CN102827770A - Color developing method for screening amylase producing strain - Google Patents
Color developing method for screening amylase producing strain Download PDFInfo
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- CN102827770A CN102827770A CN2012102962416A CN201210296241A CN102827770A CN 102827770 A CN102827770 A CN 102827770A CN 2012102962416 A CN2012102962416 A CN 2012102962416A CN 201210296241 A CN201210296241 A CN 201210296241A CN 102827770 A CN102827770 A CN 102827770A
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Abstract
A color developing method for screening an amylase producing strain comprises the following steps of: 1) collecting a sample from natural environment; 2) culture, that is, inoculating the sample in a culture dish containing a amylase producing strain selective medium, culturing for 3-4 days; 3) developing, that is, opening an upper cover of the culture dish, rapidly and uniformly spraying complex iodine on the amylase producing strain selective medium with bacterium colonies, standing for 2-4 min, observing the formation of transparent circles; 4) transferring, that is, immediately transferring the bacterium colonies with transparent circles in step 3) to a new amylase producing strain selective medium; 5) screening, that is, screening bacterium colonies with high enzyme production activity to obtain the amylase producing strain. The color developing method for screening the amylase producing strain provided by the invention preliminarily screens enzyme production microbe by a complex iodine spraying mode, can conveniently, rapidly and preliminarily screen bacterial strains producing extracellular amylase, and can be popularized for application to substitute iodine solution developing methods and iodine fumigation methods.
Description
Technical field
The present invention relates to a kind of mikrobe coloration method, especially a kind of coloration method that screens glycase generation bacterium.
Background technology
Glycase has become one of of paramount importance enzyme in the industrial application; And a large amount of mikrobes can produce highly active glycase; But the large-scale commercial applications production of enzyme still is confined to several kinds of specific fungies and bacterium, produces mikrobe from the novel high reactivity purpose glycase of occurring in nature screening and seems particularly important.Utilize the dull and stereotyped transparent circle method of starch separation screening starch enzyme-producing bacteria from physical environment, tentatively judge with Starch Indicator, can find fast that glycase produces mikrobe, the starch coloration indicator that adopts at present is iodine liquid or solid iodine.Iodine liquid, moity are iodine, potassiumiodide and zero(ppm) water.Iodine liquid developer needs oneself preparation and shelf time are of short duration; Often adopt the mode that is added drop-wise on the substratum during use, cause iodine liquid skewness, influence the consistence of colour developing, thereby prolonged the developing time of subregion, bacterium colony lethality is strengthened.
Solid iodine utilizes the colour developing of iodine subliming method, has saved the process of iodine liquid development process obtain solution, however different bacterium producing multi enzyme preparation; Consumption, tolerance time and developing time for sublimed iodine have difference [Tang Jia, Chen Chaoyin, Zhao Shenglan; Xia Jing opens great waves. and a kind of primary dcreening operation produces the simplified method of born of the same parents' exoamylases bacterial strain. biological processing, 2008; 6 (1): 37-40], and the seal degree difference of different culture ware, stifling process color developing effect also can be influenced.
Though the screening study of glycase generation bacterium is significant, the research work that the developer validity period in the amylase activity primary dcreening operation process is of short duration, the problems such as complicacy, consumption and developing time of preparing have restricted this field is extensively carried out.
Summary of the invention
Technical problem to be solved by this invention provides a kind of coloration method that adopts chelated iodine screening glycase to produce bacterium; The preliminary screening microbes producing cellulase; Preliminary screening is produced born of the same parents' exoamylases bacterial strain quickly and easily, and alternative iodine liquid development process and iodine fumigation and steaming method are promoted the use of.
For solving the problems of the technologies described above, the technical scheme that the present invention adopted is: a kind of coloration method that screens glycase generation bacterium, and this method may further comprise the steps:
1) from physical environment (soil and water body), gathers sample;
2) cultivate: with the sample employing dilution of step 1) collection or the method for quick line, be inoculated in the petridish that contains glycase generation bacterium screening culture medium, cultivated 3-4 days;
3) colour developing: chelated iodine is packed in the 30-50 milliliter plastics spray bottle; Open the petridish loam cake; Chelated iodine is sprayed to the glycase that grows bacterium colony fast and equably produces on the bacterium screening culture medium, left standstill 2-4 minute, observe the formation of transparent circle; Developing time can not oversize (being no more than 4 minutes), in order to avoid iodine volatilization influence is observed;
4) switching: the bacterium colony that step 3) is produced transparent circle is forwarded on the new glycase generation bacterium screening culture medium immediately, cultivates repeating step 3 3-4 days) colour developing, with the ratio of transparent circle diameter/colony diameter, calculate inulinase-producing activity;
5) screening: with inulinase-producing activity high the streak culture 3-4 of bacterium colony days, repeating step 3) detect the glycase production, the high bacterium colony of screening inulinase-producing activity obtains glycase and produces bacterium.
The glycase that the inulinase-producing activity that the step 5) screening is obtained is high produces bacterium, adopts 4 ℃ of test tube slants with 20 ℃ of glycerine of – are preserved.
In the step 3), the chelated iodine volume that is sprayed to glycase generation bacterium screening culture medium is the 1-2 milliliter.
In the step 5), the ratio of transparent circle diameter/colony diameter is the high bacterium colony of inulinase-producing activity greater than 1.7 bacterium colony.
A kind of coloration method that glycase produces bacterium that screens provided by the invention; Owing to adopt mode with the chelated iodine even spraying; The preliminary screening microbes producing cellulase; Have developing time weak point, reagent long preservative period, easy to operate convenience, the stable advantage of color developing effect, preliminary screening is produced born of the same parents' exoamylases bacterial strain quickly and easily, and alternative iodine liquid development process and iodine fumigation and steaming method are promoted the use of.
Embodiment
Embodiment one
A kind of coloration method that screens glycase generation bacterium, this method may further comprise the steps:
1) gathers sample:, immediately sample is taken back the laboratory near collection soil sample SanXia University's Nangyuan District students' dining hall (5 gram);
2) cultivate:
Substratum is formed (w/v): 2% Zulkovsky starch, 0.5% peptone, 0.5% NaCl, 0.05% MgSO
47H
2O, 0.05% KH
2PO
4, 1.5% agar, pH 7.0-7.2;
The sample mixing that step 1) is gathered evenly after, get the quick streak inoculation of about 0.5 gram and produce on the bacterium screening culture medium in glycase, cultivated 4 days in 28 ℃;
3) colour developing: 1-2 milliliter chelated iodine is sparged on the substratum fast and equably, leave standstill visible significantly starch hydrolysis transparent circle after 2 minutes;
4) switching: the bacterium colony that will produce transparent circle is transferred on the new screening culture medium, cultivates repeating step 34 days) detect the glycase production, estimate inulinase-producing activity with the ratio of transparent circle diameter/colony diameter;
5) screening: with the ratio of transparent circle diameter/colony diameter greater than streak culture once more 4 days of 1.7 bacterium colony, repeating step 3) detect the glycase production, the ratio that obtains transparent circle diameter/colony diameter reaches 2.2 the diastatic bacterium of product.
Embodiment two
A kind of coloration method that screens glycase generation bacterium, this method may further comprise the steps:
1) gathers sample:, immediately sample is taken back the laboratory near collection soil sample SanXia University's glad garden students' dining hall (4 gram);
2) cultivate:
Substratum is formed (w/v): 1.5% Zulkovsky starch, 0.5% peptone, 0.5% NaCl, 0.05% MgSO
47H
2O, 0.05% KH
2PO
4, 1.5% agar, pH 7.0-7.2;
Behind the sample gradient dilution with the step 1) collection, coat glycase and produce on the bacterium screening culture medium, cultivated 3 days in 38 ℃;
3) colour developing: 1-2 milliliter chelated iodine is sparged on the substratum fast and equably, leave standstill visible significantly starch hydrolysis transparent circle after 3 minutes;
4) switching: the bacterium colony that will produce transparent circle is transferred on the new screening culture medium, cultivates repeating step 33 days) detect the glycase production, estimate inulinase-producing activity with the ratio of transparent circle diameter/colony diameter;
5) screening: with the ratio of transparent circle diameter/colony diameter greater than streak culture once more 3 days of 1.7 bacterium colony, repeating step 3) detect the glycase production, the ratio that obtains transparent circle diameter/colony diameter reaches 1.9 the diastatic bacterium of product.
Embodiment three
A kind of coloration method that screens glycase generation bacterium, this method may further comprise the steps:
1) gathers sample: near the students' dining hall of garden, SanXia University east, gather water sample (5 milliliters), immediately sample is taken back the laboratory;
2) cultivate:
Substratum is formed (w/v): Zulkovsky starch 2.5%, and peptone 0.5%, NaCl 0.5%, MgSO
47H
2O 0.05%, KH
2PO
40.05%, agar 1.5%, pH 6.0-7.0;
The sample streak inoculation that step 1) is gathered produces on the bacterium screening culture medium in glycase, cultivates 4 days in 30 ℃;
3) colour developing: 1-2 milliliter chelated iodine is sparged on the substratum fast and equably, leave standstill visible significantly starch hydrolysis transparent circle after 4 minutes;
4) switching: the bacterium colony that will produce transparent circle is transferred on the new screening culture medium, cultivates repeating step 34 days) detect the glycase production, estimate inulinase-producing activity with the ratio of transparent circle diameter/colony diameter;
5) screening: with the ratio of transparent circle diameter/colony diameter greater than streak culture once more 4 days of 1.7 bacterium colony, repeating step 3) detect the glycase production, the ratio that obtains transparent circle diameter/colony diameter reaches 2.3 the diastatic bacterium of product.
Claims (4)
1. one kind is screened the coloration method that glycase produces bacterium, it is characterized in that this method may further comprise the steps:
1) from physical environment, gathers sample;
2) cultivate: the sample of step 1) collection is inoculated in the petridish that contains glycase generation bacterium screening culture medium, cultivated 3-4 days;
3) colour developing: open the petridish loam cake, chelated iodine is sprayed to the glycase that grows bacterium colony fast and equably produces on the bacterium screening culture medium, left standstill 2-4 minute, observe the formation of transparent circle;
4) switching: the bacterium colony that step 3) is produced transparent circle is forwarded on the new glycase generation bacterium screening culture medium immediately, cultivates repeating step 3 3-4 days), with the ratio of transparent circle diameter/colony diameter, calculate inulinase-producing activity;
5) screening: with inulinase-producing activity high the streak culture 3-4 of bacterium colony days, repeating step 3) detect the glycase production, the high bacterium colony of screening inulinase-producing activity obtains glycase and produces bacterium.
2. a kind of coloration method that glycase produces bacterium that screens according to claim 1 is characterized in that: in the step 1), and collected specimens from the soil of physical environment and water body.
3. a kind of coloration method that glycase produces bacterium that screens according to claim 1 is characterized in that: in the step 3), the chelated iodine volume that is sprayed to glycase generation bacterium screening culture medium is the 1-2 milliliter.
4. a kind of coloration method that glycase produces bacterium that screens according to claim 1 is characterized in that: in the step 5), the ratio of transparent circle diameter/colony diameter is the high bacterium colony of inulinase-producing activity greater than 1.7 bacterium colony.
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|---|---|---|---|
| CN2012102962416A CN102827770A (en) | 2012-08-20 | 2012-08-20 | Color developing method for screening amylase producing strain |
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| CN2012102962416A CN102827770A (en) | 2012-08-20 | 2012-08-20 | Color developing method for screening amylase producing strain |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458607A (en) * | 2014-11-24 | 2015-03-25 | 广东省生态环境与土壤研究所 | Method for quickly detecting extracellular respiratory activity of microorganisms |
| CN114480566A (en) * | 2022-03-04 | 2022-05-13 | 河南金百合生物科技股份有限公司 | Method for detecting whether bacterial strain produces enzyme |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377584A (en) * | 2002-02-28 | 2002-11-06 | 周颖 | Atomic iodine aqueous solution and its preparing method |
| CN101300036A (en) * | 2005-09-21 | 2008-11-05 | 苏尔莫迪克斯公司 | Coatings and articles including natural biodegradable polysaccharides |
-
2012
- 2012-08-20 CN CN2012102962416A patent/CN102827770A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377584A (en) * | 2002-02-28 | 2002-11-06 | 周颖 | Atomic iodine aqueous solution and its preparing method |
| CN101300036A (en) * | 2005-09-21 | 2008-11-05 | 苏尔莫迪克斯公司 | Coatings and articles including natural biodegradable polysaccharides |
Non-Patent Citations (2)
| Title |
|---|
| 卢福芝等: "淀粉酶产生菌的分离筛选", 《河池学院学报》, 30 April 2012 (2012-04-30), pages 26 - 28 * |
| 雷晓燕: "土壤中淀粉酶产生菌的筛选及产酶条件优化", 《沈阳化工大学学报》, 30 September 2010 (2010-09-30), pages 203 - 207 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458607A (en) * | 2014-11-24 | 2015-03-25 | 广东省生态环境与土壤研究所 | Method for quickly detecting extracellular respiratory activity of microorganisms |
| CN114480566A (en) * | 2022-03-04 | 2022-05-13 | 河南金百合生物科技股份有限公司 | Method for detecting whether bacterial strain produces enzyme |
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Application publication date: 20121219 |