CN103215208B - Haemophilus parasuis culture medium - Google Patents
Haemophilus parasuis culture medium Download PDFInfo
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- CN103215208B CN103215208B CN201310135288.9A CN201310135288A CN103215208B CN 103215208 B CN103215208 B CN 103215208B CN 201310135288 A CN201310135288 A CN 201310135288A CN 103215208 B CN103215208 B CN 103215208B
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Abstract
The invention discloses a haemophilus parasuis culture medium. A preparation method of the haemophilus parasuis culture medium comprises the steps of preparing a basic culture solution, treating coenzyme A, and perfecting a culture medium, wherein the basic culture solution comprises peptone, tryptone, sodium chloride, dextrose, yeast extract, glycerin and distilled water, and the basic culture solution, the coenzyme A and fetal bovine serum are uniformly mixed so as to obtain the haemophilus parasuis culture medium. The haemophilus parasuis culture medium provided by the invention can keep haemophilus parasuis, thereby facilitating the transportation of suspicious haemophilus parasuis samples; and the death of haemophilus parasuis is not caused in the process of transportation, thereby increasing the separation probability of the haemophilus parasuis.
Description
Technical field
The invention belongs to field of agricultural microbial technology, be specifically related to a kind of haemophilus parasuis substratum.
Background technology
Haemophilus parasuis (Haemophilus parasuis) can cause polyserositis and the sacroiliitis of pig.This bacterium is very tender and lovely, substratum is required relatively harsh, growth relies on Reduced nicotinamide-adenine dinucleotide (NAD or the V factor), do not need the X factor (protoheme and other porphyrin substance), the speed of growth is extremely slow, and vitro culture is preserved easily dead, and more than 72h cultivated by blood agar plate, just can grow the large small colonies of needle point, HPS is as easy as rolling off a log to be left in the basket or to be covered by other bacterium.
Haemophilus parasuis to freezing, heat is all responsive, the sample of separation of bacterial, as needed inoculation medium immediately when synovial fluid, hydrothorax etc. gather, otherwise separation and Culture often can not be successful, but in real work to collection in worksite immediately inoculation medium be a little part, poultry master or basic unit's animal doctor personnel collected specimens often need refrigerate or deliver to after freezing for some time laboratory or delivered by the mode of transport, and after sample arrives, separation and Culture plating efficiency is lower.Therefore, very large difficulty is encountered during this bacterium of separation and Culture.
Current report uses the TSA(tryptose soya agar of import) with TSB(pancreas peptone soybean broth) as the basic medium being separated haemophilus parasuis, these substratum nutrition are extremely abundant, HPS speed of growth on these substratum is very fast.If using it as the substratum transporting haemophilus parasuis suspicious specimen, because suspicious specimen may also exist the bacterium such as intestinal bacteria, suis, these bacterial classifications speed of growth on TSA or TSB is faster more than haemophilus parasuis growth, just can complete for the nutrient consumption of substratum in short period of time, and refuse of its growth generation can kill haemophilus parasuis.Therefore, TSA and TSB is also not suitable as the substratum transporting suspicious specimen, is also not suitable as the substratum preserving haemophilus parasuis.Clinically in the urgent need to a kind of substratum of applicable transport haemophilus parasuis suspicious specimen.
Summary of the invention
The invention provides a kind of haemophilus parasuis substratum, be prepared from by following preparation method, its preparation method comprise basic culture solution preparation, coenzyme A process and substratum perfect, concrete steps are as follows:
1) the basic culture solution preparation described in
A, by component 14 ~ 16g/L(weight/volume meter of basic culture solution, down together) peptone, 4 ~ 6g/L Tryptones, 4 ~ 5g/L sodium-chlor, 2 ~ 3g/L glucose, 4 ~ 6g/L yeast leach liquor and 13 ~ 18 ‰ (are counted by volume, lower same) glycerine, surplus distilled water mixes;
B, the mixed solution that step A obtains is heated to boiling and constantly stirs until mixed solution dissolves completely;
C, by the solution that obtains in step B by sodium hydroxide adjust ph to 7.0 ~ 7.2;
D, the solution packing that step C is obtained, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling;
2) the coenzyme A process described in
Get 0.14 ~ 0.16g/L coenzyme A, degerming with 8 ~ 12 ‰ sterilized water dilute filtrations;
3) substratum is perfect
Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C save backup.
The component of above-described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 5g/L, glucose is 2g/L, yeast leach liquor be 5g/L and glycerine is 15 ‰.
The component of above-described basic culture solution is peptone is 14g/L, Tryptones is 6g/L, sodium-chlor is 5g/L, glucose is 2g/L, yeast leach liquor be 5g/L and glycerine is 15 ‰.
The component protein peptone that above-described basic culture solution is is 16g/L, Tryptones is 4g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor is 4g/L and glycerine is 13 ‰.
The component of above-described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 6g/L and glycerine is 18 ‰.
The component of above-described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 5g/L, glucose is 2g/L, yeast leach liquor be 6g/L and glycerine is 16 ‰.
The component of above-described basic culture solution is peptone is 14g/L, Tryptones is 5g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 6g/L and glycerine is 14 ‰.
The component of above-described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 5g/L and glycerine is 17 ‰.
Feature of the present invention: the nutritive substances such as the peptone in substratum of the present invention just in time meet the subsistence level of haemophilus parasuis survival, and its speed of growth is slower; And basic culture solution of the present invention adds glycerine, glycerine can supplement the carbon source needed for bacterial growth on the one hand; Glycerine is a kind of hydroaropic substance on the other hand, play a part to maintain osmotic pressure, keep the integrity effect of bacteria cell wall, ensure that the haemophilus parasuis survival time is longer, make the activity maintaining bacterium in transport haemophilus parasuis sample, reach the object improving and be separated plating efficiency.Progress of the present invention: 1) be convenient to transport in aseptic technique mode, haemophilus parasuis suspicious specimen bacterial strain is put into substratum of the present invention, is loaded in test tube, at room temperature, can be transported by the mode of regular bus shipping, preservation haulage time is 12 ~ 72h.2) improve and the substratum of bacterium probability substratum of the present invention as transport haemophilus parasuis suspicious specimen, at room temperature preserve transport, after arriving laboratory, carry out separation and Culture again, the success ratio of the first haemophilus parasuis bacterial strain be separated can be significantly improved.
Embodiment
Contriver is in recent years in the research of Haemophilus parasuis, repeatedly improve the flow process of cultivating this bacterium, sum up the substratum of transport haemophilus parasuis suspicious specimen, and substratum deliver to poultry advocate peace basic unit animal doctor personnel on hand, instruct them in the mode of aseptic technique, sample is put into substratum, contriver is transported on hand by the mode of regular bus shipping when room temperature, arrive laboratory and carry out inoculation separation haemophilus parasuis again, the method increase the plating efficiency being separated haemophilus parasuis.
Embodiment 1
Get 15g/L peptone, 5g/L Tryptones, 5g/L sodium-chlor, 2g/L glucose, 5g/L yeast leach liquor and 15 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.15g/L coenzyme A, with 10 ‰ sterilized water dissolved dilution filtration sterilizations; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 2
Get 14g/L peptone, 6g/L Tryptones, 5g/L sodium-chlor, 2g/L glucose, 5g/L yeast leach liquor and 15 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.14g/L coenzyme A, with 8 ‰ sterilized water dissolved dilution filtration sterilizations; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 3
Get 16g/L peptone, 4g/L Tryptones, 4g/L sodium-chlor, 3g/L glucose, 4g/L yeast leach liquor and 13 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.15g/L coenzyme A, filter out bacterium with 10 ‰ sterilized water dissolved dilutions; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 4
Get 15g/L peptone, 5g/L Tryptones, 4g/L sodium-chlor, 3g/L glucose, 6g/L yeast leach liquor and 18 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.16g/L coenzyme A, filter out bacterium with 12 ‰ sterilized water dissolved dilutions; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 5
Get 15g/L peptone, 5g/L Tryptones, 5g/L sodium-chlor, 2g/L glucose, 6g/L yeast leach liquor and 16 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.15g/L coenzyme A, with 10 ‰ sterilized water dissolved dilution filtration sterilizations; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 6
Get 14g/L peptone, 5g/L Tryptones, 4g/L sodium-chlor, 3g/L glucose, 6g/L yeast leach liquor and 14 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.15g/L coenzyme A, with 10 ‰ sterilized water dissolved dilution filtration sterilizations; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 7
Get 15g/L peptone, 5g/L Tryptones, 4g/L sodium-chlor, 3g/L glucose, 5g/L yeast leach liquor and 17 ‰ glycerine, surplus distilled water mixes, be heated to boiling and constantly stir until mixed solution dissolves completely, by solution by sodium hydroxide adjust ph to 7.0 ~ 7.2, the solution regulating pH value is carried out packing, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling; Get 0.15g/L coenzyme A, with 10 ‰ sterilized water dissolved dilution filtration sterilizations; Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C of preservations, obtain haemophilus parasuis substratum.
Embodiment 8
When substratum transport embodiment 1 prepared is separated haemophilus parasuis sample, the tissue samples such as the blood of aseptic collection, synovial fluid, hydrothorax, lung are put into substratum, be loaded in 10 ~ 15ml test tube, build cap seal to install, vertically be placed on (add appropriate ice bag when temperature is more than 40 DEG C, prevention temperature is too high) good seal transport in suitable bubble chamber.Can not stand upside down in transport, be transported to contriver on hand when room temperature by the mode of regular bus shipping, after arriving laboratory, culture medium inoculated is separated on the substratum being separated haemophilus parasuis.
Embodiment 9
Table 1: use substratum of the present invention preservation Transportation Organization sample and freezen protective ship sample to go out bacterium effectiveness comparison
Result shows: 1, the shelf time is at below 12h, and the sample separation plating efficiency of two kinds of preserving types does not have significant difference; Shelf time, substratum of the present invention isolated haemophilus parasuis, and freezen protective occurs without haemophilus parasuis between 12 ~ 72h; Shelf time, two kinds of methods all occurred without haemophilus parasuis at more than 72h.2, substratum of the present invention is at room temperature preserved transport and is arrived laboratory, still separable go out haemophilus parasuis, and freezen protective needs cooling, and isolates bacterium situation and be obviously inferior to substratum of the present invention.
Claims (8)
1. a haemophilus parasuis substratum, is characterized in that: be prepared from by following preparation method, its preparation method comprise basic culture solution preparation, coenzyme A process and substratum perfect, concrete steps are as follows:
1) the basic culture solution preparation described in
A, by component 14 ~ 16g/L peptone of basic culture solution, 4 ~ 6g/L Tryptones, 4 ~ 5g/L sodium-chlor, 2 ~ 3g/L glucose, 4 ~ 6g/L yeast leach liquor and 13 ~ 18 ‰ (count by volume, lower with) glycerine, surplus distilled water mixes;
B, the mixed solution that step A obtains is heated to boiling and constantly stirs until mixed solution dissolves completely;
C, by the solution that obtains in step B by sodium hydroxide adjust ph to 7.0 ~ 7.2;
D, the solution packing that step C is obtained, at 121 DEG C, sterilizing 15 ~ 20min, namely obtains basic culture solution after cooling;
2) the coenzyme A process described in
Get 0.14 ~ 0.16g/L coenzyme A, degerming with 8 ~ 12 ‰ sterilized water dilute filtrations;
3) substratum is perfect
Every 100ml basic culture solution adds 1ml coenzyme A diluent and the aseptic calf serum of 5ml with gnotobasis, and mixing, be dispensed into by substratum in test tube, often prop up test tube 10 ~ 15ml, 4 ~ 5 DEG C save backup.
2. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 5g/L, glucose is 2g/L, yeast leach liquor be 5g/L and glycerine is 15 ‰.
3. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 14g/L, Tryptones is 6g/L, sodium-chlor is 5g/L, glucose is 2g/L, yeast leach liquor be 5g/L and glycerine is 15 ‰.
4. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 16g/L, Tryptones is 4g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 4g/L and glycerine is 13 ‰.
5. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 6g/L and glycerine is 18 ‰.
6. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 5g/L, glucose is 2g/L, yeast leach liquor be 6g/L and glycerine is 16 ‰.
7. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 14g/L, Tryptones is 5g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 6g/L and glycerine is 14 ‰.
8. haemophilus parasuis substratum according to claim 1, is characterized in that: the component of described basic culture solution is peptone is 15g/L, Tryptones is 5g/L, sodium-chlor is 4g/L, glucose is 3g/L, yeast leach liquor be 5g/L and glycerine is 17 ‰.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667160B (en) * | 2013-12-30 | 2015-09-23 | 山东滨州博莱威生物技术有限公司 | A kind of haemophilus parasuis high density fermentation culture medium |
| CN105018347B (en) * | 2015-07-29 | 2017-11-28 | 傅石明 | Haemophilus preiservative methods |
| CN106282075A (en) * | 2016-11-02 | 2017-01-04 | 青岛易邦生物工程有限公司 | A kind of fluid medium for cultivating haemophilus parasuis |
| CN106434480A (en) * | 2016-11-02 | 2017-02-22 | 青岛易邦生物工程有限公司 | High-density fermentation and culture method of Haemophilus parasuis |
| CN107177531B (en) * | 2017-06-15 | 2018-07-06 | 广东海大畜牧兽医研究院有限公司 | A kind of growth accelerator for improving haemophilus parasuis in vitro culture |
| CN108977392B (en) * | 2018-08-14 | 2021-08-06 | 沈阳农业大学 | Haemophilus parasuis proliferation medium and preparation method thereof |
| CN112831428A (en) * | 2019-11-25 | 2021-05-25 | 北京信得威特科技有限公司 | Haemophilus parasuis culture medium |
| CN112625969B (en) * | 2020-12-31 | 2022-04-12 | 天津瑞普生物技术股份有限公司 | Culture medium for clinical separation of haemophilus parasuis and separation method |
| CN114149951A (en) * | 2021-12-30 | 2022-03-08 | 国药集团动物保健股份有限公司 | Haemophilus parasuis culture medium and preparation method thereof |
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