CN106148480A - A kind of super resistance to selenium antibacterial research method to the reduction of selenite - Google Patents
A kind of super resistance to selenium antibacterial research method to the reduction of selenite Download PDFInfo
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Abstract
A kind of super resistance to selenium antibacterial research method to the reduction of selenite, the invention discloses a kind of super resistance to selenium antibacterial research method to the reduction of sodium selenite, and the method comprises the steps, a. activates；B. gradient coerces cultivation；C. count plate；d.Se4+Measure.The present invention uses the super resistance to selenium antibacterial of existence in selenium-rich environment, has the characteristic that sodium selenite is reduced to nano level red elemental selenium, has the highest value of exploiting and utilizing.The present invention has that material cost is relatively low, experimental technique simple and the advantage of environmentally safe, and meanwhile, biology can also be played selenium supplement effect by the Nano red elemental selenium of generation.
Description
Technical field
The invention belongs to resistance to selenium microbial technology field, be specifically related to a kind of super resistance to selenium antibacterial and sodium selenite is gone back original work
Research method.
Background technology
Selenium is a lot of biological required trace element, and in vivo, selenium is synthesized into selenium protein, and some selenium protein has
Anti-oxidation function, meanwhile, it is capable to preventing and treating certain cancers.Selenium is when content is low, useful to biology, when content height, to the mankind and
Animal pest, in micro elements needed by human, selenium is from deficiency to producing toxicity, and scope is the narrowest.
There is in soil the selenium of biological effectiveness, mainly exist with selenate and selenite form, some selenium-rich ground
The agricultural drainage in district and semi-conductor industrial waste water are the main sources of water soluble speciation in water body (selenate/selenite), and nothing
Machine selenium is virulent to human body, therefore, finds a kind of solution by having virose inorganic selenium and is converted into the nontoxic or selenium of low toxicity
The method of this problem is most important.
Report that the microorganism that inorganic selenium can be reduced to elemental selenium has at present: germ oligotrophy unit cell, spore bar
Bacterium, Rolls lead to bacterium, pottery strategic point Salmonella, desulfurization germ, hyperthermophilic Gu bacterium, root nodule bacteria, pseudomonas etc., but, the tolerance to selenium
Property is not the most the highest.Report about lysine bacillus cereus has: Li Ting etc. (2013) report that a strain has the micro-of adsorption function
The biological fixing carrier degraded to metacresol.Hayat.et.al (2013) reports Lysinibacillus
The resistance to boric acid of pakistanensis.Bahuguna.et.al (2011) report isolated spherical bad ammonia from diesel fuel contaminated soil
Acid bacillus cereus (Lysinibacillus sphaericus) can produce desulfidation to dibenzothiophenes.But, about selenium
Compound for lysine bacillus cereus (Lysinibacillus) toxicity or impact report almost without.
Microorganism can will have more supervirulent selenite or selenate reduction, and the red elemental selenium toxicity of generation reduces
And dissolubility is less, can be used for the biological treating of some selenium environmental pollution.Experiment in recent years also finds, when particle diameter is at 5-
During 200nm, red elemental selenium may have the highest using value in terms of biological selenium compensation.
Summary of the invention
According to above the deficiencies in the prior art, the technical problem to be solved is to propose a kind of super resistance to selenium antibacterial pair
The research method of the reduction of selenite, it is therefore an objective to make selenite have high percent reduction by microorganism, make selenium environment
Pollution carries out biological treating.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is:
A kind of super resistance to selenium antibacterial research method to the reduction of sodium selenite, the method comprises the steps,
A, activation: resistance to selenium antibacterial pure culture body is inoculated in LB fluid medium, be placed in shaken cultivation case cultivating；
B, gradient coerce cultivation: the resistance to selenium microbionation of activation step a obtained is 0-in the amount containing selenite
25000mg·L-1LB fluid medium in, be placed in shaken cultivation case cultivation, the inoculum concentration of this activation resistance to selenium antibacterial is
The 0.5%-1.5% of LB fluid medium volume fraction；
C, count plate: take the resistance to selenium antibacterial sterilized water dilution 10 that step b obtains5-107, take the bacterium solution after dilution afterwards
Coat LB solid medium, then count after constant temperature culture in biochemical cultivation case；
d、Se4+Measure: the resistance to selenium microbionation of activation step a obtained is 180-220mg in the amount containing selenite
L-1LB fluid medium in, the 1%-2% that inoculum concentration is LB fluid medium volume fraction of this activation resistance to selenium antibacterial, afterwards
It is placed in shaken cultivation case cultivation, divides time interval with spectrophotometer and survey the absorbance of bacterium solution, draw the growth of antibacterial
Curve；Take the bacterium solution after shaken cultivation to be performing centrifugal separation on, and with 2, Se in 3-diaminonaphthalene Spectrophotometric Determination supernatant4+Dense
Degree.
Resistance to selenium microbionation step a cultivated is in being placed with containing 200mg L-1In the LB fluid medium of sodium selenite,
Be placed in shaken cultivation case cultivation, from the beginning of 0h, every 3h, with the absorbance of spectrophotometric measurement bacterium solution, meanwhile,
Surveying bacterium solution pH value with pH meter, the bacterial load of resistance to selenium is the 1% of LB fluid medium volume fraction, and the amount of culture medium is 25mL,
The temperature of shaken cultivation case is 35 DEG C, and the rotating speed of shaken cultivation case is 180r min-1, the shaken cultivation time is 30h, light splitting light
The wavelength of degree meter is 600nm.
Described research method also includes the sign step of reduzate, and the characterizing method of reduzate includes that taking step a obtains
The activation resistance to selenium antibacterial arrived by centrifugation, sterile phosphate buffer clean, sterile water wash, then by the resuspended bacterium solution of sterilized water,
Take bacteria suspension afterwards to observe.
The amount activating resistance to selenium antibacterial described in the characterizing method of reduzate is 0.8-1.2mL, and the amount of bacteria suspension is 1-3 μ
L。
The temperature of shaken cultivation case constant temperature culture described in step a and step b is 35-37 DEG C, rotating speed 160-200r
min-1, incubation time is 10-14h.Preferably, the temperature of shaken cultivation case constant temperature culture is 35 DEG C, rotating speed 180r min-1。
The temperature of biochemical cultivation case constant temperature culture described in step c is 36-38 DEG C, incubation time 24-72h.Preferably, raw
The temperature changing incubator constant temperature culture is 37 DEG C.
In step a, the preparation method of resistance to selenium antibacterial pure culture body includes:
(1) selenium-rich soil suspension is prepared；
(2) Zengjing Granule: taking selenium-rich soil suspension prepared by step (1) and joining containing concentration is 100-200mg L-1Sub-
In the LB fluid medium of selenate, it is placed in shaken cultivation in shaken cultivation base；
(3) successive transfer culture: the bacterium solution after step (2) shaken cultivation being inoculated in containing concentration is 100-200mg L-1Sub-selenium
In the LB fluid medium of hydrochlorate, the inoculum concentration of bacterium solution is the 3%-7% of this LB fluid medium cumulative volume, be placed on vibration
Shaken cultivation in incubator, so repeats 3-5 time；
(4) dilution spread flat board: the bacterium solution sterilized water dilution 10 that step (3) is obtained1-107Times, take dilution 105、
106、107Bacterium solution is coated on the LB solid medium containing selenite, is placed in constant temperature culture in biochemical cultivation case；
(5) plate streaking；Take single bacterium colony that step (4) grows to combine plate streak and be inoculated in containing 100-200mg L-1
In the LB fluid medium of selenite, be placed on constant temperature culture in biochemical cultivation case；
The temperature of described shaken cultivation case constant temperature culture is 35-37 DEG C, rotating speed 160-200r min-1, incubation time is 8-
14h；Described in step (4) and (5), the temperature of biochemical cultivation case constant temperature culture is 36-38 DEG C, incubation time 24-72h.
Described super resistance to selenium antibacterial is lysine bacillus cereus.
The medicine have the advantages that the present invention, with natural selenium-rich soil as material, therefrom separates the two super resistance to selenium of strain
Antibacterial B1 and B2, wherein B1 is Lysinibacillus xylanilyticus, and B2 is Lysinibacillus macroides,
On the one hand, there is stronger selenite toleration, the sodium selenite half maximum effect to super resistance to selenium antibacterial-lysine bacillus cereus
Answer concentration to respectively may be about 10000,15000mg L-1, on the other hand, it is possible to sodium selenite is reduced to red elemental selenium, reduction
Rate respectively reaches 99.91% and 99.96%.The present invention has that material cost is relatively low, experimental technique simple and environmentally safe
Advantage, meanwhile, biology can also be played selenium supplement effect by the Nano red elemental selenium of generation.
Accompanying drawing explanation
Below the content expressed by this specification accompanying drawing and the labelling in figure are briefly described:
Fig. 1 is the sodium selenite half-maximal effect concentration schematic diagram to resistance to selenium antibacterial of the present invention；
Fig. 2 is the kinetic curve figure of the bacterial reduction of the resistance to selenium sodium selenite of the present invention；
Fig. 3 be the present invention the bacterial reduction of resistance to selenium sodium selenite during the variation diagram of pH；
Fig. 4 is the Characterization of The Products figure of the bacterial reduction of the resistance to selenium sodium selenite of the present invention；
Wherein, the picture left above is the Electronic Speculum figure of B1, and top right plot is the EDS figure of arrow head part in the picture left above, and lower-left figure is B2
Electronic Speculum figure, lower-left figure is the Electronic Speculum figure of B2, and bottom-right graph is the EDS figure of arrow head part in the figure of lower-left；
Fig. 5 is the two super resistance to selenium bacteria total DNA of strain of the present invention, the agarose gel electrophoresis detection figure of 16S rDNA；
Fig. 6 is the two strains super resistance to selenium antibacterial phylogenetic tree based on 16S rDNA gene order of the present invention；
Fig. 7 is the present invention two super bacteria distribution of resistance to selenium of strain, the aspect graph of purification；
Fig. 8 is the Gram’s staining result figure of the two super resistance to selenium antibacterials of strain of the present invention.
Detailed description of the invention
Below against accompanying drawing, by the description to embodiment, the present invention is further detailed explanation, to help ability
Field technique personnel have more complete, accurate and deep understanding to inventive concept, the technical scheme of the present invention.
A kind of super resistance to selenium antibacterial research method to the reduction of sodium selenite, the method comprises the steps,
A. activate
Resistance to selenium antibacterial pure culture body screening obtained is inoculated in the conical flask being placed with LB fluid medium, then will
Postvaccinal conical flask is placed in shaken cultivation case cultivation, and the amount of culture medium is 25mL, and the temperature of shaken cultivation case is 35 DEG C,
The rotating speed of shaken cultivation case is 180r min-1, the shaken cultivation time is 12h；
The screening technique of the most resistance to selenium antibacterial pure culture body is:
(1) pedotheque suspension is prepared
Weigh 2g selenium-rich soil sample, add in the conical flask being placed with sterilized 50mL phosphate buffer, be placed in
Vibrating in shaken cultivation case, the temperature of shaken cultivation case is 25 DEG C, and rotating speed is 180r min-1, duration of oscillation is 30min, then
Stand 30min, take 30mL suspension in sterilized 50mL centrifuge tube, be positioned in centrifuge centrifugal, described centrifuge
Rotating speed is 5000r min-1, centrifugation time is 10min, abandoning supernatant, adds 5mL phosphate buffer with the precipitation that suspends.
(2) Zengjing Granule
Take pedotheque suspension 1mL prepared by step (1), join and be placed with containing Na2SeO3LB fluid medium (ferment
Female extract 5g L-1, sodium chloride 10g L-1, tryptone 10g L-1, pH=7.2) conical flask in, Na2SeO3Concentration is
150mg·L-1, the amount of LB fluid medium is 50mL, is placed in shaken cultivation in shaken cultivation case, and the temperature of shaken cultivation case is
35 DEG C, rotating speed is 180r min-1, incubation time is 10h.
(3) successive transfer culture
Take step (2) bacterium solution to be inoculated into and be placed with containing Na2SeO3LB fluid medium conical flask in, bacterium solution inoculum concentration
It is 5% (volume fraction), Na2SeO3Concentration is 150mg L-1, the amount of LB fluid medium is 50mL, is placed in shaken cultivation case
Middle shaken cultivation, the temperature of shaken cultivation case is 35 DEG C, and rotating speed is 180r min-1, incubation time is 10h；So it is repeated 4 times.
(4) dilution spread flat board
Step (3) bacterium solution sterilized water is diluted 101-107Times, take 105、106、107Bacterium solution is coated containing Na2SeO3LB
On solid medium, being placed in constant temperature culture in biochemical cultivation case, the bacterium solution volume of coating is 100 μ L, Na2SeO3Concentration is
150mg·L-1, the temperature of biochemical cultivation case is 37 DEG C, and incubation time is 48h, result such as Fig. 7 (on) shown in, bacterial plaque color is red
Color, thalline is more moistening, circular edge, neat in edge, easy picking.
(5) plate streaking
The single bacterium colony grown with inoculating loop picking step (4), in conjunction with plate streak, is inoculated in containing Na2SeO3LB solid
In culture medium, it is placed in constant temperature culture in biochemical cultivation case, medium component and condition of culture same step (4), result such as Fig. 7 (under)
Shown in, scribing results shows, antibacterial list colonial morphology is regular.
(6) activation
The single bacterium colony grown with inoculating loop picking step (5), is inoculated in the conical flask being placed with LB fluid medium, connects
And postvaccinal conical flask is placed in shaken cultivation case cultivation, the amount of culture medium is 25mL, and the temperature of shaken cultivation case is
35 DEG C, the rotating speed of shaken cultivation case is 180r min-1, and the shaken cultivation time is 12h.
(7) Gram’s staining
The single bacterium colony grown with inoculating loop picking step (5), in conjunction with plate streak, is inoculated in LB solid medium, puts
Constant temperature culture in biochemical cultivation case, picking list bacterium colony is used for Gram’s staining, in 100 times of oily Microscopic observation cellular morphologies, choosing
Staphylococcus aureus (gram positive bacteria) and the escherichia coli (gram negative bacteria) of selecting known Gram’s staining result make
For comparison, the temperature of biochemical cultivation case is 37 DEG C, and incubation time is 20h, and as shown in Figure 8, result shows that B1 and B2 is to result
Gram-positive bacillus.
(8) total DNA extraction
According to step (7) result, process by gram-positive bacterium, use the extracting examination of Ezup pillar bacterial genomes DNA
Agent box (Shanghai Sheng Gong biological engineering company limited) extracts bacteria total DNA, and the DNA of extraction enters with the agarose gel electrophoresis of 1%
Row detection, 4S green dyes, and λ DNA/Hind III is as DNA Marker, and shown in result such as Fig. 5 (left), result shows, DNA
Clip size is at about 20000bp.
(9) 16S rDNA gene amplification
Bacteria total DNA bacterial 16 S rDNA universal primer step (7) extracted is Serial No. SEQ ID NO.1
The UR 5 ' of UF 5 '-AGAGTTTGATCMTGGCTCAG-3 ' (M is A or C) and Serial No. SEQ ID NO.2-
16S rDNA full length gene is expanded by ACGGTTACCTTGTTACGACTT-3 ', and the reaction system of described amplification is 30 μ l,
Including dNTPs 2 μ L, 10 × buffer 3 μ L, Mg2+2 μ L, Tap enzyme 1 μ L, each 1 μ L of primer, ddH2O 19 μ L, STb gene template 1
μ L, the reaction condition of described amplification is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 45s, 55 DEG C of annealing 4s, 72 DEG C extend 1.5min,
30 circulations；Finally extending 10min at 72 DEG C, primer is synthesized by Shanghai Sheng Gong biological engineering company limited, and pcr amplification product is used
1% agarose gel electrophoresis detection, 4S green dye, DNA Marker-F as Marker, result such as Fig. 5 (right) institute
Showing, result display amplified production specificity is preferable, and purpose band is brighter, and stripe size is at about 1500bp, according to agarose
The result of gel electrophoresis, send Sangon Biotech (Shanghai) Co., Ltd. to check order PCR primer, according to order-checking knot
Really, sequence is carried out in http://www.ezbiocloud.net sequence analysis analysis, according to comparison analysis result, choosing
Take front 10 gene orders of similarity the highest (being all higher than 96%), use ClustalX (1.83) software to carry out multiple ratio pair,
Comparison result uses MEGA5.10 to set up phylogenetic tree, with brood cell's lactobacillus (Sporolactobacillus inulinus)
As external source bacterium, as shown in Figure 6, B1 and B2 belongs to lysine bacillus cereus (Lysinibacillus) to result, wherein, B1 and
Lysinibacillus xylanilyticus homology credibility reaches 98%, B2 and Lysinibacillus
Boronitolerans, Lysinibacillus macroides homology credibility reaches 98%.
(10) low temperature resistant inspection
Take the bacterium solution that step a cultivates to be inoculated in and be placed with containing Na2SeO3LB fluid medium conical flask in, then will
Postvaccinal conical flask is placed in shaken cultivation case cultivation, Na2SeO3Concentration is 150mg L-1, the body of LB fluid medium
Amassing as 25mL, the temperature of shaken cultivation case is 35 DEG C, and the rotating speed of shaken cultivation case is 180r min-1, the shaken cultivation time is
12h, then dilutes bacterium solution, and extension rate is 101-106, then take the 10 of dilution6Bacterium solution is coated containing Na2SeO3LB solid
In culture medium, being placed in constant temperature culture in 4 DEG C of refrigerators, observe colony morphology characteristic, the bacterium solution amount of coating is 100 μ L, described
Na2SeO3Content is 150mg L-1, described incubation time is 72h, and result is as shown in table 1 below, and result shows the two equal energy of strain antibacterial
Enough growths under the conditions of 4 DEG C, have certain low temperature tolerance ability, according to Coorevits.et.al (2012) result of study, determine
B1 is Lysinibacillus xylanilyticus, and B2 is Lysinibacillus macroides.
Table 1
B. gradient coerces cultivation
By the resistance to selenium microbionation of step a cultivation in being placed with in the conical flask of the LB fluid medium containing sodium selenite,
Postvaccinal conical flask is then placed in shaken cultivation case cultivation, and the amount wherein containing sodium selenite in LB fluid medium is
0、5000、10000、15000、20000、25000mg·L-1, the bacterial load of resistance to selenium is LB fluid medium volume fraction
1%, the amount of culture medium is 25mL, and the temperature of shaken cultivation case is 35 DEG C, and the rotating speed of shaken cultivation case is 180r min-1, vibration
Incubation time is 12h.
C. count plate
The resistance to selenium antibacterial taking the cultivation of step b is positioned in the centrifuge tube of sterilizing, adds sterilized water dilution, the extension rate of bacterium solution
It is 105、106、107, the bacterium solution then taking dilution is coated on LB solid medium, and the amount of bacterium solution coating is 100 μ L, in biochemistry
Counting after constant temperature culture in incubator, the temperature of biochemical cultivation case is 37 DEG C, and incubation time is 48h, as it is shown in figure 1, result shows
Showing, sodium selenite respectively may be about 10000 to the half-maximal effect concentration of antibacterial B1 and B2,15000mg L-1。
d.Se4+Measure
Resistance to selenium microbionation step a cultivated is in being placed with containing 200mg L-1The LB fluid medium of sodium selenite
In conical flask, then postvaccinal conical flask is placed in shaken cultivation case cultivation, from the beginning of 0h, every 3h, uses light splitting
The absorbance of luminosity measurement bacterium solution, draws the growth curve of antibacterial, meanwhile, takes the bacterium solution of 1.5mL in sterilizing from conical flask
Centrifuge tube in, be positioned in centrifuge centrifugal, take 1mL supernatant in the centrifuge tube of new sterilizing, be positioned over 4 DEG C of refrigerators and protect
Deposit, treat that all samplings and sample preparation terminate, with 2, Se in 3-diaminonaphthalene Spectrophotometric Determination supernatant4+Concentration, resistance to selenium
Bacterial load is the 1.5% of LB fluid medium volume fraction, and the amount of culture medium is 150mL, and the temperature of shaken cultivation case is
35 DEG C, the rotating speed of shaken cultivation case is 180r min-1, the shaken cultivation time is 36h, and spectrophotometric wavelength is 600nm,
The rotating speed of centrifuge is 10000r min-1, the centrifugation time of centrifuge is 10min, as in figure 2 it is shown, result shows, and antibacterial B1
With B2, the percent reduction of sodium selenite is respectively reached 99.91% and 99.96%；
E.pH measures
Resistance to selenium microbionation step a cultivated is in being placed with containing 200mg L-1The LB fluid medium of sodium selenite
In conical flask, then postvaccinal conical flask is placed in shaken cultivation case cultivation, from the beginning of 0h, every 3h, uses light splitting
The absorbance of luminosity measurement bacterium solution, meanwhile, surveys bacterium solution pH value with pH meter, and the bacterial load of resistance to selenium is LB fluid medium volume
The 1% of mark, the amount of culture medium is 25mL, and the temperature of shaken cultivation case is 35 DEG C, and the rotating speed of shaken cultivation case is 180r
min-1, the shaken cultivation time is 30h, and spectrophotometric wavelength is 600nm, as it is shown on figure 3, result shows, at antibacterial B1 and
In the growth course of B2, pH is stepped up and tends towards stability.
F. reduzate characterizes
Take the bacterium solution of resistance to selenium antibacterial that 1mL step a cultivates in the centrifuge tube of sterilizing, be positioned in centrifuge centrifugal, connect
And clean with sterile phosphate buffer, then by rinsed with sterile water, then by the resuspended bacterium solution of sterilized water, then take 2 μ L bacteria suspensions,
Dropping in and polish smooth, on cleaned copper sheet, be placed in air drying, then with scanning electron microscopic observation, the rotating speed of centrifuge is
10000r·min-1, the centrifugation time of centrifuge is 10min, and as shown in Figure 4, wherein, the picture left above is the Electronic Speculum figure of B1, top right plot
Being the EDS figure of arrow head part in the picture left above, full scale 3715cts in top right plot EDS figure, cursor 0.000, lower-left figure is the electricity of B2
Mirror figure, lower-left figure is the Electronic Speculum figure of B2, and bottom-right graph is the EDS figure of arrow head part in the figure of lower-left, full scale in the EDS figure of bottom-right graph
5140cts, cursor 0.000；Scanning electron microscope it was found that in two strain bacterial cells all seen from brood cell's structure of expanding, in sample
There is high electron density granule in around thalline and outside, these granules carry out EDS (Energy Dispersive
Spectroscopy) analyzing, at the 0～2keV characteristic absorption peak occurring in that copper and selenium, wherein copper is the composition of copper sheet, it addition,
Reduction process generates with irritative gas, therefore, Se4+It is reduced to nano level red elemental selenium and volatile selenizing
Thing.
Above in conjunction with accompanying drawing, the present invention is exemplarily described, it is clear that the present invention implements not by aforesaid way
Restriction, as long as have employed the method design of the present invention and the improvement of various unsubstantialities that technical scheme is carried out, or without changing
Enter and design and the technical scheme of the present invention are directly applied to other occasion, all within protection scope of the present invention.This
Bright protection domain should be as the criterion with the protection domain that claims are limited.
Claims (7)
1. the super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that the method includes as follows
Step,
A, activation: resistance to selenium antibacterial pure culture body is inoculated in LB fluid medium, be placed in shaken cultivation case cultivating；
B, gradient coerce cultivation: the resistance to selenium microbionation of activation step a obtained is 0-25000mg in the amount containing selenite
L-1LB fluid medium in, be placed in shaken cultivation case cultivation, the inoculum concentration of this activation resistance to selenium antibacterial be LB liquid training
Support the 0.5%-1.5% of matrix fraction；
C, count plate: take the resistance to selenium antibacterial sterilized water dilution 10 that step b obtains5-107, take the coating of the bacterium solution after dilution afterwards
In LB solid medium, then count after constant temperature culture in biochemical cultivation case；
d、Se4+Measure: the resistance to selenium microbionation of activation step a obtained is 180-220mg L in the amount containing selenite-1's
In LB fluid medium, the 1%-2% that inoculum concentration is LB fluid medium volume fraction of this activation resistance to selenium antibacterial, be placed on
Shaken cultivation case is cultivated, divides time interval with spectrophotometer and survey the absorbance of bacterium solution, draw the growth curve of antibacterial；
Take the bacterium solution after shaken cultivation to be performing centrifugal separation on, and with 2, Se in 3-diaminonaphthalene Spectrophotometric Determination supernatant4+Concentration.
The super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that institute
Stating research method and also include the sign step of reduzate, it is resistance to that the characterizing method of reduzate includes taking the activation that step a obtains
Selenium antibacterial by centrifugation, sterile phosphate buffer clean, sterile water wash, then by the resuspended bacterium solution of sterilized water, take bacterium afterwards and hang
Liquid is observed.
The super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that also
The amount activating resistance to selenium antibacterial described in the characterizing method of original thing is 0.8-1.2mL, and the amount of bacteria suspension is 1-3 μ L.
The super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that step
Described in rapid a and step b, the temperature of shaken cultivation case constant temperature culture is 35-37 DEG C, rotating speed 160-200r min-1, incubation time
For 10-14h.
The super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that step
Described in rapid c, the temperature of biochemical cultivation case constant temperature culture is 36-38 DEG C, incubation time 24-72h.
The super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that step
In rapid a, the preparation method of resistance to selenium antibacterial pure culture body includes:
(1) selenium-rich soil suspension is prepared；
(2) Zengjing Granule: taking selenium-rich soil suspension prepared by step (1) and joining containing concentration is 100-200mg L-1Monohydrated selenium dioxide
In the LB fluid medium of salt, it is placed in shaken cultivation in shaken cultivation base；
(3) successive transfer culture: the bacterium solution after step (2) shaken cultivation being inoculated in containing concentration is 100-200mg L-1Selenite
LB fluid medium in, the inoculum concentration of bacterium solution is the 3%-7% of this LB fluid medium cumulative volume, be placed on shaken cultivation
Shaken cultivation in case, so repeats 3-5 time；
(4) dilution spread flat board: the bacterium solution sterilized water dilution 10 that step (3) is obtained1-107Times, take dilution 105、106、107
Bacterium solution is coated on the LB solid medium containing selenite, is placed in constant temperature culture in biochemical cultivation case；
(5) plate streaking；Take single bacterium colony that step (4) grows to combine plate streak and be inoculated in containing 100-200mg L-1Sub-selenium
In the LB fluid medium of hydrochlorate, be placed on constant temperature culture in biochemical cultivation case；
The temperature of described shaken cultivation case constant temperature culture is 35-37 DEG C, rotating speed 160-200r min-1, incubation time is 8-14h；
Described in step (4) and (5), the temperature of biochemical cultivation case constant temperature culture is 36-38 DEG C, incubation time 24-72h.
The super resistance to selenium antibacterial research method to the reduction of sodium selenite, it is characterised in that institute
Stating super resistance to selenium antibacterial is lysine bacillus cereus.
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| CN106884000A (en) * | 2017-04-25 | 2017-06-23 | 安徽天邦生物技术有限公司 | A kind of preparation and application of spindle lysine bacillus selenium-rich microbial agent |
| CN107435030A (en) * | 2017-06-29 | 2017-12-05 | 浙江大学 | A kind of secondary bacillus licheniformis and the method for preparing rich biological nano selenium probiotics |
| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN111411060A (en) * | 2020-04-29 | 2020-07-14 | 广西大学 | Selenium-resistant bacterium screening method and application |
| CN112760249A (en) * | 2020-12-31 | 2021-05-07 | 武汉华硒生物科技有限公司 | Selenium reducing bacteria, nano-selenium-organic selenium nutrient solution and preparation method thereof |
| CN113481191A (en) * | 2021-04-02 | 2021-10-08 | 中国地质大学（武汉） | Immobilized particle embedded with selenite reducing bacteria and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106884000A (en) * | 2017-04-25 | 2017-06-23 | 安徽天邦生物技术有限公司 | A kind of preparation and application of spindle lysine bacillus selenium-rich microbial agent |
| CN107435030A (en) * | 2017-06-29 | 2017-12-05 | 浙江大学 | A kind of secondary bacillus licheniformis and the method for preparing rich biological nano selenium probiotics |
| CN107435030B (en) * | 2017-06-29 | 2018-07-17 | 浙江大学 | A kind of pair bacillus licheniformis and the method for preparing rich biological nano selenium probiotics |
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| CN111411060A (en) * | 2020-04-29 | 2020-07-14 | 广西大学 | Selenium-resistant bacterium screening method and application |
| CN112760249A (en) * | 2020-12-31 | 2021-05-07 | 武汉华硒生物科技有限公司 | Selenium reducing bacteria, nano-selenium-organic selenium nutrient solution and preparation method thereof |
| CN113481191A (en) * | 2021-04-02 | 2021-10-08 | 中国地质大学（武汉） | Immobilized particle embedded with selenite reducing bacteria and preparation method and application thereof |
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