CN1970787A - Method for detecting number of effective bacillus - Google Patents
Method for detecting number of effective bacillus Download PDFInfo
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- CN1970787A CN1970787A CN 200510126211 CN200510126211A CN1970787A CN 1970787 A CN1970787 A CN 1970787A CN 200510126211 CN200510126211 CN 200510126211 CN 200510126211 A CN200510126211 A CN 200510126211A CN 1970787 A CN1970787 A CN 1970787A
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- bacillus
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 19
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims description 21
- 239000003337 fertilizer Substances 0.000 claims description 19
- 230000000813 microbial effect Effects 0.000 claims description 18
- 241000726221 Gemma Species 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 238000000053 physical method Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 39
- 238000004519 manufacturing process Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000005059 dormancy Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quantitative detecting method of microbe bacillus, which comprises the following steps: disposing sample through physical method; adsorbing according to normal method; coating utensil; growing visible bacteria on the board after 2-3d; counting and calculating normal bacteria; obtaining the quantity of pure germ.
Description
Technical field
The present invention relates to a kind of microbe quantity quantity measuring method, specifically relate to a kind of detection method of number of effective bacillus.
Background technology
Microbial fertilizer (microbial inoculum) is a kind of novel capital goods in the agricultural sector, promotes and show fine application prospect in China.For the effect of stabilised microorganism fertilizer, must at first guarantee the quality product of microbial fertilizer.The number of viable of effective microbe in the microbial fertilizer is the quality core of product.Therefore the accuracy of effective microbe number of viable detection method is produced instructing, is guaranteed that quality product has very important meaning.For this reason, China Ministry of Agriculture has formulated and issued with the number of viable of effective microbe is the target level of product quality " microbial fertilizer industry standard (NY-227-94) " of core, and its relevant quality determining method has been made concrete regulation.Wherein about the detection of effective microbe quantity, selected conventional microorganism colony counting method for use, with effective microbe in the product under prescribed condition, formed colony counts is represented with hundred million of living bacteria count amounts (the colony-forming unit)/gram (milliliter) that contains in the sample as the standard of counting.
Current, in the microbial fertilizer product, the stability of effective microbe quantity (factory inspection is to the validity period of product regulation) is a problem that directly influences quality product and effect.Because the microbial cells of most of kinds, can not withstand high temperatures and exsiccant influence, therefore in product through losing viability after a while.Even select for use have high temperature resistance, the genus bacillus of resist drying, also because the production technique difference, under big working condition, also there are very big-difference in the quantity and the ratio that really form gemma.And now during the genus bacillus in detecting microbial fertilizer production and product, use conventional microorganism dilution plate assay method to detect, though the sum of measuring the effective microbe viable bacteria in the sample that can be relatively accurate, but can't distinguish the formed bacterium number of different thalli morphologies, promptly can't tell bacterium colony is that the stable gemma of do as one likes shape is sprouted and formation, or the unsettled nourishing body direct growth formation of do as one likes shape.So, use existing method to be difficult to measure the effective microbe quantity that has stability in the product, bring difficulty for the real quality of estimating product.
Summary of the invention
The objective of the invention is to overcome the result of existing detection method, the true quantity that can not reflect effective viable bacteria in the product, can not accurately estimate the defective of the real quality of product, thereby on the basis of existing universal test method NY-227-94, according to the biological characteristics of genus bacillus, provide a kind of detection method that is used for the number of effective bacillus of microbial fertilizer.
The objective of the invention is to realize by the following technical solutions:
The detection method of number of effective bacillus in the microbial fertilizer product provided by the invention comprises following step:
1), carries out sampling, weighing, dilution, Tu ware, cultivation, counting and the calculating of product according to the regulation of microbial fertilizer industry standard NY-227-94;
2) above-mentioned dilution of sample liquid is packed in triangular flask or the test tube, place 80～90 ℃ of waters bath with thermostatic control to carry out pyroprocessing 10～20 minutes;
3) simultaneously, adopt and step 2) in identical triangular flask or test tube, add and step 2) in dilute sample with the distilled water of volume, the reference standard liquid as Temperature Treatment places same water bath, carries out pyroprocessing 10～20 minutes in 80～90 ℃;
4) take out sample diluting liquid, draw and carry out the Tu ware, put into 30 ℃ of incubators then and cultivate according to ordinary method through pyroprocessing;
5) grow the visible bacterium colony on the rear plate in 2～3 days, take out and carry out conventional enumeration and calculating, obtain the bacterium number that pure gemma sprouting forms in this sample.
Detection method provided by the invention is applicable to that the microbial fertilizer of employing effective bacillus production and the quality product of other microbial quotient product fertilizer detect use.Can also further recognize the ratio of " gemma " and nourishing body in the product, make its detected result when product export meet product actual effectively bacterium number before the deadline more, help instructing and produce, guarantee quality product.
The detection method of number of effective bacillus provided by the invention, be on the basis of existing universal test method, according to the biology characteristics of microorganism and " gemma " bacillus, and the detection method that can directly detect grown spore and colony counts that other vegetative cell forms in the sample that designs, uses.This method is by pyroprocessing, and the dormancy that not only can break grown spore in the product makes it very fast sprouting and forms bacterium colony (without the processing of heating, the part gemma is arranged promptly, because be in dormancy, can not sprout immediately, can not form bacterium colony and can't count); Can also kill the thalline of failing to form gemma in the sample, make it dead in advance, avoid counting total viable count, interference detection results because nourishing body forms bacterium colony.Accomplish early stage discriminating, " eliminating the false and retaining the true " makes the detected result when dispatching from the factory can meet the actual number of viable of product in effective service life more.It is more accurate to detect data with this result as dispatching from the factory of product, meets truth more, helps guaranteeing the quality of product more.And, to compare with existing method, detection method provided by the invention is easy, accurately easily goes, and is applicable to that the microbial fertilizer product of all employing genus bacillus productions and the commodity of microbial fertilizer product detect.
Embodiment
Identical sampling is 2 parts from the liquid product with a collection of bacillus megaterium (phosphorus bacteria), 1 part of ordinary method that " the microbial fertilizer industry standard NY-227-94 " that issues according to the Ministry of Agriculture stipulates wherein, detect the preparation and the dull and stereotyped making of substratum, and the sampling of sample, weighing, dilution, Tu ware, cultivation, counting and calculating, obtain effective bacterium sum of sample, the results are shown in table 1.
Another duplicate samples is used method provided by the invention, carries out the detection of number of effective bacillus, and concrete steps are as follows:
1) ordinary method of " microbial fertilizer industry standard NY-227-94 " regulation of issuing according to the Ministry of Agriculture equally detects the preparation and the dull and stereotyped making of substratum, and the sampling of sample, weighing, dilution, Tu ware, cultivation, counting and calculating;
2) above-mentioned sample diluting liquid is packed in triangular flask or the test tube, place 80 ℃ of waters bath with thermostatic control to carry out pyroprocessing 10 minutes;
3) simultaneously, adopt and step 2) in identical triangular flask or test tube, add and step 2) in dilute sample with the distilled water of volume, place same water bath simultaneously, as the reference standard liquid of Temperature Treatment, in 80 ℃, carried out pyroprocessing 10 minutes;
4) will according to the Hang Tu ware of the identical method Jin of step 1), put into 30 ℃ of incubators then and cultivate through the sample diluting liquid of pyroprocessing;
5) grow the visible bacterium colony on the rear plate in 2～3 days, take out and to carry out conventional enumeration and calculating, then can obtain in this sample pure gemma and sprout formed living bacteria count, the results are shown in table 1.
The detected result of table 1,2 kinds of methods
| Detection method | Product export detects | Product stock detected in 6 months |
| Ordinary method | 9.51 hundred million/ml | 1.82 hundred million/ml |
| The inventive method | 1.84 hundred million/ml | 1.83 hundred million/ml |
By the listed result of table 1 as can be seen:
1, owing to contain a large amount of nourishing bodys and a small amount of gemma in the product, therefore when product export, adopts the conventional sense method, recorded the bacterium number of comparatively high amts.But identical product after 6 months, adopts ordinary method to detect through room temperature preservation again, then because a large amount of nourishing body death in the product cause number of viable to descend significantly.The detected result of this moment only is 19% of the detected result of dispatching from the factory.Quantity descends significantly, the result shows: though ordinary method can be measured the total viable count in the product relatively accurately, but detection for active bacteria formulation and commercial fertilizer thereof, very big sum of errors limitation is then arranged, can not really reflect active bacteria formulation number of viable before the deadline, make troubles for the quality-guarantee and the application of product, cause confusion.
2, adopt the result that the present invention detected, owing to adopted pyroprocessing, killed the nourishing body in the product ahead of time, detected result when therefore dispatching from the factory and product are preserved the detection data basically identical after 6 months, show that the result that this method is measured more tallies with the actual situation.
3, according to the requirement of microbial fertilizer commodity and the biological nature of genus bacillus, adopt detection method of the present invention, can get rid of the sample intrinsic and disturb, measurement result tallies with the actual situation more, more accurate, more have in the quality and the effect that guarantee product.
Claims (4)
1, a kind of detection method of number of effective bacillus comprises following step:
1), carries out sampling, weighing, dilution, Tu ware, cultivation, counting and the calculating of product according to the regulation of microbial fertilizer industry standard NY-227-94;
2) above-mentioned dilution of sample liquid is packed in triangular flask or the test tube, place water bath with thermostatic control to carry out pyroprocessing 10～20 minutes;
3) simultaneously, adopt and step 2) in identical triangular flask or test tube, add and step 2) in dilute sample with the distilled water of volume, as the reference standard liquid of Temperature Treatment, place same water bath to carry out pyroprocessing 10～20 minutes;
4) take out sample diluting liquid, draw and carry out the Tu ware, put into incubator then and cultivate according to ordinary method through pyroprocessing;
5) grow the visible bacterium colony on the rear plate in 2～3 days, take out and carry out conventional enumeration and calculating, obtain the bacterium number that pure gemma sprouting forms in this sample, then can represent the living bacteria count amount of this sample.
2, the detection method of number of effective bacillus as claimed in claim 1 is characterized in that: the temperature of water bath with thermostatic control described step 2) is 80～90 ℃.
3, the detection method of number of effective bacillus as claimed in claim 1 is characterized in that: the temperature of the water bath with thermostatic control of described step 3) is 80～90 ℃.
4, the detection method of number of effective bacillus as claimed in claim 1 is characterized in that: the incubator temperature of described step 4) is 30 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005101262110A CN1970787B (en) | 2005-11-25 | 2005-11-25 | Method for detecting number of effective bacillus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005101262110A CN1970787B (en) | 2005-11-25 | 2005-11-25 | Method for detecting number of effective bacillus |
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| Publication Number | Publication Date |
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| CN1970787A true CN1970787A (en) | 2007-05-30 |
| CN1970787B CN1970787B (en) | 2011-09-07 |
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| CN2005101262110A Expired - Fee Related CN1970787B (en) | 2005-11-25 | 2005-11-25 | Method for detecting number of effective bacillus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127518A (en) * | 2010-12-31 | 2011-07-20 | 广州市容川饲料有限公司 | Selective culture medium used for quantitative detection of bacillus |
| CN101586144B (en) * | 2009-07-03 | 2011-12-28 | 武汉科诺生物科技股份有限公司 | Method for detecting the number of live spore in Bacillus subtilis powder of 5 hundred billion live spores/gram |
| CN104152376A (en) * | 2014-07-30 | 2014-11-19 | 湖南豫园生物科技有限公司 | Spore protective agent and application thereof, and detection method of sporeformer producing content in microbial fertilizer |
| CN108676841A (en) * | 2018-05-25 | 2018-10-19 | 广州南侨食品有限公司 | The method of inspection of gemma and heat resistant spores |
| CN108949589A (en) * | 2018-08-24 | 2018-12-07 | 泸州众康农业检测有限公司 | The effective detection method of bacterium in a kind of liquid microbe bacterial manure |
-
2005
- 2005-11-25 CN CN2005101262110A patent/CN1970787B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586144B (en) * | 2009-07-03 | 2011-12-28 | 武汉科诺生物科技股份有限公司 | Method for detecting the number of live spore in Bacillus subtilis powder of 5 hundred billion live spores/gram |
| CN102127518A (en) * | 2010-12-31 | 2011-07-20 | 广州市容川饲料有限公司 | Selective culture medium used for quantitative detection of bacillus |
| CN104152376A (en) * | 2014-07-30 | 2014-11-19 | 湖南豫园生物科技有限公司 | Spore protective agent and application thereof, and detection method of sporeformer producing content in microbial fertilizer |
| CN104152376B (en) * | 2014-07-30 | 2017-04-05 | 湖南豫园生物科技股份有限公司 | The detection method of spore production bacteria content in spore protective agent, application and microbial manure |
| CN108676841A (en) * | 2018-05-25 | 2018-10-19 | 广州南侨食品有限公司 | The method of inspection of gemma and heat resistant spores |
| CN108949589A (en) * | 2018-08-24 | 2018-12-07 | 泸州众康农业检测有限公司 | The effective detection method of bacterium in a kind of liquid microbe bacterial manure |
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| Publication number | Publication date |
|---|---|
| CN1970787B (en) | 2011-09-07 |
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