CN103966141B - A kind of quick screening antibacterial activity bacterium and the method for actinomyces - Google Patents
A kind of quick screening antibacterial activity bacterium and the method for actinomyces Download PDFInfo
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- CN103966141B CN103966141B CN201410212912.5A CN201410212912A CN103966141B CN 103966141 B CN103966141 B CN 103966141B CN 201410212912 A CN201410212912 A CN 201410212912A CN 103966141 B CN103966141 B CN 103966141B
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Abstract
The present invention relates to a kind of quick screening antibacterial activity bacterium and the method for actinomyces, its step includes（1）Sample collection；（2）It is prepared by culture medium；（3）Bacterium and actinomyces separate；（4）Intercept the preparation of suction nozzle；（5）Antibacterial activity bacterial strain screening.Extracting method of the present invention rapidly screens antibacterial activity bacterium and actinomyces by way of co-culturing, compared with antibacterial activity bacterium and actinomyces are screened in purebred culture, the probability of novel anti-bacterial active bacterial strain and antibacterial substance can be increased, and whole screening process is quick, it is simple to operate, labour intensity is smaller, can quickly, intuitively, efficiently screen the bacterium and actinomyces of antibacterial activity, for the screening study of antibacterial activity bacterium and actinomyces is provided greatly conveniently.
Description
Technical field
In particular it is that a kind of quick screening antibacterial activity is thin the present invention relates to a kind of fast separating process of microorganism
Bacterium and the method for actinomyces.
Background technology
Long-term the widely using of antibiotic causes the increase of drug resistance pathogenetic bacteria species, or even " superbacteria " occurs, newly
Infectious diseases also continue to bring out, cause the research and development of novel antibacterial material extremely urgent（Li Yuezhong, Chen Qi seas
The research of foreign microbial resources and its generation bioactive metabolite, bioengineering progress, 2000,20 (5): 28-31）.
Found in the development process that studies for a long period of time to microbial resources, new antibacterial material is found from Secondary Metabolites of Microorganisms
Difficulty is increasing.The screening technique of antibiotic directly affects the screening efficiency of antibiotics.（Wang Xing, Wu Wenhui, Chen Zhi
China, Zhang Jie wraps the architectural feature of refined marine microorganisms secondary metabolite and the progress of bioactivity, and China is natural
Medicine, 2010,8 (4): 309-320）.Protocols in Molecular Biology is fast-developing, new by building Metagenomic library screening
Natural products can avoid the separation process of microorganism, expand natural products screening scope.But, current environment large fragment
The extraction of DNA is difficult, gene is not expressed in host cell or expression quantity is low, cannot carry out high-throughout screening bioactive compoundses
The problems such as constrain the practicality of grand genomic library.Being synthesized by chemical modification or full chemistry can obtain antibiotic compounds,
But building-up process is complicated, and causes environmental pollution.Being excavated by genome can improve the discovery efficiency of natural products, but
The Function Identification of natural products gene cluster is always still unsolved problem（Zhu Meng is grand, what Jing actinomyces genome large fragment
Cloning vector and the progress of heterogenous expression host transformation, microbiology circular, 2013,40 (10): 1920-1928）.By
This is visible, and microorganism is investigated, and screens new type natural product producing bacterial strain, is still a kind of new antibiosis of important exploitation
The approach of element.
Recent studies suggest that, the co-cultivation of two or more microorganisms can stimulate microorganism to produce novel cometabolism
Product, the exploitation to novel materials is significant, there is provided the screening efficiency of novel anti-bacterial material is improved by co-culturing
Possibility.（Huang Bing, Liu Ning, Huang Ying, Chen Jing spring actinomyces are with the co-cultivation of bacillus subtilis and its to active secondary metabolism
The influence of product, bioengineering journal, 2009,25 (6): 932-940）.But, at present to the screening side of novel anti-bacterial material
The screening technique that fado is carried out with single pure culture.Bacterium and actinomyces are produced by co-culturing screening novel anti-bacterial material,
According to the conventional method, separation of bacterial and actinomyces first, to all bacterial strains and other bacterial strain common fluid cultivation and fermentations, prepare
Fermented supernatant fluid or zymotic fluid organic solvent extract, further determine bacteriostatic activity with filter paper enzyme, cylinder-plate method, punch method
（The new method that Li little Jun, Cheng Lixia, Wu Yanbin, the gorgeous blue or green Antagonistic Fungis antimicrobial spectrum in side and zymotic fluid antagonistic ability are determined, biological skill
Art, 2007,17 (1): 55-58）.The method high cost, the cycle is long, and labour intensity is big.
The content of the invention
The technical problems to be solved by the invention are directed to the deficiencies in the prior art, there is provided a kind of low cost, it is simple to operate,
Cycle is short, efficiency high by co-culturing the method that mode screens antibacterial activity bacterium and actinomyces.
The technical problems to be solved by the invention are realized by following technical scheme.The present invention is a kind of quick
Screening antibacterial activity bacterium and the method for actinomyces, are characterized in, its step is as follows：：
（1）Sample collection：The samples such as seawater, ooze, soil and river are gathered with sterile sampling bottle, laboratory is delivered to rapidly
And bacterium and actinomyces separation are carried out as early as possible.
（2）It is prepared by culture medium：2216E culture mediums：Peptone 5g, yeast extract 1g, FePO40.1g, Chen Haishui
1000ml；Gause I culture medium：Soluble starch 20.0g, KNO3 1.0g, K2HPO4 0.5g, MgSO4·7H2O 0.5g.
NaCl 0.5g, FeSO4·7H2O 0.01g, distilled water 1000 ml, pH7.2；Nutrient agar：Beef extract 3g, albumen
Peptone 10g, NaCl 5g, distilled water 1000 ml, pH7.2；PDA culture medium：Potato 200g, glucose 20g, distilled water 1000
Ml, natural pH；If above-mentioned culture medium is solid medium, addition agar 20g/L；In addition to 2216E culture mediums, if using Yu Haiyang sample
The separation of product bacterial strain, then distilled water is replaced with seawater in culture medium;
（3）Bacterium and actinomyces separate：Samples taken is carried out into gradient dilution, the dilution point of 50 μ L difference dilution factors is taken
Not coating the flat board of the potassium bichromate solution for adding final concentration of 50 mg/ml is used for bacterium and actinomyces separation.Ocean sample
The Gause I flat board that the bacterium and actinomyces of product are prepared with 2216E flat boards and seawater；The bacterium of Lu Yuan samples and actinomyces are used
Nutrient agar panel and Gause I flat board；30 DEG C are inverted culture, Bacteria Culture 2-7d, actinomyces culture 7-14d, according to bacterium colony
Judge different strain after micro- sem observation after feature and Gram's staining；
（4）Intercept the preparation of suction nozzle：1ml suction pipette heads are taken, are defined by wide mouth end, intercept 12mm, remainder is amputated,
Edge is polished smooth, 121 DEG C of sterilizing 20min；
（5）Antibacterial activity bacterial strain screening：The picking from inclined-planeEscherichia coli Strain is preserved, is about in thickness
Three rides on 3mm nutrient agar flat boards, 37 DEG C are inverted culture to there is single bacterium colony；Inhaled with aseptic interception in super-clean bench
Head punches above single bacterium colony corresponding points, and culture medium is retained in suction nozzle；With the other end of this suction nozzle in dilution spread point
From bacterium or actinomyces single bacterium colony on punch, and the culture medium in suction nozzle is gently pressed with aseptic nipper, make upper and lower ends in suction nozzle
Culture medium contact, i.e.,Escherichia coliFormed with bacterium to be screened or actinomyces and co-cultured.This co-cultivation is micro-
Biological interception suction nozzle is placed inStaphylococcus aureus、Escherichia coli、Saccharomyces cerevisiae、Penicillium expansumFlat board is indicated Deng containing bacterium, bacterial indicator culture medium is nutrient agar culture
Base, fungi is PDA culture medium.In observation after the temperature culture that indicator bacteria is adapted to, and the size of inhibition zone is determined with slide measure；
Inhibition zone is bigger, and bacterial strain antibacterial activity is stronger；So as to quickly screening obtains antibacterial activity bacterium and actinomyces.
The bacterial strain being related in the present invention both from China General Microbiological DSMZ,Escherichia coli
Preserving number be CGMCC 1.487,Staphylococcus aureusPreserving number be CGMCC 1.2465,Saccharomyces cerevisiaePreserving number be CGMCC 2.114,Penicillium expansumPreserving number be
CGMCC 3.3703。
The step of method of a kind of quick screening antibacterial activity bacterium of the present invention and actinomyces（5）In, by cutting
Taking suction nozzle willEscherichia coliCo-cultured with bacterium to be screened or actinomyces, and be put in indicator bacteria flat board, indicator bacteria training
Antibacterial circle diameter is determined after supporting, antibacterial activity bacterial strain is quickly screened according to the presence or absence of inhibition zone and size.
Compared with prior art, the beneficial effects of the invention are as follows, extracting method of the present invention by way of co-culturing rapidly
Screening antibacterial activity bacterium and actinomyces, compared with antibacterial activity bacterium and actinomyces are screened in purebred culture, can increase novelty
The probability of antibacterial activity bacterial strain and antibacterial substance, and whole screening process is quick, and simple to operate, labour intensity is smaller,
The bacterium and actinomyces of antibacterial activity can quickly, intuitively, be efficiently screened, is the screening of antibacterial activity bacterium and actinomyces
Research provides greatly convenient.
Specific embodiment
Technical scheme is further illustrated by the following examples, without constituting the limitation to its right.
A kind of method of embodiment 1, quick screening antibacterial activity bacterium and actinomyces, its step is as follows：
（1）Sample collection：The samples such as seawater, ooze, soil and river are gathered with sterile sampling bottle, laboratory is delivered to rapidly
And bacterium and actinomyces separation are carried out as early as possible；
（2）It is prepared by culture medium：2216E culture mediums：Peptone 5g, yeast extract 1g, FePO40.1g, Chen Haishui
1000ml；Gause I culture medium：Soluble starch 20.0g, KNO3 1.0g, K2HPO4 0.5g, MgSO4·7H2O 0.5g.
NaCl 0.5g, FeSO4·7H2O 0.01g, distilled water 1000 ml, pH7.2；Nutrient agar：Beef extract 3g, albumen
Peptone 10g, NaCl 5g, distilled water 1000 ml, pH7.2；PDA culture medium：Potato 200g, glucose 20g, distilled water 1000
Ml, natural pH；If above-mentioned culture medium is solid medium, addition agar 20g/L；In addition to 2216E culture mediums, if using Yu Haiyang sample
The separation of product bacterial strain, then distilled water is replaced with seawater in culture medium；
（3）Bacterium and actinomyces separate：Samples taken is carried out into gradient dilution, the dilution point of 50 μ L difference dilution factors is taken
Not coating the flat board of the potassium bichromate solution for adding final concentration of 50 mg/ml is used for bacterium and actinomyces separation.Ocean sample
The Gause I flat board that the bacterium and actinomyces of product are prepared with 2216E flat boards and seawater；The bacterium of Lu Yuan samples and actinomyces are used
Nutrient agar panel and Gause I flat board；30 DEG C are inverted culture, Bacteria Culture 2-7d, actinomyces culture 7-14d, according to bacterium colony
Judge different strain after micro- sem observation after feature and Gram's staining；
（4）Intercept the preparation of suction nozzle：1ml suction pipette heads are taken, are defined by wide mouth end, intercept 12mm, remainder is amputated,
Edge is polished smooth, 121 DEG C of sterilizing 20min；
（5）Antibacterial activity bacterial strain screening：The picking from inclined-planeEscherichia coli Strain is preserved, is about in thickness
Three rides on 3mm nutrient agar flat boards, 37 DEG C are inverted culture to there is single bacterium colony；Inhaled with aseptic interception in super-clean bench
Head punches above single bacterium colony corresponding points, and culture medium is retained in suction nozzle.With the other end of this suction nozzle in dilution spread point
From bacterium or actinomyces single bacterium colony on punch, and the culture medium in suction nozzle is gently pressed with aseptic nipper, make upper and lower ends in suction nozzle
Culture medium contact, i.e.,Escherichia coliFormed with bacterium to be screened or actinomyces and co-cultured.This co-cultivation is micro-
Biological interception suction nozzle is placed inStaphylococcus aureus、Escherichia coli、Saccharomyces cerevisiae、Penicillium expansumFlat board is indicated Deng containing bacterium, bacterial indicator culture medium is nutrient agar culture
Base, fungi is PDA culture medium.In observation after the temperature culture that indicator bacteria is adapted to, and the size of inhibition zone is determined with slide measure；
Inhibition zone is bigger, and bacterial strain antibacterial activity is stronger；So as to quickly screening obtains antibacterial activity bacterium and actinomyces.
（6）Cultural characteristic and the Physiology and biochemistry identification of bacterial strain
The identification reference of the bacterium and actinomyces of antibacterial activity《Bergey’s manual of detrerminative
bacteriology》（ninth edition）Carry out.
The step of embodiment 2, method of a kind of quick screening antibacterial activity bacterium and actinomyces described in embodiment 1（5）
In, will by intercepting suction nozzleEscherichia coliCo-cultured with bacterium to be screened or actinomyces, and be put in indicator bacteria flat board,
Antibacterial circle diameter is determined after indicator bacteria culture, antibacterial activity bacterial strain is quickly screened according to the presence or absence of inhibition zone and size.
Example example 3, contrast experiment：
1. art methods screening.
Seawater, ooze are taken from Lianyun Harbour marine site Lian Dao, soil and freshwater sample is gathered on flowers and fruits mountain, by method of dilution butteron on plate
Using bacterium and actinomyces in the Gause I culture medium separating marine sample that 2216E culture mediums and seawater are prepared, nutrition is used
Agar medium and Gause I culture medium separate bacterium and actinomyces in the sample of land.Single bacterium colony after purification is seeded to
4 DEG C of preservations after inclined-plane culture.Isolate 691 plants of bacterium of acquisition, 93 plants of actinomyces.Different marine bacterias and land derived bacterium are inoculated with
To 2216E culture mediums and nutrient agar, marine actinomycete and Lu Yuan actinomyces are inoculated with seawater and distilled water preparation respectively
Gause I culture medium, culture fermented equipped with 25 mL to after cell pack 2%, taking during 0.2mL is inoculated with 100 mL triangular flasks
Culture medium, ferment 10 d, and 8,000g, 10 min are centrifuged, it is testing sample that supernatant is crossed after 0. 22 μm of miillpore filters, is adopted
Bacteriostatic activity is determined with cylinder-plate method.200 μ L fermented samples are added in each Oxford cup, using containingStaphylococcus aureus、Escherichia coli、Penicillium expansumFlat board is indicated Deng containing bacterium, is observed after 30 DEG C of cultures, altogether
ResistedStaphylococcus aureus139 plants of activated bacterial, 25 plants of actinomyces；It is anti-Escherichia coliIt is active thin
67 plants of bacterium, 15 plants of actinomyces；It is anti-Penicillium expansum96 plants of activated bacterial, 16 plants of actinomyces.
2. the inventive method screening.
With above-mentioned same sample, by method of dilution butteron on plate, the dilution for taking 50 μ L difference dilution factors is respectively coated on and adds
Plus the flat board of the potassium bichromate solution of final concentration of 50 mg/ml is used for bacterium and actinomyces separate.The bacterium of Oceanic Samples and
The Gause I flat board that actinomyces are prepared with 2216E flat boards and seawater.The bacterium and actinomyces of Lu Yuan samples are flat with nutrient agar
Plate and Gause I flat board.The picking from inclined-planeEscherichia coli Strain is preserved, 3mm nutrient agars are about in thickness
Three ride on culture medium flat plate, 37 DEG C are inverted culture to there is single bacterium colony；With aseptic interception suction nozzle in single bacterium colony in super-clean bench
The punching of corresponding points top, and culture medium is retained in suction nozzle.The bacterium that in dilution spread separate with the other end of this suction nozzle or
Punched in actinomyces single bacterium colony, and the culture medium in suction nozzle is gently pressed with aseptic nipper, connect the culture medium of upper and lower ends in suction nozzle
Touch, i.e.,Escherichia coliFormed with bacterium to be screened or actinomyces and co-cultured.By the interception of this co-cultivation microorganism
Suction nozzle is placed inStaphylococcus aureus、Escherichia coli、Penicillium expansumIndicate flat board,
Observed after 30 DEG C of cultures, resisted altogetherStaphylococcus aureus185 plants of activated bacterial, 33 plants of actinomyces；It is anti-Escherichia coli73 plants of activated bacterial, 18 plants of actinomyces；It is anti-Penicillium expansum121 plants of activated bacterial,
25 plants of actinomyces.
The inventive method can be seen that by above contrast experiment screen the resistant strain quantity that obtains and significantly improve.Value
Must be concerned with, by co-culturing, single bacterial strain of the culture without antibacterial activity is produced antibacterial activity, but not over co-cultivation
The bacterial strain for having antibacterial activity is set to lose the generation of antibacterial activity phenomenon.
Claims (2)
1. a kind of method for screening antibacterial activity bacterium and actinomyces, it is characterised in that its step is as follows：
(1) sample collection：Seawater, ooze, soil or river sample are gathered with sterile sampling bottle, laboratory is delivered to rapidly；
(2) prepared by culture medium：2216E culture mediums：Peptone 5g, yeast extract 1g, FePO40.1g, Chen Haishui 1000ml；Gao Shi mono-
Number culture medium：Soluble starch 20.0g, KNO3 1.0g,K2HPO4 0.5g,MgSO4·7H2O 0.5g.NaCl0.5g,
FeSO4·7H2O 0.01g, distilled water 1000ml, pH7.2；Nutrient agar：Beef extract 3g, peptone 10g, NaCl
5g, distilled water 1000ml, pH7.2；PDA culture medium：Potato 200g, glucose 20g, distilled water 1000ml, natural pH；If on
Culture medium is stated for solid medium, addition agar 20g/L；In addition to 2216E culture mediums, if for the separation of Oceanic Samples bacterial strain,
Then distilled water is replaced with seawater in culture medium；
(3) separation of bacterium and actinomyces：Samples taken is carried out into gradient dilution, the dilution difference of 50 μ L difference dilution factors is taken
Coating the flat board of the potassium bichromate solution for adding final concentration of 50mg/ml is used for the separation of bacterium and actinomyces；Oceanic Samples
Bacterium and the Gause I flat board prepared with 2216E flat boards and seawater of actinomyces；The bacterium of Lu Yuan samples and actinomyces are sought
Support agar plate and Gause I flat board；30 DEG C are inverted culture, Bacteria Culture 2-7d, actinomyces culture 7-14d, according to bacterium colony spy
Judge different strain after the micro- sem observation of Gram's staining of seeking peace；
(4) preparation of suction nozzle is intercepted：1ml suction pipette heads are taken, is defined by wide mouth end, intercept 12mm, remainder is amputated, edge
Polish smooth, 121 DEG C of sterilizing 20min；
(5) screening of antibacterial activity bacterial strain：Picking Escherichia coli preserve strain from inclined-plane, are 3mm's in thickness
Three rides on nutrient agar flat board, 37 DEG C are inverted culture to there is single bacterium colony；With aseptic interception suction nozzle in super-clean bench
Punched above single bacterium colony corresponding points, and culture medium is retained in suction nozzle；Separated in dilution spread with the other end of this suction nozzle
Bacterium or actinomyces single bacterium colony on punch, and the culture medium in suction nozzle is gently pressed with aseptic nipper, make upper and lower ends in suction nozzle
Culture medium contact, i.e. Escherichia coli and bacterium to be screened or actinomyces form and co-culture；This co-cultivation is micro-
Biological interception suction nozzle is placed in Staphylococcus aureus, Escherichia coli, Saccharomyces
Cerevisiae, Penicillium expansum indicate flat board containing bacterium, and bacterial indicator culture uses nutrient agar, very
Bacterium culture uses PDA culture medium；In observation after the temperature culture that indicator bacteria is adapted to, and the big of inhibition zone is determined with slide measure
It is small；Inhibition zone is bigger, and bacterial strain antibacterial activity is stronger；Antibacterial activity bacterium and actinomyces are obtained so as to screen.
2. a kind of method for screening antibacterial activity bacterium and actinomyces according to claim 1, it is characterised in that：Step
(5) Escherichia coli and bacterium to be screened or actinomyces are co-cultured by intercepting suction nozzle in, and is put in indicator bacteria and put down
Plate, antibacterial circle diameter is determined after indicator bacteria culture, and antibacterial activity bacterial strain is screened according to the presence or absence of inhibition zone and size.
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| CN105462839A (en) * | 2016-01-18 | 2016-04-06 | 浙江大学 | Fast screening method of pathogenic-bacterium-resistant bacillus |
| CN108624534A (en) * | 2018-05-17 | 2018-10-09 | 辽宁兆友生物科技有限公司 | Method for discovering novel antibiotic actinomycetes and antibacterial and antiviral product from natural product |
| CN109576341A (en) * | 2018-12-05 | 2019-04-05 | 海南省海洋与渔业科学院（海南省海洋开发规划设计研究院） | A kind of lead compound screening technique inhibiting grouper pathogenic vibrio |
| CN113215068A (en) * | 2021-06-29 | 2021-08-06 | 广东医科大学 | Culture medium for separating mangrove rhizosphere soil actinomycetes and application thereof |
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| CN101050438A (en) * | 2007-03-09 | 2007-10-10 | 吉林大学 | New method for separating and purifying strain of bacteria of sulfate reducting bacteria |
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| 海洋链霉菌GB-2的分离筛选及其抗菌物质的研究;刘姝;《南京农业大学博士学位论文》;20071231;第二章1.1-1.10节 * |
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