CN103571756B - Test tube screening method of isaria cicadae strain and culture medium - Google Patents
Test tube screening method of isaria cicadae strain and culture medium Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, and in particular discloses a test tube screening method of an isaria cicadae strain. The method comprises the following steps: collecting wild cordyceps cicadae or conidium thereof, and separating and purifying through a panel culture medium to obtain isaria cicadae single colony; picking the isaria cicadae single colony from the panel culture medium in the former step to a slope culture medium, and culturing to obtain conidium; collecting the conidium obtained from the former step, preparing spore solution, and inoculating to a test tube equipped with a test tube culture medium made of wheat; culturing and observing sporocarp, spore or hypha production condition. The test tube culture is adopted by the test tube screening method of the isaria cicadae strain, the occupied culture area is small, the operation is convenient, the culture period is short, the seed culture process in a shake flaks is not needed, the labor intensity is greatly reduced, and the energy consumption in the screening process is reduced; the wheat culture medium has good isaria cicadae strain selective culture effect.
Description
Technical field
The present invention relates to microorganism field, specifically disclose a kind of test tube screening method and substratum of cicada Isaria bacterial strain.
Background technology
Cicada fungus is medicinal entomogenous fungi, is China's tradition rare traditional Chinese medicine, has another name called cicada pupa grass, cicada is fine and soft, cicada, cicada etc. recklessly, and its natural host has bamboo cicada, a kind of cicadahun, mountain cicada, little ring cicada etc.Its medicinal beginning is loaded in the Lei Gong's Treatise on Preparation and Broiling of Materia Medica in Northern and Southern Dynasties Liu Song epoch.Traditional medicine is thought, cicada fungus has effect of dispelling wind and heat pathogens, promoting eruption, relievng spasm by subduing liver-wind, improving acuity of vision and removing nebula.Modern medicine study shows, it has immunomodulatory, improves body nutritional status, improves renal function, antitumor, antiviral, the different physiological roles such as antipyretic-antalgic, tranquilizing soporific, strengthening by means of tonics.Research confirms that cicada fungus contains abundant biologically active substance and nutritive ingredient, and cicada fungus contains the nutritive substances such as rich in protein, amino acid, trace element, lipid acid, VITAMIN and carbohydrate, is rare nutritive health-care food; In addition containing physiologically active ingredients such as polysaccharide, cordycepic acid, adenosine, ergosterol, hyaluronic acid, myriocin.
Cicada fungus is extensively approved by common people because of its pharmaceutical use, but wild cicada fungus resource rare and the restriction in season that produces, its output far can not be met the need of market.The artificial culture of cicada fungus will be the important channel of meeting the need of market.But, due to impacts such as host, environment, weathers, define the species diversity (Liu Aiying of cicada Isaria bacterial strain, Guizhou Agricultural Sciences, 2007,35 (2): 9 ~ 11), some bacterial strains are applicable to forming sporophore, some bacterial strains are applicable to producing conidium, and the bacterial strain also had is applicable to producing mycelia.How from abundant cicada Isaria bacterial strain, rapid screening goes out object bacterial strain, is the important step of artificial culture cicada Isaria.
Summary of the invention
The object of the invention is to the defect overcoming prior art, there is provided a kind of easy to operate, cultivation period is short, save the test tube screening method of cicada Isaria bacterial strain and the substratum of the energy, by Tube propagation to the product sporophore of bacterial strain, produce spore or produce mycelium ability and tentatively judge.
First aspect present invention discloses a kind of test tube sieve method of cicada Isaria bacterial strain, and step is as follows:
1) gathering wild cicada fungus or its conidium, by carrying out separation and purification containing antibiotic Potato-dextrose or containing antibiotic potato-sucrose plate substratum to it, obtaining cicada Isaria list bacterium colony;
2) from step 1) plate culture medium picking cicada Isaria list bacterium colony to Potato-dextrose or potato-sucrose slant medium, cultivate obtain conidium;
3) step 2 is collected) conidium that obtains, add aqua sterilisa and fully shake mixing, preparation spore liquid; Wheat is soaked in water after abundant imbibition and drains, the wheat after imbibition is loaded in test tube, and according to the wheat after imbibition: the mass ratio of water=1:1 ~ 1:1.5 adds water in test tube, tampon beyond the Great Wall, and sterilizing, prepares test-tube culture medium;
4) by step 3) spore liquid be inoculated in test-tube culture medium, cultivate and observe test tube sub-entities, spore or mycelia production.
Preferably, step 1) described containing antibiotic Potato-dextrose slat chain conveyor based formulas be:
The described antibiotic potato-sucrose plate culture medium prescription that contains is:
Preferably, step 1) culture condition of plate culture medium is 23 ~ 26 DEG C, dark culturing 4 ~ 6 days.
Preferably, step 2) described Potato-dextrose slant culture based formulas is: potato 15 ~ 25wt%, glucose 1.5 ~ 2.5wt%, agar 1.5 ~ 2.5wt%, surplus is water; Described potato-sucrose slant culture based formulas is: potato 15 ~ 25wt%, sucrose 1.5 ~ 2.5wt%, agar 1.5 ~ 2.5wt%, and surplus is water.
Preferably, step 2) culture condition of described slant medium is 23 ~ 25 DEG C, dark culturing 10 ~ 15 days.
Preferably, step 3) concentration of described spore liquid is 10
6~ 10
7individual/ml.
Preferably, step 3) in, often prop up the wheat after the imbibition of test tube loading 1/7 ~ 1/3 test tube volume.
Step 3) sterilising conditions is 121 DEG C of sterilizings 40 ~ 60 minutes.
Preferably, step 4) the spore liquid inoculum size that is inoculated into test-tube culture medium is 5% ~ 10v/w%; The culture condition of described test-tube culture medium is at 20 ~ 25 DEG C, and intensity of illumination 50 ~ 200Lux cultivates 20 ~ 25 days.
This law second aspect, discloses the application of a kind of wheat broth in screening cicada Isaria bacterial strain, described wheat broth consist of abundant imbibition and the wheat drained and water, and abundant imbibition the mass ratio of the wheat drained and water is 1:1 ~ 1:1.5.
Preferably, described abundant imbibition the preparation method of the wheat drained, for wheat is immersed in the water immersion 8 ~ 12h, takes out wheat and also drain.
The invention has the advantages that: 1) Tube propagation method of the present invention takies that culture area is little, easy to operate, cultivation period is short, cultivate sieve method compared to traditional Cans, test tube sieve method of the present invention decreases shake-flask seed culturing process, labour intensity reduces greatly, reduce energy consumption simultaneously; 2) growth type of wheat broth to cicada Isaria bacterial strain that have employed of novelty of the present invention is distinguished, and achieves cicada Isaria bacterial classification and selects culture effect preferably, can distinguish from field acquisition to the suitable Cultural type of cicada Isaria bacterial strain.
Accompanying drawing explanation
Fig. 1: the present invention is from embodiment 1 numbering 1 strain tube culture
Fig. 2: the present invention is from embodiment 2 numbering 2 strain tube culture
Fig. 3: the present invention is from embodiment 3 numbering 3 strain tube culture
Fig. 4: the present invention is from embodiment 4 numbering 1 bacterial strain Cans culture
Fig. 5: the present invention is from embodiment 4 numbering 2 bacterial strain Cans culture
Fig. 6: the present invention is from embodiment 4 numbering 3 bacterial strain Cans culture
Fig. 7: the present invention is from embodiment 5 numbering 1 bacterial strain Cans culture
Fig. 8: the present invention is from embodiment 5 numbering 2 bacterial strain Cans culture
Fig. 9: the present invention is from embodiment 5 numbering 3 bacterial strain Cans culture
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, should understand example not for limiting the scope of the invention.
Embodiment 1
From the wild cicada fungus of field acquisition (numbering 1), take back laboratory, with inoculating needle picking conidium to containing the antibiotic potato glucose plate culture medium of antibacterium, the composition of plate culture medium is: potato 20wt%, glucose 2wt%, agar 2wt%, microbiotic 0.01%.25 DEG C of dark culturing 4 days.From plate culture medium, picking cicada Isaria bacterium colony is in potato glucose slant medium, consisting of of slant medium: potato 15wt%, glucose 2.5wt%, agar 2.5wt%, cultivates 10 days for 25 DEG C, obtains numbering 1 bacterial strain.With the conidium of transfering loop from inclined-plane scraping maturation, add in aqua sterilisa and fully shake mixing, be mixed with 10
6the spore suspension of individual/ml.Drain after wheat soaked overnight, and in test tube, add the imbibition wheat (4g) of 1/4 test tube volume, and the 6ml that adds water, beyond the Great Wall tampon, 121 DEG C of sterilizings 50 minutes, cooling.Spore suspension is received on wheat broth according to 5v/w% inoculum size (0.5ml), repeats 3 pipes.20 DEG C of conditions, intensity of illumination 50 ~ 200Lux, cultivates 25 days.Culture taken out observation, describe.Experimental result as shown in Figure 1, all grows 1 ~ 2 root entity in every root test tube.
Embodiment 2
From the wild cicada fungus of field acquisition (numbering 2), take back laboratory, with inoculating needle picking conidium to containing in the antibiotic potato sucrose plate culture medium of antibacterium, consisting of of plate culture medium: 25wt%, glucose 1.5wt%, agar 2.5wt%, microbiotic 0.02%, 23 DEG C of dark culturing 5 days.From plate culture medium, picking cicada Isaria bacterium colony is in potato sucrose slant medium, consisting of of slant medium: potato 20wt%, sucrose 1.5wt%, agar 1.5wt%, cultivates 15 days for 23 DEG C, obtains numbering 2 bacterial strain.With the conidium of transfering loop from inclined-plane scraping maturation, add in aqua sterilisa and fully shake mixing, be mixed with 10
7the spore suspension of individual/ml.Drain after wheat soaked overnight, and in test tube, add the imbibition wheat (2.5g) of 1/7 test tube volume, and the 3.75ml that adds water, beyond the Great Wall tampon, 121 DEG C of sterilizings 50 minutes.Spore suspension is inoculated on wheat broth according to 10v/w% (0.625ml) inoculum size, repeats 3 pipes.25 DEG C of conditions, intensity of illumination 50 ~ 200Lux, cultivates 20 days.Culture taken out observation, describe.Experimental result as shown in Figure 2, all only produces conidium in every root test tube.
Embodiment 3
From the wild cicada fungus of field acquisition (numbering 3), take back laboratory, with inoculating needle picking conidium to containing the antibiotic potato sucrose plate culture medium of antibacterium, consisting of of plate culture medium: 15wt%, sucrose 2.5wt%, agar 1.5wt%, microbiotic 0.015%, 24 DEG C of dark culturing 5 days.From plate culture medium, picking cicada Isaria bacterium colony is in potato sucrose slant medium, consisting of of slant medium: potato 25wt%, sucrose 2.0wt%, agar 2.5wt%, cultivates 13 days for 24 DEG C, obtains numbering 3 bacterial strain.With the conidium of transfering loop from inclined-plane scraping maturation, add in aqua sterilisa and fully shake mixing, be mixed with 5 × 10
6the spore suspension of individual/ml.Drain after wheat soaked overnight, and in test tube, add the imbibition wheat (5g) of 1/3 test tube volume, and add 5ml water, tampon beyond the Great Wall, 121 DEG C of sterilizings 50 minutes.Spore suspension is inoculated on wheat broth according to 7.5v/w% (0.75ml) inoculum size, repeats 3 pipes.Temperature 23 DEG C, intensity of illumination 50 ~ 200Lux, cultivates 23 days.Culture taken out observation, describe.Experimental result as shown in Figure 3, all only long mycelia in every root test tube.
Embodiment 4
By numbering 1 bacterial strain, numbering 2 bacterial strain, the ripe inclined-plane of numbering 3 strain culturing, with inoculation transfering loop scraping conidium, access in potato sucrose Shake flask medium respectively, 25 DEG C, 150rmp, shaking table cultivates 3 days.
After being cleaned by wheat, soak, draining, add the water of geometric ratio, 121 DEG C of sterilizings 50 minutes, cooling.Shake-flask seed liquid is inoculated on wheat broth in 5v/w% ratio respectively, fully mixes, repeat 5 bottles.Culture temperature 23 DEG C, intensity of illumination 50 ~ 200Lux, cultivates 25 days.Culture taken out observation, describe, and comparative examples 1, embodiment 2, embodiment 3 are evaluated.
Result shows, and numbering 1 bacterial strain every bottle has all grown more sporophore, and experimental result is shown in Fig. 4; Numbering 2 bacterial strain every bottle all only produces conidium, and experimental result is shown in Fig. 5; Numbering 3 bacterial strain every bottle all only produces mycelia, and experimental result is shown in Fig. 6; Above-mentioned experimental result shows, it is all consistent with the present embodiment that embodiment 1-3 method obtains strain growth characteristic, confirm that cicada Isaria strain tube sieve method of the present invention accurately can obtain the growth characteristics of gathered wild cicada fungus, and experimental result has repeatability rapid screening can go out object bacterial strain, for the artificial culture of cicada Isaria from abundant cicada Isaria bacterial strain.
Control experiment
By numbering 1 bacterial strain, numbering 2 bacterial strain, the ripe inclined-plane of numbering 3 strain culturing, with inoculation transfering loop scraping conidium, access in potato sucrose Shake flask medium respectively, 25 DEG C, 150rmp, shaking table cultivates 3 days.The water of 1.5 times of weight is added, 121 DEG C of sterilizings 30 minutes, cooling in rice.Shake-flask seed liquid is inoculated on rice medium in 5v/w% ratio respectively, fully mixes, repeat 5 bottles.Culture temperature 23 DEG C, intensity of illumination 50 ~ 200Lux, cultivates 25 days.By culture take out observe, describe, and with embodiment 4 comparative evaluation.
Result shows, and numbering 1 bacterial strain every bottle all grows a large amount of conidium, and sporophore almost can not grow, and experimental result is shown in Fig. 7; Numbering 2 bacterial strain every bottle all only produces conidium, and experimental result is shown in Fig. 8; Numbering 3 bacterial strain every bottle all produces a small amount of mycelia, and with a large amount of conidium, experimental result is shown in Fig. 9; Above-mentioned experimental result shows, it is all relevant with producing conidium that embodiment 1-3 method obtains strain growth characteristic, and the bacterial strain that some can not be had particular growth characteristic by the substratum showing in embodiment 5 distinguishes.
Claims (10)
1. a test tube sieve method for cicada Isaria bacterial strain, step is as follows:
1) gathering wild cicada fungus or its conidium, by carrying out separation and purification containing antibiotic Potato-dextrose or containing antibiotic potato-sucrose plate substratum to it, obtaining cicada Isaria list bacterium colony;
2) from step 1) plate culture medium picking cicada Isaria list bacterium colony to Potato-dextrose or potato-sucrose slant medium, cultivate obtain conidium;
3) step 2 is collected) conidium that obtains, add aqua sterilisa and fully shake mixing, preparation spore liquid; Wheat is soaked in water after abundant imbibition and drains, the wheat after imbibition is loaded in test tube, and according to the wheat after imbibition: the mass ratio of water=1:1 ~ 1:1.5 adds water in test tube, tampon beyond the Great Wall, and sterilizing, prepares test-tube culture medium;
4) by step 3) spore liquid be inoculated in test-tube culture medium, cultivate and observe test tube sub-entities, spore or mycelia production.
2. sieve method as claimed in claim 1, is characterized in that, step 1) the described formula containing antibiotic Potato-dextrose plate culture medium is:
The described formula containing antibiotic potato-sucrose plate substratum is:
3. sieve method as claimed in claim 1, is characterized in that, step 1) culture condition of described plate culture medium is 23 ~ 26 DEG C, dark culturing 4 ~ 6 days.
4. sieve method as claimed in claim 1, is characterized in that, step 2) formula of described Potato-dextrose slant medium is: potato 15 ~ 25wt%, glucose 1.5 ~ 2.5wt%, agar 1.5 ~ 2.5wt%, surplus is water; The formula of described potato-sucrose slant medium is: potato 15 ~ 25wt%, sucrose 1.5 ~ 2.5wt%, agar 1.5 ~ 2.5wt%, and surplus is water.
5. sieve method as claimed in claim 1, is characterized in that, step 2) culture condition of described slant medium is 23 ~ 25 DEG C, dark culturing 10 ~ 15 days.
6. sieve method as claimed in claim 1, is characterized in that, step 3) concentration of described spore liquid is 10
6~ 10
7individual/ml.
7. sieve method as claimed in claim 1, is characterized in that, step 3) in, often prop up the wheat after the imbibition of test tube loading 1/7 ~ 1/3 test tube volume.
8. sieve method as claimed in claim 1, is characterized in that, step 4) the spore liquid inoculum size that is inoculated into test-tube culture medium is 5 ~ 10v/w%; The culture condition of described test-tube culture medium is at 20 ~ 25 DEG C, and intensity of illumination 50 ~ 200Lux cultivates 20 ~ 25 days.
9. the application of wheat broth in screening cicada Isaria bacterial strain, is characterized in that, described wheat broth consist of abundant imbibition and the wheat drained and water, and abundant imbibition the mass ratio of the wheat drained and water is 1:1 ~ 1:1.5.
10. apply as claimed in claim 9, it is characterized in that, described abundant imbibition the preparation method of the wheat drained, for wheat is immersed in the water immersion 8 ~ 12h, takes out wheat and also drain.
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| CN106912293B (en) * | 2015-12-24 | 2020-08-25 | 浙江泛亚生物医药股份有限公司 | Artificial culture method of cordyceps sobolifera |
| CN106190861A (en) * | 2016-07-19 | 2016-12-07 | 湖州新驰医药科技有限公司 | The bacterial screening of a kind of artificial isaria cicadae miq, preparation and productive culture method |
| CN106978468B (en) * | 2017-05-27 | 2020-03-24 | 福建省农业科学院植物保护研究所 | Isaria fumosorosea conidia culture medium for greenhouse collection and separation and preparation method thereof |
| CN107176697A (en) * | 2017-07-01 | 2017-09-19 | 贵州大学 | Cicada fungus flocculant |
| CN108040752B (en) * | 2017-12-01 | 2020-03-31 | 南京中医药大学 | Artificial cultivation method of isaria cicadae and traditional Chinese medicine cordyceps sobolifera |
| CN111269840B (en) * | 2020-03-06 | 2022-02-22 | 山西农业大学 | Isaria fumosorosea fermentation and spore powder preparation process |
| CN114214199B (en) * | 2022-01-07 | 2023-08-22 | 沈阳农业大学 | Preservation method of Isaria cicadae spore powder |
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