CN104894213A - Fn (fusobacterium nucleatum) culture medium for tartar specimen - Google Patents

Abstract

The invention discloses an Fn (fusobacterium nucleatum) culture medium for a tartar specimen and belongs to the field of culture of test microorganisms. The Fn culture medium is characterized in that 1,000 ml of the prepared culture medium comprises casein tryptone, pea starch, yeast powder, sodium chloride, agar, ethylenediamine tetraacetic acid dipotassium magnesium salt, valine, L-homoarginine, sterile thermal deformation calf serum and the balance of distilled water. Compared with the prior art, the Fn culture medium has the characteristics of detection reliability and high separation rate of Fn.

Description

For the Fusobacterium nucleatum substratum of tartar sample

Technical field

The present invention relates to inspection microorganism culturing field, be specifically related to the Fusobacterium nucleatum Selective agar medium for tartar sample.

Background technology

Microorganism culturing is a kind of technology making microbial growth by artificial means, and the substratum that selective medium is used to promote or suppress the organism (as cell or bacterium etc.) of certain type and designs, utilize this substratum required microorganism can be separated from the microorganism mixed.Periodontopathy is one of large class principal disease in oral cavity two, very high sickness rate is had in China, Fusobacterium nucleatum (Fusobacterium nucleatum, Fn) gram-negative obligate anaerobic bacillus(cillus anaerobicus) is belonged to, closely related with the inflammatory reaction of periodontal disease, being active microorganism in periodontopathy, is also one of putative pathogen of adult periodontitis and juvenile periodontitis.This bacterium, except can causing oral disease, also can cause the infection at other positions of health, comprises brain, lung and liver, blood and joint, chest and belly.At present, Fusobacterium nucleatum measuring means mainly subgingival plaque sampling carries out polymerase chain reaction (polymerase chain reaction, PCR) this kind of method needs to carry out DNA extraction and PCR primer design to sample, and need to be equipped with PCR related equipment, cost intensive, complicated operation.Laboratory is commonly used CDC anaerobic agar and is cultivated Fusobacterium nucleatum, because Clinical Laboratory In Grass Root Hospital lacks anaerobic inspection machine mostly, therefore multiplex greatly " anaerobism cylinder culture method ", so-called anaerobism cylinder is common dry cylinder, the cultivation of dry cylinder put into by flat board or the liquid nutrient medium test tube of having inoculated sample, exhaust oxygen in cylinder by physicochemical method, cause anaerobic environment.The oxygen depletion of anaerobism cylinder method needs the time, and Fusobacterium nucleatum is obligate anaerobic bacillus, therefore there is following defect in this kind of culture technique: the Fusobacterium nucleatum poor growth 1. in anaerobism cylinder on CDC anaerobic agar substratum, and positive rate is low, and within one week, qualification can be made more accurately in right and left; 2. due to non-absolute oxygen free condition, therefore in tartar sample, aerophil and facultative anaerobe have life condition, and substratum separating power is poor.In addition, be difficult to the Detection of Porphyromonas in tartar sample, Pu Shi bacillus, peptostreptococcus in existing CDC anaerobic agar culture medium culturing process, and Streptococcus hemolyticus, streptococcus mutans, Veillonella, streptococcus aureus, Yi Shi and the oral cavity such as actinomyces naeslundii, Candida albicans are resided bacterium and are separated, the growth affecting Fusobacterium nucleatum be separated.

Summary of the invention

Technical assignment of the present invention is for above the deficiencies in the prior art, provides a kind of Selective agar medium being suitable for Fusobacterium nucleatum growth.

The technical scheme that the present invention solves its technical problem is: for the Fusobacterium nucleatum substratum of tartar sample, it is characterized in that, contains in the formula of preparation 1000ml substratum:

Pancreas casein peptone 5.0 ~ 10.0g;

Pea starch 2.0 ~ 5.0g;

Yeast powder 5.0 ~ 10.0g;

Sodium-chlor 5.0g;

Agar 15.0 ~ 20.0g;

Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2 ~ 3g;

α-amino-isovaleric acid 0.1 ~ 0.5g;

L-homoarginine 0.1 ~ 0.5g;

Aseptic thermal distortion calf serum 50 ~ 100ml;

Distilled water adds to 1000ml.

Above-mentioned aseptic thermal distortion calf serum preparation method is: heat 10min at getting aseptic calf serum 80 DEG C, add sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.

The preparation method of above-mentioned substratum is:

(1) pea starch is taken at 50 ~ 70 DEG C, gelatinization in 100ml distilled water;

(2) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; L-homoarginine, mix with the pasted starch that distilled water dissolves afterwards and step (1) obtains, 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Described pancreas casein peptone contains various saccharides, amino acid, VITAMIN and trace element provides energy for Fusobacterium nucleatum grows, pea starch provides carbon source, the nutritive ingredients such as yeast powder rich in proteins, amino acid, polypeptide, Nucleotide, VITAMIN, somatomedin, trace element, promote the Growth and Reproduction of Fusobacterium nucleatum; Sodium-chlor maintains balanced osmotic pressure; Agar is the peptizer of substratum; α-amino-isovaleric acid is anerobe nutrition agent; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt provides the inorganic salt needed for Fusobacterium nucleatum growth, and the ethylenediamine tetraacetic acid dipotassium magnesium salt salt under this concentration is attached to bacteria cell wall by electrical complexing action, cellularstructure is caused to be out of shape, carry out playing antibacterial sterilization functions, and outer membrane protein OMP on Fusobacterium nucleatum cell walls and fadA adhesion protein make the cell wall area iso-electric point of Fusobacterium nucleatum not mate with ethylenediamine tetraacetic acid dipotassium magnesium salt salt, electrical complexing action is caused to lose efficacy, therefore antibacterial inefficacy.By experiment respectively by common for oral cavity anaerobe porphyromonas gingivalis Zymomonas mobilis, Pu Shi bacillus, Veillonella, Fusobacterium nucleatum inoculation is on the ethylenediamine tetraacetic acid dipotassium magnesium salt salt CDC anaerobic selection substratum adding 3g/L, control group is common CDC anerobe Selective agar medium, in 37 DEG C, observe after cultivating 48h in anaerobic jar, the substratum of interpolation EDTA Dipotassium salt finds small-sized Fusobacterium nucleatum translucent colony, do not find Detection of Porphyromonas, Pu Shi bacillus, Veillonella bacterium colony, control group substratum finds Detection of Porphyromonas, Pu Shi bacillus, Veillonella, Fusobacterium nucleatum bacterium colony, confirm that EDTA Dipotassium salt is to Detection of Porphyromonas thus, Pu Shi bacillus, Veillonella has good sterilization functions, and Fusobacterium nucleatum is had no significant effect.

Thermal distortion calf serum has optionally antibacterial effect, and its thermal distortion method is: heat 10min at getting aseptic calf serum 80 DEG C, adds sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.Thermal distortion calf serum antibacterial properties is for aerophil, change aerophil cell wall structure on the one hand, cause permeability increase and dead, the redox-potential of substratum can be reduced on the other hand, promote oxygen-free environment and accelerate aerophil death, and this kind of anti-microbial effect is only directed to aerophil, then invalid for anerobe.By experiment same coli strain and Fusobacterium nucleatum bacterial strain are inoculated into respectively and with the addition of on the optimization CDC Selective agar medium of the thermal distortion calf serum of 80ml/L in CDC Selective agar medium and formula, cultivate at 37 DEG C, hermetically drying cylinder ring border, 24h, 48h identify, adding thermal distortion calf serum intestinal bacteria positive rate during 24h is 50%, and on the substratum not adding thermal distortion calf serum, intestinal bacteria positive rate is 80%; 48h detected result is add the substratum Fusobacterium nucleatum positive rate 70% of thermal distortion calf serum, Fusobacterium nucleatum positive rate 20% on the substratum not adding thermal distortion calf serum; Confirm that thermal distortion calf serum has good inhibition to aerophil in anaerobism cylinder thus, and promote that anerobe grows by accelerating oxygen-free environment.L-homoarginine is activator, promotes the absorption of α-amino-isovaleric acid; γ-aminobutyric acid is nutrient intensifier, promotes Fusobacterium nucleatum growth; Rhizome of Grass leaf Sweelflag (formal name used at school: Acorus gramineus) belongs to Acoraceae, and be graminoid per nnial herb, medicinal effects is rhizome position.Modern medicine study shows, Rhizome of Grass leaf Sweelflag is containing compositions such as volatile oil and carbohydrate, organic acid, amino acid, inorganic elements such as α-trans-Isoasarones, there are the effects such as swelling and pain relieving, waking up the patient from unconsciousness by dissipating phlegm, ulcer mange, Rhizome of Grass leaf Sweelflag Aqueous extracts is good nutrition-fortifying agent, Rhizome of Grass leaf Sweelflag water logging agent (1:3), all has restraining effect in various degree to dermatophytess such as trichophyton, Trichophyton concentricum, star-shaped nocardias in vitro.Have report to point out, Rhizome of Grass leaf Sweelflag decoction has the effect of killing ascites cells.The research such as Masuda T finds that the α-trans-Isoasarone in Rhizome of Grass leaf Sweelflag all has restraining effect to streptococcus aureus, Candida albicans, intestinal bacteria etc.Experiment confirms, Rhizome of Grass leaf Sweelflag water cooking liquid under 200g/L concentration there is no killing action for anerobe, take anaerobic culture medium as contrast, to add 200g/L Rhizome of Grass leaf Sweelflag water cooking liquid in Anaerobic culturel based formulas for experimental group, inoculation Fusobacterium nucleatum bacterial strain, the positive rate of 96h is respectively 60% and 70%, and the two compares no significant difference (P>0.05).

The present invention compared with prior art, has and detects the feature reliable, Fusobacterium nucleatum separation rate is high.

Embodiment

Below in conjunction with practical situation, the specific embodiment of the present invention is elaborated.

Embodiment 1, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 10.0g; Pea starch 2.0g; Yeast powder 5.0g; Sodium-chlor 5.0g; Agar 15.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 3g; α-amino-isovaleric acid 0.1g; Aseptic thermal distortion calf serum 100ml; Distilled water adds to 1000ml.Preparation method: (1) takes pea starch at 70 DEG C, gelatinization in 100ml distilled water; (2) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid, mix with the pasted starch that distilled water dissolves afterwards and step (1) obtains, 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Described aseptic thermal distortion calf serum preparation method heats 10min at getting aseptic calf serum 80 DEG C, adds sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.Aseptic thermal distortion calf serum preparation method in other embodiments is in the same manner as in Example 1.

Embodiment 2, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 8.0g; Pea starch 5.0g; Yeast powder 8.0g; Sodium-chlor 5.0g; Agar 15.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2.5g; α-amino-isovaleric acid 0.3g; L-homoarginine 0.1g; Aseptic thermal distortion calf serum 50ml; Distilled water adds to 1000ml.Preparation method: (1) takes pea starch at 50 DEG C, gelatinization in 100ml distilled water; (2) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; L-homoarginine, mix with the pasted starch that distilled water dissolves afterwards and step (1) obtains, 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 3, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 10.0g; Pea starch 3.0g; Yeast powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 3g; α-amino-isovaleric acid 0.5g; γ-aminobutyric acid 0.01g; Aseptic thermal distortion calf serum 80ml; Distilled water adds to 1000ml.Preparation method: (1) takes pea starch at 70 DEG C, gelatinization in 100ml distilled water; (2) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; γ-aminobutyric acid, mix with the pasted starch that distilled water dissolves afterwards and step (1) obtains, 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 4, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 5.0g; Pea starch 5.0g; Yeast powder 8.0g; Sodium-chlor 5.0g; Agar 20.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2g; α-amino-isovaleric acid 0.1g; Aseptic thermal distortion calf serum 80ml; 200g/L Rhizome of Grass leaf Sweelflag Aqueous extracts 200ml; Distilled water adds to 1000ml.Preparation method: (1) is got 40g Rhizome of Grass leaf Sweelflag and added water boil 30min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 200ml; (2) pea starch is taken at 60 DEG C, gelatinization in 100ml distilled water; (3) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid, mix with the pasted starch that distilled water dissolves afterwards and step (2) obtains, then add in the Rhizoma Acori Gramineii extract liquid of step (1) gained and mix 121 DEG C of sterilizing 15min, be cooled to 45 DEG C, add the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 5, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 10.0g; Pea starch 2.0g; Yeast powder 5.0g; Sodium-chlor 5.0g; Agar 18.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2.5g; α-amino-isovaleric acid 0.3g; L-homoarginine 0.3g; γ-aminobutyric acid 0.05g; Aseptic thermal distortion calf serum 80ml; Distilled water adds to 1000ml.Preparation method: (1) takes pea starch at 50 DEG C, gelatinization in 100ml distilled water; (2) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; L-homoarginine; γ-aminobutyric acid, mix with the pasted starch that distilled water dissolves afterwards and step (1) obtains, 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 6, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 5.0g; Pea starch 5.0g; Yeast powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2g; α-amino-isovaleric acid 0.3g; L-homoarginine 0.3g; Aseptic thermal distortion calf serum 80ml; The Rhizome of Grass leaf Sweelflag Aqueous extracts 250ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 50g Rhizome of Grass leaf Sweelflag and added water boil 45min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 250ml; (2) pea starch is taken at 70 DEG C, gelatinization in 100ml distilled water; (3) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; L-homoarginine, mix with the pasted starch that distilled water dissolves afterwards and step (2) obtains, then add in the Rhizoma Acori Gramineii extract liquid of step (1) gained and mix 121 DEG C of sterilizing 15min, be cooled to 45 DEG C, add the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 7, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 8.0g; Pea starch 5.0g; Yeast powder 8.0g; Sodium-chlor 5.0g; Agar 15.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2.5g; α-amino-isovaleric acid 0.3g; γ-aminobutyric acid 0.1g; Aseptic thermal distortion calf serum 50ml; The Rhizome of Grass leaf Sweelflag Aqueous extracts 300ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 60g Rhizome of Grass leaf Sweelflag and added water boil 60min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 300ml; (2) pea starch is taken at 60 DEG C, gelatinization in 100ml distilled water; (3) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; γ-aminobutyric acid, mix with the pasted starch that distilled water dissolves afterwards and step (2) obtains, then add in the Rhizoma Acori Gramineii extract liquid of step (1) gained and mix 121 DEG C of sterilizing 15min, be cooled to 45 DEG C, add the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 8, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 8.0g; Pea starch 3.0g; Yeast powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 3g; α-amino-isovaleric acid 0.3g; L-homoarginine 0.5g; γ-aminobutyric acid 0.01g; Aseptic thermal distortion calf serum 100ml; The Rhizome of Grass leaf Sweelflag Aqueous extracts 300ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: preparation method: (1) is got 60g Rhizome of Grass leaf Sweelflag and added water boil 60min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 300ml; (2) pea starch is taken at 60 DEG C, gelatinization in 100ml distilled water; (3) pancreas casein peptone is taken; Yeast powder; Sodium-chlor; Agar; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; α-amino-isovaleric acid; L-homoarginine; The pasted starch distilled water that γ-aminobutyric acid and step (2) obtain dissolves, mixes, be dissolved in the Rhizoma Acori Gramineii extract liquid of step gained and mix 121 DEG C of sterilizing 15min, be cooled to 45 DEG C, add the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Described Rhizome of Grass leaf Sweelflag Aqueous extracts concentration 200g/L refers to often liter containing crude drug 200g.

The Fusobacterium nucleatum substratum of gained of the present invention is used for tartar sample, and have and detect the feature reliable, Fusobacterium nucleatum separation rate is high, be clinical data sufficient proof, pertinent data is as follows.

1 object and method.

1.1 medium preparing: experimental group establishes 5 groups altogether, are respectively embodiment 1 scheme group, embodiment 2 scheme group, embodiment 3 scheme group, embodiment 6 scheme group, embodiment 8 scheme group.Control group is common CDC anaerobic agar, and its formula is: pancreas casein peptone 15g/L, soy peptone 5g/L, yeast powder 5g/L; CYSTINE hydrochloride 0.4g/L; Agar 15g/L, sodium-chlor 5g/L, aseptic vitamin K 0.01g/L, aseptic protohemine 0.005g/L, aseptic Sheep Blood 50ml/L.

1.2 bacterial plaques gather cultural method: patients with periodontal disease 32 example, and every wet saliva, supragingival scaler removes supragingival calculus, after iodine tincture disinfection, gathers 2 diseased tooth subgingival plaques with aseptic Gracey curettage instrument.The bacterial plaque of collection is suspended in 1ml sterile saline, is inoculated in each experimental group and control group substratum with transfering loop sectional streak.At 37 DEG C, anaerobism cylinder is cultivated, and carries out identification of bacteria respectively at the tiny bacterium colony of 48h, 96h picking.Each sample number censorship PCR does molecular biology identification.

1.3 detect positive criteria: take out culture dish, and observe colony growth situation on substratum, thalline is elongated, and without gemma, two ends point, sometimes in long filament shape, bacterium colony is translucent, and edge is irregular.Check according to " clinical microbiology diagnosis and diagram ", net result gold standard is PCR qualification.

2 results: it is 29 examples that 32 parts of patients with periodontal disease tartar samples cultivate what be finally accredited as the Fusobacterium nucleatum positive through PCR.Each group of 48h detects the positive, false positive, feminine gender, false negative bacterial strain number of cases see the following form,

Grouping	Sum	PCR is positive	Positive	False positive	Negative	False negative
							Embodiment 1	32	29	18	2	14	13
Embodiment 2	32	29	20	1	12	10
							Embodiment 3	32	29	21	1	11	9
Embodiment 6	32	29	20	1	12	10
							Embodiment 8	32	29	22	0	10	7
Control group (CDC)	32	29	5	1	27	25

The above results can be found out, cultivate after 48 hours, each embodiment group and control group positive rate more all have pole notable difference (P<0.001).

Each group of 96h detects the positive, false positive, feminine gender, false negative bacterial strain number of cases see the following form,

Grouping	Sum	PCR is positive	Positive	False positive	Negative	False negative
							Embodiment 1	32	29	24	1	8	6
Embodiment 2	32	29	24	1	8	6
							Embodiment 3	32	29	24	0	8	5
Embodiment 6	32	29	25	0	7	4
							Embodiment 8	32	29	26	0	10	3
Control group (CDC)	32	29	16	2	16	15

The above results can be found out, cultivate after 96 hours, the positive rate of control group (CDC) increases, but each embodiment group compares with control group positive rate, all has notable difference (P<0.05).Each embodiment group compares with PCR detected result, positive rate no significant difference (P>0.05), and control group positive rate is then starkly lower than PCR detected result (P<0.001).

Above result shows, tartar sample is after cultivating in the dry cylinder of embodiment of the present invention substratum, occur during 48h rising appreciably, separation rate is apparently higher than existing substratum (CDC), after 96 hours, each embodiment and control group positive rate difference reduce, but difference still has statistical significance, each embodiment true positives recall rate detects with PCR and compares, no difference of science of statistics, and in common CDC anaerobic agar substratum anaerobism cylinder, cultivate under-nutrition, miscellaneous bacteria breeding rapidly, Fusobacterium nucleatum poor growth, although there is obvious bacterial strain Growth positive during 96h, but positive rate is still starkly lower than PCR detected result, do not play diagnostic effect.Substratum of the present invention is used for cultivating Fusobacterium nucleatum under dry cylinder ring border, and detect reliable, separation rate is high, thus the time is made a definite diagnosis in shortening.

Claims (1)

1. for the Fusobacterium nucleatum substratum of tartar sample, it is characterized in that: contain in often liter of substratum: pancreas casein peptone 5.0 ~ 10.0g; Pea starch 2.0 ~ 5.0g; Yeast powder 5.0 ~ 10.0g; Sodium-chlor 5.0g; Agar 15.0 ~ 20.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2 ~ 3g; α-amino-isovaleric acid 0.1 ~ 0.5g; L-homoarginine 0.1 ~ 0.5g; Aseptic thermal distortion calf serum 50 ~ 100ml; Distilled water adds to 1000ml; Wherein said aseptic thermal distortion calf serum preparation method is: heat 10min at getting aseptic calf serum 80 DEG C, add sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.