inoculation in liquid nutrient medium with sterile razor blade, put 37 ℃, 80rpm and shake cultivation, take out culture 10 times dilution methods by red stain when yellow to the liquid nutrient medium color and be diluted to 10
-4, obtain diluent; Get this diluent of 0.2mL and be seeded to the dull and stereotyped continuation cultivation of solid medium, obtain the mycoplasma feature bacterium colony of purifying.
3) repeating step 2, and the mycoplasma bacterium colony that obtains is successively carried out purifying, more than the triplicate, obtain the ocean jacket class animal mycoplasma of purifying, can preserve standby in-80 ℃ of very low temperature glycerine.
The content part that does not relate in the various embodiments described above is same as the prior art.
Claims (5)
1. a marine crustacean mycoplasma culture medium comprises liquid nutrient medium and solid medium, it is characterized in that:
Will
Mycoplasma broth culture 12.0～15.0g
0.4-1wt% phenolsulfonphthalein 2.35～3.00mL
Redistilled water 470～600mL
At pH is the 10-20min that sterilizes under 7.0-8.0,110 ℃-121 ℃ the condition, add the 5-30mL calf serum after being cooled to 50-60 ℃ at 56 ℃ of deactivation 30min, and then add 10-40v% fresh yeast leach liquor 5-20mL, 5-10wt% glucose solution 0.5-3mL, 5-15wt% thaliium acetate aqueous solution 0.1-0.5mL and 5-20mg/mL penicillin 0.5-2mL and mix and obtain described liquid nutrient medium;
Will
Mycoplasma broth culture 12.0～15.0g
Redistilled water 470～600mL
Agar powder 0.94～1.2wt%
10-20min sterilizes under pH7.0-8.0,110 ℃ of-121 ℃ of conditions, to be cooled after 55～60 ℃, the calf serum 5-30mL, 10-40v% fresh yeast leach liquor 5-20mL, 5-15wt% thaliium acetate aqueous solution 0.1-0.5mL and the 5-15mg/mL penicillin 0.5-2mL that add filtration sterilization after mixing are poured into mixed solution on the sterilization flat board and make solid medium;
Wherein, the preparation method of described fresh yeast leach liquor is as follows:
Take by weighing the high reactivity yeast of working, be suspended in the 2-6 distilled water doubly, 20-40 ℃ stir 5-30min after, the 15-60min that at room temperature ferments is heated to boiling point then, cooling obtains fermented liquid; Fermented liquid with the centrifugal 15-30min of 2000-4000rpm, is got supernatant liquor,, then supernatant liquor is placed 90～95 ℃ of water-bath 10-20min with NaOH adjust pH to 7.0～8.0; After the cooling,, get supernatant liquor and promptly obtain fresh yeast leach liquor with the degerming of 0.22-0.45 μ m membrane filtration again with the centrifugal 15-30min of 7000～8000rpm.
2. the separation purification method of a marine crustacean mycoplasma is characterized in that comprising the steps:
1) the sick sample organ of aseptic clip, add the mycoplasma broth culture of 3～6 times of sick sample organ volumes and the quartz sand of the doubly sick sample organ of 0.5-1 volume, the aseptic emulsion that grinds to form, the centrifugal 10-20min of 3000-5000rpm, with supernatant liquor through the sterile filtration of 0.22-0.45 μ m film, remove the impurity elimination bacterium, obtain aseptic supernatant liquor;
2) getting aseptic supernatant liquor is inoculated in 10 times of described liquid nutrient mediums of volume, placing 22-41 ℃, concentration is that the CO2gas incubator of 5%～10v% is cultivated, the 48-72h blind passage, in blind passage generation, be inoculated on the described solid medium, placing 22-41 ℃, concentration is that the CO2gas incubator of 5%～10v% is cultivated, carry out colony growth, obtain the feature bacterium colony;
3) described feature bacterium colony is inoculated incubation growth on the described liquid nutrient medium, the culture that obtains is inoculated into carries out colony growth on the described solid medium; Repeat this step, until the feature bacterium colony that obtains required mycoplasma;
4) with sterile razor blade picking described feature colony inoculation in described liquid nutrient medium, shake cultivations in 22-41 ℃, 50-100rpm, to liquid nutrient medium color taking-up culture during by the red stain Huang; This culture and described liquid nutrient medium are carried out 10 times of dilution methods, be diluted to desired concn, obtain diluent; Get the above-mentioned diluent of 0.1-1mL and be inoculated in solid medium continuation cultivation, isolate mycoplasma feature germ;
5) repeating step obtains the ocean crustaceans mycoplasma of purifying more than 4 three times.
3. the separation purification method of a marine crustacean mycoplasma is characterized in that comprising the steps:
1) the sick sample organ of aseptic clip, add the mycoplasma broth culture of 3～6 times of sick sample organ volumes and the quartz sand of the doubly sick sample organ of 0.5-1 volume, the aseptic emulsion that grinds to form, the centrifugal 10-20min of 3000-5000rpm, supernatant liquor is filtered through 0.22-0.45 μ m sterile filters, remove the impurity elimination bacterium, obtain aseptic supernatant liquor;
2) get the aseptic supernatant liquor of 0.2-1mL and coat on the described solid medium, place the wet environment of CO2gas incubator to cultivate CO under 22-41 ℃
2Concentration range is 5%～10v%, cultivates 3～5 days, obtains mycoplasma feature bacterium colony; Choose described feature colony inoculation in liquid nutrient medium with sterile razor blade, concussion is cultivated under 22-41 ℃, 50-100rpm condition, obtains culture during to its colour changed into yellow of liquid nutrient medium; In this culture, add described liquid nutrient medium and carry out 10 times of dilution methods, obtain the diluent of desired concn; This diluent is seeded to described solid medium continues to cultivate, isolate mycoplasma feature bacterium colony;
3) the feature bacterium colony repeating step that obtains more than 2 three times, is obtained the ocean crustaceans mycoplasma of purifying.
4. the separation purification method of a marine crustacean mycoplasma is characterized in that comprising the steps:
1) gets 3-5mm with aseptic the picking of transfering loop
3The sick sample tissue block of size is directly streak inoculation on described solid medium, places the CO2gas incubator of wet environment, 22-41 ℃ to cultivate CO
2Concentration range is 5%～10v%, cultivates 3～14 days, grows bacterium colony;
2) choose required colony inoculation in liquid nutrient medium with sterile razor blade, put 22-41 ℃, 50-100rpm and shake cultivation, obtain the mycoplasma culture during to the liquid nutrient medium its colour changed into yellow; In this culture, add described liquid nutrient medium and carry out 10 times of dilution methods, obtain the diluent of desired concn; This diluent is seeded to described solid medium continues to cultivate, isolate mycoplasma feature bacterium colony;
3) the feature bacterium colony repeating step that obtains more than 2 three times, is obtained the ocean crustaceans mycoplasma of purifying.
5. the separation purification method of a marine crustacean mycoplasma is characterized in that comprising the steps:
1) gets the sick sample of live body, the sick sample of aseptic condition down cut, effusive tissue juice is inoculated in 10 times of described liquid nutrient mediums of volume, placing 22-41 ℃, concentration is that the CO2gas incubator of 5%～10v% is cultivated, the 48-72h blind passage, blind passage is carried out colony growth for being inoculated on the described solid medium, obtain the feature bacterium colony;
2) described feature bacterium colony is inoculated incubation growth on the described liquid nutrient medium, the culture that obtains is inoculated into carries out colony growth on the described solid medium; Repeat this step, until the feature bacterium colony that obtains required mycoplasma;
3) with sterile razor blade picking described feature colony inoculation in described liquid nutrient medium, shake cultivations in 22-41 ℃, 50-100rpm, to liquid nutrient medium color taking-up culture during by the red stain Huang; This culture and described liquid nutrient medium are carried out 10 times of dilution methods, be diluted to desired concn, obtain diluent; Get the above-mentioned diluent of 0.1-1mL and be inoculated in solid medium continuation cultivation, isolate mycoplasma feature germ;
4) repeating step obtains the ocean crustaceans mycoplasma of purifying more than 4 three times.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1042836A (en) * | 1988-12-01 | 1990-06-13 | 中国农业科学院哈尔滨兽医研究所 | The preparation method of deactivation vaccine emulsion for chicken septic mycoplasma |
| CN1128214C (en) * | 1999-06-25 | 2003-11-19 | 江苏省农业科学院畜牧兽医研究所 | Cloned weakening strain of chicken virus mycoplasma |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1042836A (en) * | 1988-12-01 | 1990-06-13 | 中国农业科学院哈尔滨兽医研究所 | The preparation method of deactivation vaccine emulsion for chicken septic mycoplasma |
| CN1128214C (en) * | 1999-06-25 | 2003-11-19 | 江苏省农业科学院畜牧兽医研究所 | Cloned weakening strain of chicken virus mycoplasma |
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