CN101648013B - Preparation method of inactivated vaccine of infectious coryza of chicken - Google Patents
Preparation method of inactivated vaccine of infectious coryza of chicken Download PDFInfo
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Abstract
The invention discloses a preparation method of an inactivated vaccine of infectious coryza of chicken, which comprises the following steps: firstly, diluting haemophilus paragallinarum strains, inoculating a chicken agar plate, culturing at 37 DEG C for 22-24h; selecting a typical colony to inoculate a chicken agar slant culture-medium, culturing to obtain first class slant strains; secondly, getting the first class slant strains to inoculate into a chicken soup culture medium, vibrating and culturing at 37 DEG C for 12h, obtaining second class strains; thirdly, inoculating the second class strains into a semisynthetic culture medium, culturing by a biological fermentation cylinder; and inactivating to obtain the inactivated vaccine. Aiming at defects of complex process, high production cost, low yield and the like existing in the prior preparation method of the inactivated vaccine of infectious coryza of chicken, the research work of an improved production process is developed, and the important breakout on the culture method is obtained. The method has simple and feasible operation; compared with the prior art, the invention greatly improves the culture strains, shortens the culture time by 10-12h, and effectively lowers the production cost.
Description
Technical field
The present invention relates to a kind of method for preparing of inactivated vaccine, relate in particular to the method for preparing of inactivated vaccine of infectious coryza of chicken, belong to the preparation field of inactivated vaccine.
Background technology
Infectious coryza of chicken is a kind of acute respiration systematic infection disease that is caused by haemophilus paragallinarum; Principal character is that inflammation in various degree takes place for eye and nasal mucosa; Sickness rate is very high, can cause that young chicken growth retardation and laying hen egg yield significantly descend, and the chicken and the bird inlay in age in 8-12 week the most often take place.This disease is distributed in the whole world, reduces 10%-40% because the laying hen that infects is laid eggs, and the chicken number is stagnated and eliminated in the weightening finish of growth chicken to be increased, and often causes serious economic loss.Though this disease can adopt antibiotic and sulfa drugs to treat, and can cause drug residue, all adopting vaccinated mode to prevent and treat according to the large-scale chicken farm of terrain investigation more than 90% should disease.
At present, the infectious coryza of chicken vaccine of more domestic biological product manufacturers productions is inactivated vaccine.These manufacturers all adopt " veterinary biologics rules " technology to produce, and its main process is following:
1, first class inoculum preparation: strain streak inoculation Carnis Gallus domesticus agar plate; At 5%-10%CO
2Concentration is cultivated 16-18h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, for use after pure inspection affirmation is aseptic;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 5%-10%CO
2Cultivate 16-18h for 37 ℃; Select 37 ℃ of cultivation 16-20h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% inserts semisynthetic medium; Cultivate 18-20h, shake culture bottle secondary sample count plate therebetween for 37 ℃; After pure inspection affirmation was aseptic, deactivation promptly got.
The culture bacteria number of producing the prepared inactivated vaccine that obtains according to " veterinary biologics rules " is generally all at 30-40 hundred million/ml, and 5,000,000,000/ml joins the Seedling requirement according to " veterinary biologics rules ", need carry out concentration to bacterium liquid.So there is complex process in the method for preparing of existing inactivated vaccine of infectious coryza of chicken, production cost is high, and defective such as yield poorly haves much room for improvement.
Summary of the invention
Technical problem to be solved by this invention is the existing complex process of method for preparing that overcomes existing inactivated vaccine of infectious coryza of chicken; Production cost is high; Defective such as yield poorly provides a kind of method for preparing of new inactivated vaccine of infectious coryza of chicken, and it is simple and direct that this method for preparing has technology; Production cost is low, the output advantages of higher;
Technical problem to be solved by this invention realizes through following technical scheme:
A kind of method for preparing of inactivated vaccine of infectious coryza of chicken may further comprise the steps:
(1), preparation primary inclined plane strain:, cultivate 22-24h for 37 ℃ with haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate; Select colonies typical inoculation Carnis Gallus domesticus agar slant culture-medium, cultivate 24h, obtain the primary inclined plane strain for 37 ℃;
(2), the preparation second class inoculum: get the primary inclined plane bacterial classification inoculation 37 ℃ of shaken cultivation 12h that in chicken soup culture medium, (adopt full temperature agitator), obtain second class inoculum, subsequent use;
(3), bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Confirm aseptic after, deactivation promptly gets.
Wherein, in order to reach better technique effect, in step (1), select 10-20 colonies typical inoculation Carnis Gallus domesticus agar slant culture-medium;
In the step (3), described fermentation culture conditions is preferably: cultivation temperature is 37 ℃; Rotating speed is 100-200r/min; Incubation time is 8h; The inventor in incubation, adopts the method that strengthens ventilation gradually through further test discovery, can significantly improve the culture bacteria number.
The present invention is directed to the existing complex process of method for preparing of existing inactivated vaccine of infectious coryza of chicken, production cost is high, and defective such as yield poorly has been carried out the work of improvement Research on Process, finally on cultural method, has obtained important breakthrough.The inventive method is easy and simple to handle feasible, and the culture bacteria number is improved largely than existing method, and incubation time has also shortened 10-12h, effectively reduces production cost.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
1, the haemophilus paragallinarum strain is available from China Veterinery Drug Inspection Office.
2, the concrete constituent of each culture medium: (1) Carnis Gallus domesticus agar plate: chicken juice 100ml, casein peptone 1g, sodium chloride 0.5g, agar powder 1.5g, nadide 0.05g, healthy chicken serum 10ml; (2) Carnis Gallus domesticus agar slant culture-medium: composition is identical with the Carnis Gallus domesticus agar plate; (3) chicken soup culture medium: chicken juice 100ml, casein peptone 1g, sodium chloride 0.5g, nadide 0.05g, healthy chicken serum 10ml; (4) semisynthetic medium: polypepton 5g, casein peptone 30g, sodium glutamate 15g, yeast soak powder 5g, glucose 3g, sodium chloride 15g, distilled water 3000ml; Transfer pH7.2-7.4,115 ℃ of sterilizations 40 minutes, add the healthy chicken serum 30-60ml and the 0.5% nadide aqueous solution 8-10ml of deactivation before using.
Embodiment 1
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 22h for 37 ℃, selects 10 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 100r/min, incubation time 8h; After the pure inspection affirmation of the dull and stereotyped count plate of taking a sample was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 2
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 24h for 37 ℃, selects 20 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 200r/min, incubation time 8h; In the fermentation culture process, adopt the method that strengthens ventilation gradually; After the pure inspection affirmation of the dull and stereotyped count plate of taking a sample was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 3
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 23h for 37 ℃, selects 15 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 150r/min, incubation time 8h; After the pure inspection affirmation of the dull and stereotyped count plate of taking a sample was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 4
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 22h for 37 ℃, selects 20 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 120r/min, incubation time 8h; The dull and stereotyped count plate of taking a sample, pure inspection confirm aseptic after, deactivation promptly gets.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 5
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 24h for 37 ℃, selects 10 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 180r/min, incubation time 8h; The dull and stereotyped count plate of taking a sample, pure inspection confirm aseptic after, deactivation promptly gets.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 6
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 23h for 37 ℃, selects 16 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 140r/min, incubation time 8h; The dull and stereotyped count plate of taking a sample, pure inspection confirm aseptic after, deactivation promptly gets.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 7
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 24h for 37 ℃, selects 17 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 190r/min, incubation time 8h; The dull and stereotyped count plate of taking a sample, pure inspection confirm aseptic after, deactivation promptly gets.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Embodiment 8
1, primary inclined plane strain preparation: haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate is cultivated 24h for 37 ℃, selects 18 inoculations of colonies typical Carnis Gallus domesticus agar slant culture-medium, cultivates 24h for 37 ℃, obtains the primary inclined plane strain, and it is for use to place 4 ℃ of refrigerators;
2, second class inoculum preparation: get primary inclined plane bacterial classification inoculation full temperature agitator in chicken soup culture medium and cultivate 12h for 37 ℃, after pure inspection affirmation is aseptic, obtain second class inoculum, subsequent use;
3, bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Cultivation temperature: 37 ℃, rotating speed: 200r/min, incubation time 8h; In the fermentation culture process, adopt the method that strengthens ventilation gradually; The dull and stereotyped count plate of taking a sample, pure inspection confirm aseptic after, deactivation promptly gets.The testing result of culture bacteria number is seen table 1.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 1
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 5%CO
2Concentration is cultivated 16h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 5%CO
2Cultivate 16h for 37 ℃; Select 37 ℃ of cultivation 16h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 18h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 2
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 10%CO
2Concentration is cultivated 18h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 10%CO
2Cultivate 18h for 37 ℃; Select 37 ℃ of cultivation 20h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 20h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 3
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 8%CO
2Concentration is cultivated 17h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 8%CO
2Cultivate 18h for 37 ℃; Select 37 ℃ of cultivation 16-20h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 19h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 4
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 5%%CO
2Concentration is cultivated 18h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 5%CO
2Cultivate 18h for 37 ℃; Select 37 ℃ of cultivation 20h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 20h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 5
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 10%CO
2Concentration is cultivated 16h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 10%CO
2Cultivate 16h for 37 ℃; Select 37 ℃ of cultivation 16h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 18h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 6
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 10%CO
2Concentration is cultivated 17h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 6%-CO
2Cultivate 17h for 37 ℃; Select 37 ℃ of cultivation 17h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 19h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 7
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 9%CO
2Concentration is cultivated 18h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 9%CO
2Cultivate 16h for 37 ℃; Select 37 ℃ of cultivation 16-20h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 20h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 8
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 7%CO
2Concentration is cultivated 18h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 10%CO
2Cultivate 18h for 37 ℃; Select 37 ℃ of cultivation 17h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 19h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Control Example 9
1, first class inoculum preparation: haemophilus paragallinarum strain streak inoculation Carnis Gallus domesticus agar plate; At 5%%CO
2Concentration is cultivated 17h for following 37 ℃; Select colonies typical to inoculate in the 5 age in days chick embryo yolk sacs, 37 ℃ are continued to hatch, and collect 30h with interior dead Embryo Gallus domesticus yolk liquid, and pure inspection confirms that aseptic back is for use;
2, second class inoculum preparation: Embryo Gallus domesticus yolk liquid streak inoculation Carnis Gallus domesticus agar plate, 9%CO
2Cultivate 18h for 37 ℃; Select 37 ℃ of cultivation 18h in the colonies typical inoculation chicken soup culture medium, pure inspection confirms that aseptic back is for use;
3, bacterium liquid is cultivated: the inoculum concentration by 2.5% is linked in the semisynthetic medium; Cultivate 19h for 37 ℃, shake culture bottle secondary therebetween, sampling count plate; After pure inspection affirmation was aseptic, deactivation promptly got.
Adopt " veterinary biologics rules " viable bacteria counting method, the Carnis Gallus domesticus agar plate count calculates the culture bacteria number, and the testing result of culture bacteria number is seen table 1.
Table 1 adopts " veterinary biologics rules " technology to cultivate and the contrast of the inventive method culture bacteria number
Claims (1)
1. the method for preparing of an inactivated vaccine of infectious coryza of chicken may further comprise the steps:
(1), preparation primary inclined plane strain:, cultivate 22-24h for 37 ℃ with haemophilus paragallinarum strain dilution inoculation Carnis Gallus domesticus agar plate; Select 10-20 colonies typical inoculation Carnis Gallus domesticus agar slant culture-medium, cultivate 24h, obtain the primary inclined plane strain for 37 ℃;
(2), the preparation second class inoculum: get primary inclined plane bacterial classification inoculation 37 ℃ of shaken cultivation 12h in chicken soup culture medium, obtain second class inoculum, subsequent use;
(3), bacterium liquid is cultivated: second class inoculum is inoculated in the semisynthetic medium cultivates with biological fermentation tank; Described condition of culture is: cultivation temperature is 37 ℃; Rotating speed is 100-200r/min; Incubation time is 8h; In incubation, adopt the method that strengthens ventilation gradually to cultivate; Confirm aseptic after, deactivation promptly gets.
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| CN104212736A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Haemophilus paragallinarum, inactivated vaccine and preparation method of inactivated vaccine |
| CN106267176B (en) * | 2015-05-12 | 2020-03-10 | 普莱柯生物工程股份有限公司 | Infectious coryza vaccine composition, preparation method and application thereof |
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