CN101050438B - New method for separating and purifying strain of bacteria of sulfate reducing bacteria - Google Patents
New method for separating and purifying strain of bacteria of sulfate reducing bacteria Download PDFInfo
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- CN101050438B CN101050438B CN200710055389XA CN200710055389A CN101050438B CN 101050438 B CN101050438 B CN 101050438B CN 200710055389X A CN200710055389X A CN 200710055389XA CN 200710055389 A CN200710055389 A CN 200710055389A CN 101050438 B CN101050438 B CN 101050438B
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Abstract
This invention relates to a novel method for separating and purifying pure strain of sulfate reducing bacteria. The method can be used in microbial antisepsis, wastewater treatment, ore extraction, food industry and medicine. The method comprises: rapidly, precisely and simply obtaining single pure culture colonies of sulfate reducing bacteria, utilizing a plate solid culture medium as the carrier for bacteria growth, spraying the bacteria solution onto the surface of the culture medium with an injector with pinhead to obtain single bacteria adhered to the surface of the culture medium, culturing at constant temperature and moisture in bacteria-free environment to obtain colonies with single bacteria proliferation, selecting a single colony, placing in a liquid culture medium, and proliferating to obtain pure strain of sulfate reducing bacteria with a large amount and high bioactivity. The precision and efficiency of the method are higher than those of traditional bacteria separation and purification method.
Description
Technical field:
The present invention relates to a kind of novel method that this non-strictly anaerobic bacterium of sulphate reducing bacteria is separated and purifies, it is flat board spraying partition method that present method is named.This method adopts the API solid medium to cultivate, and is mainly used in the extraction strain of bacteria of sulfate reducting bacteria, and picks out the strongest bacterial strain of biological activity from a plurality of pure strain bacterial strains.This method also extends to the extraction of other oxytolerant type, facultative type and non-strictly anaerobic type bacterium pure strain, but is not suitable for strictly anaerobic bacterium.
Background technology
The separation of bacterium pure strain and purifying are meant separates different types of microorganism mixed in together to obtain producing and studying needed single culture, and this needs special separation method.The extraction of microorganism pure strain is that microbiological industry is used and the gordian technique of research, is widely used in that microorganism is anticorrosion, sewage disposal, mineral carry fields such as smelting, foodstuffs industry, pharmaceuticals production.Traditionally, the separation of bacterium pure strain and purifying mainly adopt ten times of isolation by dilution method, plate streak, fall flat board and spread plate, puncture partition method, unicellular partition method and ultra micro membrane separation process etc.But these methods often relate to, and technology is meticulous, program is loaded down with trivial details, especially require operator that quite high, skilled operant level is arranged, and causes very big difficulty and inconvenient for the extraction of microorganism pure strain.At present, separate and the universal method of purifying adopts is the puncture partition method at sulphate reducing bacteria: promptly extract the bacterium bacterium liquid of mixing with syringe, syringe needle is thrust certain depth in the solid medium, injection bacterium liquid, prolong former road then and extract syringe needle, add whiteruss then substratum is carried out anaerobic sealing, solid medium carries out constant temperature culture in anaerobic environment, finally grows one pure culture bacterium colony at the position along syringe needle.This method operation easier is big, often needs to control well agar content, injection bacterium liquid quantity in the solid medium, also will create anaerobic environment, and the unusual difficulty of the single bacterium colony of picking, and success rate of extracting is extremely low.The misunderstanding that this kind method is based on traditionally the physiological property of sulphate reducing bacteria produces, usual earlier thinks that sulphate reducing bacteria is a strictly anaerobic bacterium, but recent document shows that sulphate reducing bacteria has stronger oxygen-resistant ability, can carry out normal vital movement in air.
Technology contents
The object of the present invention is to provide the separation and the purifying novel method of the strain of bacteria of sulfate reducting bacteria that a kind of bacterial classification extracts accurately, method is simple, success ratio is high.
The technological line of this invention is: guaranteeing fast, accurately, obtain easily under the prerequisite of single pure culture bacterium colony of sulfuric acid sample reduction bacterium, adopt the carrier of plate solid medium as bacterial growth, with the band syringe needle syringe with bacterium liquid with the surface of vaporific splash at substratum, to obtain one bacterial adhesion in media surface, under the fixed temperature and humidity gnotobasis, cultivate, acquisition has single vegetative bacterium colony, the single bacterium colony of picking is put into liquid nutrient medium and is bred then, final acquisition is a large amount of, biological activity is good, the pure strain of single sulphate reducing bacteria.Concrete technology side is by being:
A kind of separation of strain of bacteria of sulfate reducting bacteria and purifying novel method, be under aerobic environment, to extract and turn out strain of bacteria of sulfate reducting bacteria and to pick out the bacterial classification of the strongest purifying bacterial classification of vital activity as secondary separation and purifying, concrete processing step is as follows:
A) with the sulphate reducing bacteria obligate liquid nutrient medium of selecting for use, in gnotobasis, be heated to when at a simmer, stir after adding an amount of agar, be condensed into solid-state then;
B) in gnotobasis, the asepsis injector that at first will load onto syringe needle contains bacterium and handles one section air of back extraction, to contain the bacterium air then with the vaporific surface that evenly is ejected into solid medium, in constant incubator, turn out bacterium colony, the single bacterium colony of picking is put into the liquid nutrient medium of sterilization and is turned out active best sulphate reducing bacteria purifying bacterial classification therein;
C) be bacterial classification with sulphate reducing bacteria purifying bacterial classification, flash liberation and the purification step by bacterial classification repeats flash liberation and purifying again, and the liquid nutrient medium of gained promptly is the pure strain of the sulphate reducing bacteria that will obtain.
Said sulphate reducing bacteria obligate liquid nutrient medium is packed into and is heated in the Erlenmeyer flask of sterilising treatment by composition weighing, preparation and after regulating pH value and slightly being slight alkalinity to neutrality; Said condensation process is in gnotobasis, and the substratum that will contain agar joins in the plate while hot, and add-on is no more than 1/3 of the plate degree of depth, and cooled and solidified is stand-by solid medium then.
The said asepsis injector of loading onto syringe needle contains bacterium to be handled and to be: with asepsis injector and load onto syringe needle, from the sulphate reducing bacteria bacterial classification that contains assorted bacterium, extract an amount of bacterium liquid, and then bacterium liquid injected back in the seed bottle, emptying bacterium liquid makes syringe and needle tubing by the sulfate reduction fungi pollution.
Temperature is about 37 ℃ in the said constant incubator.
The liquid nutrient medium that is said sterilization is for adding the ferrous ammonium sulphate and the xitix of sterilization in substratum.
The method that the invention provides is directly carried out bacterium liquid spraying to the solid culture primary surface and is adhered to, and directly operates in aerobic environment and cultivates, and the result shows that this method bacterial classification extracts accurately, method is simple, success ratio is high.
Description of drawings
Fig. 1 adopts the contrast of the bacterium colony that dull and stereotyped spraying partition method and traditional puncture partition method obtain.
The pattern of the sulphate reducing bacteria under Fig. 2 transmission electron microscope.
Embodiment
Further specify particular content of the present invention and embodiment thereof below in conjunction with example.
1. separate and required equipment and the starting material of purifying
Equipment and vessel: clean bench, constant incubator, autoclave sterilization pot, electric furnace, heating asbestos pad, culture dish, inoculating needle, glass stick, spirit lamp, 2ml needle and syringe head.
Material: the SRB special culture media that API (API) is recommended, agar powder contains the sulphate reducing bacteria bacterial classification of assorted bacterium.
2. separate and the technical essential of purifying
(1) preparation of API liquid nutrient medium
With the API liquid nutrient medium by composition weighing, preparation and after regulating pH value and slightly being slight alkalinity to neutrality, the API liquid nutrient medium is installed in the Erlenmeyer flask, utilize electric furnace (on add asbestos pad) heating then, by the time when at a simmer, add an amount of agar powder, stir with glass stick, good with the tampon plug then, wrap to put into behind the kraft paper and prepare sterilization in the autoclave sterilization pot.
(2) glassware is sterilized: culture dish, syringe and syringe needle are wrapped to put into kraft paper respectively prepare sterilization in the autoclave sterilization pot.
(3) sterilization: the standard sterilising method that adopts API to recommend.
(4) preparation of API solid medium
The ultraviolet sterilizing lamp of opening clean bench is with the worktable certain hour of sterilizing, light spirit lamp, worktable is put in the taking-up of article in the Autoclave, while hot nutrient agar is joined in the plate, add-on is no more than the appropriate location of the plate degree of depth, cooling is stand-by after the culture medium solidifying then.
(5) separation of pure strain and purifying
Operate in the clean bench and carry out, and light spirit lamp:
A. take out syringe and load onto syringe needle, after activation, contain the sulphate reducing bacteria bacterial classification of assorted bacterium and extract an amount of bacterium liquid, and then bacterium liquid is injected back in the seed bottle emptying bacterium liquid.Its objective is and make syringe and needle tubing by the sulfate reduction fungi pollution.
B. extract one section air with needle tubing, then syringe needle is aimed at the top of plate substratum, syringe needle promotes needle tubing then apart from the certain distance of media surface, will contain the bacterium air and fall the surface of solid medium with atomized spray.
C. repeat to go on foot b repeatedly, on other surface of solid medium, spray, to obtain the tangible pure strain bacterium colony of personal feature.
D. cover culture dish ware lid, put into 37 ℃ of constant incubators and cultivate, until growing single bacterium colony.
(6) wait to grow single bacterium colony after, in clean bench,, put into sterilising liq API substratum with the aseptic inoculation ring single bacterium colony of picking respectively, carry out 37 ℃ of constant temperature culture respectively.
(7) pick out the strongest purifying bacterial classification of vital activity
Liquid nutrient medium at first fully blackening be the strongest sulphate reducing bacteria purifying bacterial classification of vital activity.
(8) be bacterial classification with the strongest sulphate reducing bacteria purifying bacterial classification of activity then, repeating step (1)～(6) are carried out purifying again one time, and the liquid nutrient medium of gained promptly is the pure strain of the sulphate reducing bacteria that will obtain.
Example 1:
Adopt dull and stereotyped spraying partition method, the sulphate reducing bacteria bacterial classification that contains assorted bacterium is separated and purifying.Adopt puncture method to compare (consulting Fig. 1) in addition.The result shows: spraying with flat board, this novel method of partition method is separated and the bacterium colony of purifying disperses, and bacterium colony is single distribution, identification easily, easy extraction; And the bacterium colony that adopts traditional puncture partition method to obtain fuses, and is difficult to select one pure culture bacterium colony.The small colonies individuality is bigger, and very difficult explanation is formed by single bacterial reproduction.Therefore use the separation of carrying out bacterial classification of new method and accuracy rate, efficient and the success ratio of purifying to be higher than traditional puncture partition method far away.With the strain of bacteria of sulfate reducting bacteria of novel method separation and purifying, through ferrous ammonium sulphate discriminating, H
2S gas differentiates that common staining is identified, the Gram-negative dientification of bacteria, and pattern evaluation several different methods such as (consulting Fig. 2) is comprehensively identified under the transmission electron microscope, the result shows that the bacterial classification with novel method separation and purifying is strictly the pure strain of sulphate reducing bacteria.
Example 2:
Adopt dull and stereotyped spraying partition method, the lactobacterium casei bacterial classification that contains assorted bacterium is separated and purifying, obtained the pure strain of lactobacterium casei exactly.Compare with plate streak, accuracy rate, success ratio and the working efficiency of using dull and stereotyped spraying partition method to extract bacterial classification are better than the latter.
Example 3:
Adopt dull and stereotyped spraying partition method, the Rhizopus oryzae bacterial classification that contains assorted bacterium is separated and purifying, obtained the pure strain of Rhizopus oryzae exactly.Compare with plate streak, accuracy rate, success ratio and the working efficiency of using dull and stereotyped spraying partition method to extract bacterial classification are better than the latter.
Claims (3)
1. the separation of a strain of bacteria of sulfate reducting bacteria and purifying novel method, it is characterized in that extracting under aerobic environment and turning out strain of bacteria of sulfate reducting bacteria and pick out the bacterial classification of the best purifying bacterial classification of biological activity as secondary separation and purifying, concrete processing step is as follows:
A) with the pure bacterium API of the sulphate reducing bacteria liquid nutrient medium of selecting for use, in gnotobasis, be heated to when at a simmer, stir after adding an amount of agar, be condensed into solid-state then; The ferrous ammonium sulphate and the xitix that add sterilization in the described API liquid nutrient medium;
B) in gnotobasis, the asepsis injector that at first will load onto syringe needle contains bacterium and handles one section air of back extraction, to contain the bacterium air then with the vaporific surface that evenly is ejected into solid medium, in constant incubator, turn out bacterium colony, the single bacterium colony of picking therein, the API liquid nutrient medium of putting into sterilization is turned out active best, even the liquid nutrient medium sulfatereducting bacteria purifying bacterial classification of blackening fully at first; Describedly contain bacterium and handle and to be: with asepsis injector and load onto syringe needle, extract an amount of bacterium liquid from the sulphate reducing bacteria bacterial classification that contains assorted bacterium, and then bacterium liquid is injected back in the seed bottle, emptying bacterium liquid makes syringe and needle tubing by the sulfate reduction fungi pollution;
C) be bacterial classification with the described sulphate reducing bacteria purifying of step b) bacterial classification, flash liberation and the purification step by bacterial classification repeats flash liberation and purifying again, and the liquid nutrient medium of gained promptly contains the pure strain of the sulphate reducing bacteria that will obtain.
2. the separation of strain of bacteria of sulfate reducting bacteria according to claim 1 and purifying novel method, it is characterized in that said sulphate reducing bacteria API liquid nutrient medium by composition weighing, preparation and after regulating pH value and slightly being slight alkalinity to neutrality, packs into and heat in the Erlenmeyer flask of sterilising treatment; Said condensation process is in gnotobasis, and the substratum that will contain agar joins in the plate while hot, and add-on is no more than 1/3 of the plate degree of depth, and cooled and solidified is stand-by solid medium then.
3. the separation of strain of bacteria of sulfate reducting bacteria according to claim 1 and purifying novel method is characterized in that temperature is 37 ℃ in the said constant incubator.
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| CN102260642A (en) * | 2011-07-20 | 2011-11-30 | 天津亿利科能源科技发展股份有限公司 | Method for separating and purifying sulfate reducing bacteria in oil field production water |
| CN102424812B (en) * | 2011-12-30 | 2013-04-10 | 黑龙江八一农垦大学 | Method for separating cellulose-decomposing anaerobic bacteria from cellulose-decomposing composite bacterium strain |
| CN104560771B (en) * | 2013-10-29 | 2017-08-22 | 中国石油化工股份有限公司 | A kind of isolated culture method of anaerobic bacteria |
| CN103966108B (en) * | 2014-05-20 | 2017-06-27 | 淮海工学院 | A kind of method of quick screening antibacterial activity filamentous fungi |
| CN103966141B (en) * | 2014-05-20 | 2017-06-16 | 淮海工学院 | A kind of quick screening antibacterial activity bacterium and the method for actinomyces |
| CN104312962A (en) * | 2014-10-27 | 2015-01-28 | 中国石油化工股份有限公司 | Separation and purification method for sulfate reducing bacteria in sewage of oil field |
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