CN1970787B - Method for detecting number of effective bacillus - Google Patents
Method for detecting number of effective bacillus Download PDFInfo
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- CN1970787B CN1970787B CN2005101262110A CN200510126211A CN1970787B CN 1970787 B CN1970787 B CN 1970787B CN 2005101262110 A CN2005101262110 A CN 2005101262110A CN 200510126211 A CN200510126211 A CN 200510126211A CN 1970787 B CN1970787 B CN 1970787B
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Abstract
The present invention discloses a quantity detecting method of microbe, specifically relating to a quantity detecting method for bacillus of microbe fertilizer. The method is characterized in that: disposing a sample to be detected through simple and practical physical method; adsorbing according to the normal method; coating utensil; growing visible bacterial colony on the board after 2-3d; counting and calculating normal bacterial colony; and obtaining the quantity of pure germ in the sample. The detecting method provided by the present invention is suitable for detecting product quality of microbial fertilizer produced by bacillus and other microbial fertilizer product. Moreover, the proportion between germ and trophozoite in the product can be better learned about so that the detection result of the manufactured product can be more conformed to the practical viable count at expiration of the product, thus being good in guiding production and guaranteeing product quality. Comparing with the prior art, the detecting method is more simple, convenient, accurate, and practical.
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 genus bacillus quantity.
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 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 genus bacillus quantity of microbial fertilizer.
The objective of the invention is to realize by the following technical solutions:
The detection method of genus bacillus quantity in the microbial fertilizer product provided by the invention comprises following step:
1) according to the regulation of microbial fertilizer industry standard NY-227-94, carry out product sampling, weighing and dilution;
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 handle 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, handles in 80～90 ℃ 10～20 minutes;
4) take out sample diluting liquid, draw and be coated with 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 genus 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 bacterium number before the deadline more, help instructing and produce, guarantee quality product.
The detection method of genus bacillus quantity 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 the total viable count of sample, the results are shown in table 1.
Another duplicate samples is used method provided by the invention, carries out the detection of living spores bacillus quantity, 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 viable 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 (2)
1. the detection method of a genus bacillus quantity comprises following step:
1), carries out sampling, weighing and the dilution 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 handle 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 80～90 ℃ of water baths to handle 10～20 minutes;
4) take out sample diluting liquid, draw and be coated with 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, be the number of viable of this sample.
2. the detection method of genus bacillus quantity as claimed in claim 1 is characterized in that: the incubator temperature of described step 4) is 30 ℃.
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| CN2005101262110A CN1970787B (en) | 2005-11-25 | 2005-11-25 | Method for detecting number of effective bacillus |
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| CN2005101262110A CN1970787B (en) | 2005-11-25 | 2005-11-25 | Method for detecting number of effective bacillus |
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| CN1970787B true CN1970787B (en) | 2011-09-07 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127518B (en) * | 2010-12-31 | 2013-02-20 | 广州市容川饲料有限公司 | Selective culture medium used for quantitative detection of bacillus |
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| 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 |
| 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 |
-
2005
- 2005-11-25 CN CN2005101262110A patent/CN1970787B/en active Active
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| .饲料微生物污染检测法中接菌方法的改进.饲料博览 5.1996,(5),全文. |
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| 陈洪岩;辛晓光;宋小华;南锡;张永江;张爱国;.饲料微生物污染检测法中接菌方法的改进.饲料博览 5.1996,(5),全文. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127518B (en) * | 2010-12-31 | 2013-02-20 | 广州市容川饲料有限公司 | Selective culture medium used for quantitative detection of bacillus |
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