CN115369052B - Improved streptococcus inoculation liquid - Google Patents
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- 241000194017 Streptococcus Species 0.000 title claims abstract description 74
- 239000007788 liquid Substances 0.000 title claims abstract description 19
- 238000011081 inoculation Methods 0.000 title abstract description 15
- 210000004369 blood Anatomy 0.000 claims description 35
- 239000008280 blood Substances 0.000 claims description 35
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 30
- 239000001963 growth medium Substances 0.000 claims description 27
- 210000002966 serum Anatomy 0.000 claims description 26
- 239000000654 additive Substances 0.000 claims description 24
- 241001465754 Metazoa Species 0.000 claims description 22
- 239000002054 inoculum Substances 0.000 claims description 21
- 230000000996 additive effect Effects 0.000 claims description 20
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 15
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 15
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 15
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 15
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 15
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 15
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 150000002500 ions Chemical class 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 239000012091 fetal bovine serum Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 6
- 238000004806 packaging method and process Methods 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000000463 material Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 7
- 230000002195 synergetic effect Effects 0.000 abstract description 4
- 230000003042 antagnostic effect Effects 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 230000012010 growth Effects 0.000 description 26
- 230000000052 comparative effect Effects 0.000 description 16
- 230000035755 proliferation Effects 0.000 description 8
- 244000025254 Cannabis sativa Species 0.000 description 7
- 241000194046 Streptococcus intermedius Species 0.000 description 6
- 241000194024 Streptococcus salivarius Species 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000006781 columbia blood agar Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 241000194021 Streptococcus suis Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000194019 Streptococcus mutans Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 241001505901 Streptococcus sp. 'group A' Species 0.000 description 1
- 241000193990 Streptococcus sp. 'group B' Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000007478 blood agar base Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- ZXJXZNDDNMQXFV-UHFFFAOYSA-M crystal violet Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1[C+](C=1C=CC(=CC=1)N(C)C)C1=CC=C(N(C)C)C=C1 ZXJXZNDDNMQXFV-UHFFFAOYSA-M 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- OOYIOIOOWUGAHD-UHFFFAOYSA-L disodium;2',4',5',7'-tetrabromo-4,5,6,7-tetrachloro-3-oxospiro[2-benzofuran-1,9'-xanthene]-3',6'-diolate Chemical compound [Na+].[Na+].O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC(Br)=C([O-])C(Br)=C1OC1=C(Br)C([O-])=C(Br)C=C21 OOYIOIOOWUGAHD-UHFFFAOYSA-L 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229960002413 ferric citrate Drugs 0.000 description 1
- 229960001235 gentian violet Drugs 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 1
- 239000009384 kangtai Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to the field of IPC C12N1, and more particularly relates to an improved streptococcus inoculation liquid. By improving and culturing the traditional inoculation liquid formula, the streptococcus which grows weakly or does not grow originally can be cultured in a reasonable time, so that the culture capacity of the inoculation liquid is greatly improved, and meanwhile, the synergistic or antagonistic effect on the acting medicine is avoided.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to the field of IPCC12N1, and more particularly relates to an improved streptococcus inoculation liquid.
Background
The drug-sensitive inoculation culture solution is mainly used in an in-vitro drug sensitivity test and has the function of enrichment culture. 2.5-5wt% of streptococcus inoculating liquid for cracking horse blood is added as a traditional formula for in vitro drug sensitivity of streptococcus, and although most of the streptococcus growth problems such as group A streptococcus, group B streptococcus, streptococcus pneumoniae and the like can be solved, the situation that the growth of part of the streptococcus with more severe nutritional requirements such as constellation streptococcus, intermediate streptococcus, saliva streptococcus and the like in grass green streptococcus is weak, even no growth exists. Affecting clinical drug susceptibility testing of such samples.
In the prior art, the patent document with the application publication number of CN 104195216A discloses a KF streptococcus agar culture medium and application thereof, wherein the preparation method of the culture medium is simple, the cost is low, the culture medium has a promoting effect on the growth of faecal streptococcus, but has a small influence on the growth of fastidious bacteria such as constellation streptococcus, intermediate streptococcus, salivary streptococcus and the like.
Patent publication No. CN 104651468A discloses a Peak broth basal medium and application thereof, which can improve survival and proliferation of hemolytic streptococcus, but has no obvious promotion effect on the survival of strenuous streptococcus such as constellation streptococcus, intermediate streptococcus, salivary streptococcus and the like.
Therefore, there is a need for an improved Streptococcus inoculant that has improved inoculant culture capacity, no synergistic or antagonistic effects on the active agents, high stability, and low batch-to-batch variability.
Disclosure of Invention
In order to solve the above problems, the first aspect of the present invention provides an improved Streptococcus inoculant solution, comprising: culture medium, lysed blood, animal serum and ionic additives.
Preferably, the culture medium is one or more of camdb broth, eosin blue agar (EMB), middle Brook 7H10 broth, middle Brook 7H9 agar, MH broth (MHB), gentian violet blood agar base; further preferred is CAMHB broth.
The applicant has unexpectedly found that the selection of CAMHB broth as a culture medium not only promotes the growth of streptococcus mutans on streptococcus herbicolus, but also increases the stability of the inoculum. This is probably due to the fact that the CarMB broth contains appropriate amounts of beef extract, acid hydrolyzed casein, soluble starch, etc., which provides a sufficient carbon and nitrogen source for the growth of streptococci, and also contains trace amounts of calcium chloride which act synergistically with other components of the system to improve the stability and batch-to-batch reproducibility of the inoculum, but cells are not viable in simple media and require the addition of certain amounts of nutrients and growth factors to survive and proliferate.
Preferably, the lysed blood is one or more of defibrinated sheep blood, defibrinated goat blood, anticoagulated calf blood, anticoagulated horse blood and anticoagulated donkey blood; further preferred is anticoagulated lysed horse blood.
Preferably, the animal serum is one or more of chicken serum, fetal bovine serum, calf serum, newborn bovine serum, adult bovine serum, rabbit serum, pig serum and horse serum; further preferred is fetal bovine serum.
In some preferred embodiments, the use of fetal bovine serum as animal serum promotes the survival and proliferation of Streptococcus viridae, streptococcus intermedius, streptococcus salivarius, and the like. This is probably due to the fact that fetal bovine serum not only provides growth factors and low molecular nutrients to sustain cell growth, but also recognizes vitamins, metal ions and other hormones and coordinates their activities so that streptococcus suis, streptococcus intermedius, streptococcus salivarius, etc. can exhibit linear rapid proliferation. In addition, the fetal bovine serum contains copper ions with higher concentration, can be compounded with an ion additive to act as an enzyme auxiliary factor to drive neurotransmitter transmission and resist oxidative stress, eliminates the toxic action of free radicals on green grass streptococcus such as constellation streptococcus, intermediate streptococcus and salivary streptococcus, and promotes the growth and development of green grass streptococcus such as constellation streptococcus, intermediate streptococcus and salivary streptococcus. However, animal serum may carry a risk of contamination due to source problems, thereby causing batch-to-batch variation of the inoculum.
Preferably, the ionic additive is an aqueous solution composed of one or more of sodium chloride, calcium nitrate, manganese sulfate, magnesium sulfate, potassium hydrogen phosphate, ferric chloride, zinc chloride, sodium nitrate, ammonium chloride, ferric citrate, potassium chloride and sodium bicarbonate; further preferred is an aqueous solution composed of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate, and ferric chloride.
Preferably, the weight ratio of the sodium chloride to the calcium nitrate to the magnesium sulfate to the potassium hydrogen phosphate to the ferric chloride is 1: (0.1-0.5): (0.5-1): (0.5-1): (0.1-0.2); further preferably, it is 5:2:4:4:1.
the applicant has found unexpectedly that the weight ratio is 1: (0.1-0.5): (0.5-1): (0.5-1): the aqueous solution consisting of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride (0.1-0.2) is used as an ion additive, so that the culture capability of the inoculation liquid on streptococcus constellation, streptococcus intermedius, streptococcus salivarius and other green grass streptococcus can be improved. This is probably due to the synergistic effect of the positive and negative ions in the ionic additive, which not only regulates the transmembrane transport of many nutrients and macromolecules, but also serves as a cofactor for the enzymatic reaction, participating in the metabolism of the cells, helping the cells maintain osmotic pressure balance, thus allowing the green streptococcus such as streptococcus constellation, streptococcus intermedius, streptococcus salivarius and the like to survive and reproduce in the inoculum.
Preferably, the preparation steps of the ionic additive are as follows: weighing sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride according to the weight ratio, adding a proper amount of deionized water, and stirring for 5-20min to obtain the finished product.
Preferably, the total concentration of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride in the ionic additive is 0.1-5g/L; further preferably, it is 2g/L.
In a second aspect, the present invention provides a method of preparing an improved Streptococcus inoculant comprising the steps of:
s1, weighing 20-30g of culture medium, adding 1L of deionized water, heating to 30-50 ℃, stirring for 5-10min to dissolve the culture medium to obtain a culture medium solution, and sub-packaging into triangular bottles;
s2, heating the liquid in the triangular flask in the step S1 to 110-140 ℃, sterilizing at high temperature for 5-20min, cooling to below 50 ℃, adding the lysed blood, the animal serum and the ion additive, stirring for 1-10min, and packaging into sterile test tubes.
Preferably, the addition amount of the lysed blood in the step S2 is 2.5-5wt% of the culture medium solution; further preferably, it is 4wt%.
Preferably, the weight ratio of the lysed blood, the animal serum and the ionic additive in step S2 is (1-8): (2-10): 1, a step of; further preferably, it is 4:5:1.
the applicant has unexpectedly found that the addition of a certain amount of anticoagulated lysed horse blood as the lysed blood, and that the weight ratio of lysed blood, animal serum and ionic additives is (1-8): (2-10): 1, the proliferation of green streptococcus such as constellation streptococcus, intermediate streptococcus, salivary streptococcus and the like can be stimulated, and the proliferation rate can be accelerated. In addition, the addition of the lysed blood can reduce the use of animal serum, so that pollution and batch-to-batch differences caused by the instability of the animal serum can be avoided as much as possible, and the method can inhibit the growth of other bacterial colonies such as escherichia coli, salmonella and the like.
Preferably, the maximum volume of the flask in the step S1 is 250mL, and the volume of the culture medium solution in each flask is 100mL.
The beneficial effects are that:
1. by selecting the CAMHB broth as a culture medium, the growth of streptococcus inoculation liquid on streptococcus grass green can be promoted, and the stability of the inoculation liquid can be improved.
2. The survival and proliferation of streptococcus suis such as streptococcus constellation, streptococcus intermedius, streptococcus salivarius and the like can be promoted by selecting the fetal bovine serum as animal serum.
3. The weight ratio of the components is 1: (0.1-0.5): (0.5-1): (0.5-1): the aqueous solution consisting of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride (0.1-0.2) is used as an ion additive, so that the culture capability of the inoculation liquid on streptococcus constellation, streptococcus intermedius, streptococcus salivarius and other green grass streptococcus can be improved.
4. By selecting anticoagulated lysed horse blood as the lysed blood, the weight ratio of the lysed blood, animal serum and ion additive is (1-8): (2-10): 1, the proliferation of green streptococcus such as constellation streptococcus, intermediate streptococcus, salivary streptococcus and the like can be stimulated, and the proliferation rate can be accelerated. In addition, the addition of the lysed blood can reduce the use of animal serum, so that pollution and batch-to-batch differences caused by the instability of the animal serum can be avoided as much as possible, and the method can inhibit the growth of other bacterial colonies such as escherichia coli, salmonella and the like.
5. The method improves and cultures the traditional inoculation liquid formula, can culture streptococcus which grows weakly or does not grow in a reasonable time, greatly improves the culture capacity of the inoculation liquid, and has no synergistic or antagonistic effect on the acting medicine.
Detailed Description
Examples
Example 1
Example 1 provides an improved Streptococcus inoculant composition comprising: culture medium, lysed blood, animal serum and ionic additives.
The culture medium is CAMHB broth.
The camdb broth was purchased from camdb broth produced by the supplier peninsula maritime organisms.
The lysed blood is anticoagulated lysed horse blood.
The animal serum is fetal bovine serum.
The ion additive is an aqueous solution composed of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride.
The weight ratio of the sodium chloride, the calcium nitrate, the magnesium sulfate, the potassium hydrogen phosphate and the ferric chloride is 5:2:4:4:1.
the preparation steps of the ionic additive are as follows: weighing sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride according to the weight ratio, adding a proper amount of deionized water, and stirring for 15min to obtain the aqueous solution.
The total concentration of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride in the ion additive is 2g/L.
In a second aspect, the present invention provides a method of preparing an improved Streptococcus inoculant comprising the steps of:
s1, weighing 25g of culture medium, adding 1L of deionized water, heating to 40 ℃, stirring for 8min to dissolve the culture medium to obtain a culture medium solution, and sub-packaging into triangular flasks;
s2, heating the liquid in the triangular flask in the step S1 to 120 ℃, sterilizing at high temperature for 15min, cooling to 40 ℃, adding the lysed blood, the animal serum and the ion additive, stirring for 5min, and packaging into sterile test tubes.
The addition amount of the lysed blood in the step S2 is 4wt% of the culture medium solution.
The weight ratio of the pyrolysis blood, the animal serum and the ion additive in the step S2 is 4:5:1.
The maximum volume of the triangular flask in the step S1 is 250mL, and the volume of the culture medium solution in each triangular flask is 100mL.
Comparative example 1
Comparative example 1 provides an improved Streptococcus inoculum, which differs from example 1 in that: the raw materials comprise: culture medium, lysed blood. Only lysed blood was added in step S2.
Comparative example 2
Comparative example 2 provides an improved Streptococcus inoculum, which differs from example 1 in that: the culture medium is based on Columbia blood agar.
The Columbia blood agar base was purchased from Columbia blood agar base produced by the supplier Qingdao sea Bo organism.
Comparative example 3
Comparative example 3 provides an improved Streptococcus inoculum, which differs from example 1 in that: the weight ratio of the sodium chloride to the calcium nitrate to the magnesium sulfate to the potassium hydrogen phosphate to the ferric chloride is 10:1:1:1:1.
comparative example 4
Comparative example 4 provides an improved Streptococcus inoculum, which differs from example 1 in that: the weight ratio of the pyrolysis blood, the animal serum and the ion additive in the step S2 is 1:1:1.
performance test method
The Streptococcus inoculum prepared in example 1 and comparative example 1 was evaluated for the ability to cultivate by a growth test on 153 samples, wherein the same Streptococcus was a parallel test, and there was a possibility that there was a difference in the results and growth vigor between different strains of the same bacterium. All strains were from kangtai biotechnology limited in wenzhou.
And 52 samples which can grow in the inoculation liquid before and after the improvement are extracted for drug sensitivity test comparison, so that the improved inoculation liquid is ensured to have no obvious influence on drug sensitivity results.
TABLE 1 Vaccinability of the culture fluids prepared in comparative example 1 and example 1 against Streptococcus
Remarks: "+" represents "growth"; "-" means "no growth"; "+ -" stands for "weak growth"
Table 2 summary of the results in table 1
The data in Table 2 are for the number of growth, weak growth, no growth of Streptococcus in Table 1, and the number of growth not in example 1 is 29 for comparative example 1, which means that 29 Streptococcus samples which are not prone to growth can grow in the Streptococcus inoculum modified in the present application.
The results of counting 153 samples of growth in the Streptococcus inoculum prepared in example 1 and comparative examples 1-4, respectively, are reported in Table 3.
TABLE 3 statistical table of growth of Streptococci
| Growth | Weak growth | Does not grow | Totalizing | |
| Example 1 | 147 | 5 | 1 | 153 |
| Comparative example 1 | 98 | 20 | 35 | 153 |
| Comparative example 2 | 125 | 16 | 12 | 153 |
| Comparative example 3 | 139 | 8 | 6 | 153 |
| Comparative example 4 | 123 | 11 | 19 | 153 |
Analysis of results: by analyzing 153 samples, the modified streptococcus inoculating liquid CAMHB+LHB can better promote the growth of streptococcus, especially for the slow-growing grass green streptococcus.
Claims (4)
1. An improved Streptococcus inoculant comprising the following materials: culture medium, lysed blood, animal serum and ionic additives;
the culture medium is CAMHB broth;
the lysed blood is anticoagulated lysed horse blood;
the animal serum is fetal bovine serum;
the ionic additive is an aqueous solution composed of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride, and the weight ratio of the sodium chloride to the calcium nitrate to the magnesium sulfate to the potassium hydrogen phosphate to the ferric chloride is 1: (0.1-0.5): (0.5-1): (0.5-1): (0.1-0.2);
the weight ratio of the lysed blood, the animal serum and the ion additive is (1-8): (2-10): 1.
2. an improved Streptococcus inoculant solution according to claim 1, wherein the total concentration of sodium chloride, calcium nitrate, magnesium sulfate, potassium hydrogen phosphate and ferric chloride in the ionic additive is 0.1-5g/L.
3. A method of preparing an improved Streptococcus inoculant solution according to any one of claims 1-2, comprising the steps of:
s1, weighing 20-30g of culture medium, adding 1L of deionized water, heating to 30-50 ℃, stirring for 5-10min to dissolve the culture medium to obtain a culture medium solution, and sub-packaging into triangular bottles;
s2, heating the liquid in the triangular flask in the step S1 to 110-140 ℃, sterilizing at high temperature for 5-20min, cooling to below 50 ℃, adding the lysed blood, the animal serum and the ion additive, stirring for 1-10min, and packaging into sterile test tubes.
4. A method of preparing an improved Streptococcus inoculant solution as claimed in claim 3, wherein the amount of the blood to be lysed in step S2 is 2.5-5% by weight of the culture medium solution.
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| CN103820310A (en) * | 2014-02-15 | 2014-05-28 | 江苏中新医药有限公司 | Concentration gradient detection kit for drug sensitivity tests and measuring method thereof |
| CN108018244A (en) * | 2018-01-12 | 2018-05-11 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | A kind of novel neisseria gonorrhoeae fluid nutrient medium and its method applied to gonococcus medicament sensitivity test |
| CN112029815A (en) * | 2020-08-25 | 2020-12-04 | 惠州市阳光生物科技有限公司 | Group B streptococcus culture medium and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103820310A (en) * | 2014-02-15 | 2014-05-28 | 江苏中新医药有限公司 | Concentration gradient detection kit for drug sensitivity tests and measuring method thereof |
| CN108018244A (en) * | 2018-01-12 | 2018-05-11 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | A kind of novel neisseria gonorrhoeae fluid nutrient medium and its method applied to gonococcus medicament sensitivity test |
| CN112029815A (en) * | 2020-08-25 | 2020-12-04 | 惠州市阳光生物科技有限公司 | Group B streptococcus culture medium and preparation method thereof |
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