CN1796568A - Quick detecting coliform group and medium for culturing coliforms and preparation method - Google Patents
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- CN1796568A CN1796568A CN 200410081655 CN200410081655A CN1796568A CN 1796568 A CN1796568 A CN 1796568A CN 200410081655 CN200410081655 CN 200410081655 CN 200410081655 A CN200410081655 A CN 200410081655A CN 1796568 A CN1796568 A CN 1796568A
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Abstract
This invention relates to a culture medium for the rapid detection of coliform and coliform groups, which is mixed from distilled water solution containing peptone, yeast extract, lactose, 4-methylumbelliferone-beta-D-galactoside, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and agar (or agar-free), the solution of indophenol-beta-D-glucoside and solution of cefsulodin. Peptone, yeast extract, lactose, 4-methylumbelliferone-beta-D-galactoside, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and agar (or agar-free) are weighed according to a certain criterion and added to distilled water for heat dissolution. The consequent solution is adjusted to a certain pH value, put into a high pressure sterilizer for sterilization and then added with filtrated and sterilized indophenol-beta-D-glucoside solution and cefsulodin solution after the temperature lowered to 50 deg.C+/-5 deg.C. This is useful in either qualitative or quantitative detection of coliform and coliform groups in drinking water, environmental water and foods, high efficiency and accurate detection result.
Description
Technical field
The present invention relates to health and epidemic prevention detection and environmental water quality monitoring technology, be specifically related to a kind of coliform that is used for rapid detection tap water, ambient water or food and colibacillary substratum and preparation method thereof.
Background technology
In water or food, coliform and intestinal bacteria are used as the index bacterium that sample is polluted by pathogen enterobacteria, it is one of important indicator of evaluating water quality and food hygiene quality, so the coliform and the intestinal bacteria that can detect quickly and accurately in water outlet or the food have crucial meaning.At present, coliform in the food and colibacillary detection there are two standards in China: the one, national standard promptly 9 is managed fermentation methods, and the detection of only having stipulated coliform detects step and was divided into for three steps, and two steps of back are confirmatory experiment, 3 days consuming time; The 2nd, the import-export commodity inspection industry standard, be SN0169-92, intestinal bacteria are detected requirement is, on the basis of carrying out the detection of coliform and excrement colibacillus group, carry out plate isolation again, with suspicious or colonies typical be connected on the inclined-plane cultivate after, after carrying out colibacillary every biochemical test with culture, carry out colibacillary judgement, about ten days consuming time according to the result of its biochemical test.And to the detection of ambient water quality, detection method and standard have only been stipulated at present to coliform, be filter membrane method and 15 pipe fermentation methods, filter membrane method is earlier with behind the water sample process filter membrane suction filtration, again the filter membrane sheet is put in the substratum and cultivates, typical case or suspicious bacterium colony are carried out reaching a conclusion behind the confirmatory experiment 2 days consuming time; 15 pipe fermentation method operations were divided into for three steps, and two steps of back are confirmatory experiment, 3 days consuming time.Be not difficult to find out that above the whole bag of tricks exists obviously that experimental procedure is cumbersome, the kinds of culture medium of usefulness is many, it is long to expend time in, detect the high deficiency of cost, need be improved.
In recent years, a lot of work have been done by various countries aspect rapid detection, import-export commodity inspection industry standard SN0333-94 regulation as China, 4-methyl umbelliferone-β-D-glucoside joined carry out colibacillary maximum possible value (MPN) in LST (lauryl sulfate Tryptones) meat soup and detect, though saved the cumbersome step of traditional detection method, but still need confirmatory experiment, and can only single detection intestinal bacteria for the not fluorescent test tube of aerogenesis.And for example, be mainly used in Violet Red Bile Agar (VRBA) substratum that detects intestinal bacteria and coliform in the milk, this substratum as assorted bacteria inhibitor, adds the developer that toluylene red causes the medium pH value to change as the object bacteria growth metabolism with cholate; After the object bacteria ferment lactose produced acid, the medium pH value descended, and bacterium colony becomes brick-red, can judge that it is the coliform bacterium colony.This red bacterium colony forms and needs 24-28 hour, carry out colibacillary determine then to need again red bacterium colony to be connected in the BGLB on fermentation or EMB (eosin methylene blue agar) flat board just can reach a conclusion after streak culture, as can be seen, this method needs confirmatory experiment equally, and consumed time is also long.
The Colilert system a kind ofly carries out the substratum of rapid detection to coliform and intestinal bacteria, and this substratum contains ortho-nitrophenyl base β-D-galactoside and 4-methyl umbelliferone-β-two kinds of enzyme substratess of D-glucoside; The former is at coliform, and coliform can produce β-D-galactase in growth, and this enzyme can produce yellow after decomposing ONPG; The latter is at intestinal bacteria, and intestinal bacteria can produce β-D-glucuroide in growth, after this enzyme decomposes MUG, produces bluish voilet fluorescence under UV-light.When sample joins in this substratum, in 24-28 hour, just can carry out coliform and colibacillary judgement to sample according to the situation that produces yellow and fluorescence.Though this method is fast and convenient, but also there are some defectives: the one, the ONPG back xanchromatic neighbour-oil of mirbane that is decomposed easily is diffused in the agar and has influence on result's observation, the 2nd, the fluorescence generation diffusion after MUG is decomposed is very fast, be not easy to observe, thereby, when nineteen ninety Clark proposes to use this method that tap water is carried out coliform and colibacillary detection, exist higher false negative rate.In addition, also have some other rapid detection coliform and colibacillary method, but much be that an experiment can only detect a kind of object bacteria, promptly in substratum, add the enzyme substrates that a kind of bacterium produces enzyme, detect another kind of object bacteria and then can only select another kind of substratum again for use, the workload that not only increase method detects, and cause raw material and time waste.
Summary of the invention
At the above problem that in coliform and intestinal bacteria detection, exists at present, the invention provides a kind of rapid detection coliform and colibacillary substratum and preparation method thereof, adopt this substratum, only needed 24 hours just can carry out qualitative or quantitative analysis to coliform in water or the food sample and intestinal bacteria, can also suppress the growth of the Gram-negative bacteria of gram-positive microorganism and non-coliform, reduce the probability that false positive and false negative occur, improve the accuracy of detection efficiency and detected result.
Rapid detection coliform of the present invention and colibacillary substratum, indolol-β-D-glucoside solution and the mixing of 4.5-5.0mL cefsulodin solution by 1000mL distilled water solution that contains peptone 3.5-5.0g, yeast extract 2.0-3.0g, lactose 0.6-1.2g, 4-methyl umbelliferone-β-D-galactoside 0.05-0.25g, sodium-chlor 6.0-7.5g, dipotassium hydrogen phosphate 2.5-3.4g, potassium primary phosphate 0.7-1.2g, Sodium Lauryl Sulphate BP/USP 0.1-0.2g, agar 0-15.0g and 15-25mL are formed, and the final pH value is 6.9 ± 0.2.When basal culture medium is used for coliform and intestinal bacteria are qualitative or MPN value when detecting, wherein do not add agar.
Peptone and yeast extract belong to conventional medium component, for coliform and colibacillary growth provide organic nitrogenous source, promote its growth, and yeast extract can also promote the reparation of injured bacterium.Sodium Lauryl Sulphate BP/USP joins in the substratum as fungistat, can suppress gram-positive cocci and sporogenic varied bacteria growing.The adding cefsulodin can suppress the growth of the Gram-negative bacteria of gram-positive microorganism and non-coliform.False positive and false-negative appearance have been controlled in the adding of these fungistats effectively, make detected result accurately and reliably.Lactose provides organic carbon source not only for the growth of object bacteria, or inductor, and this is because beta-D-galactosidase is a kind of inducible enzyme, and the adding of lactose makes colibacillus group energy greater amount ground produce this kind of enzyme, thereby makes reacting phenomenon more obvious, is convenient to observe.The adding of sodium-chlor, dipotassium hydrogen phosphate and potassium primary phosphate provides growth required mineral substance for coliform and intestinal bacteria, keeps stable osmotic pressure, and keeps nutrient solution near neutral pH value.Because lactose and other nutrition can produce acid after growth is decomposed, thereby cause the nutrient solution peracid, hinder the growth of bacterium, the adding of dipotassium hydrogen phosphate and potassium primary phosphate can be kept stable p H value, is beneficial to coliform and colibacillary growth.
The preparation method of described detection coliform and colibacillary substratum, its step is as follows:
(1) component content by above-mentioned culture medium solution takes by weighing peptone, yeast extract, lactose, 4-methyl umbelliferone-β-D-galactoside, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, Sodium Lauryl Sulphate BP/USP and agar, adds heating for dissolving in the distilled water;
Perhaps the component content by above-mentioned culture medium solution takes by weighing peptone, yeast extract, lactose, 4-methyl umbelliferone-β-D-galactoside, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, Sodium Lauryl Sulphate BP/USP, adds heating for dissolving in the distilled water;
(2) treat the cooling of above solution after, supply this solution after heat front volume with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add 15-25mL indolol-β-D-glucoside and 4.5-5.0mL cefsulodin preparation just and filtration sterilization respectively.
Since coliform in process of growth, can produce beta-D-galactosidase (β-D-galactosidase), so, in substratum, just added a kind of substrate---4-methyl umbelliferone-β-D-galactoside of this enzyme.If there is coliform in sample, then 4-methyl umbelliferone-β-D-galactoside will be decomposed by beta-D-galactosidase, and the 4-methyl umbelliferone group that dissociates out can produce fluorescence under the 366mm UV-light, thereby shows and have coliform in the sample.
Because intestinal bacteria can produce β-D-glucuroide (β-D-glucosidase) in process of growth, so, in substratum, added a kind of substrate of this enzyme---indolol-β-D-glucoside, decomposed the back by this enzyme and produce insoluble indigo precipitation, thereby show and have intestinal bacteria in the sample.
Because above-mentioned two kinds of enzymes have stronger specificity, this is as white powder, do not fluoresce, and after they are applied simultaneously, reacting phenomenon can not interfere with each other or be subjected to the interference of external environment, therefore, can the coliform in water or the food sample be detected, can the intestinal bacteria in water or the food sample be detected again simultaneously.
The present invention develops according to enzymatic fluorescence and enzymatic colour developing principle that external developed recently gets up, contrast has the following advantages with prior art: can detect simultaneously coliform in tap water, ambient water or the food and intestinal bacteria, the time of detection is short, efficient is high; Can carry out qualitative detection to coliform and intestinal bacteria, can carry out quantitative analysis to coliform and intestinal bacteria again; Can reduce the probability that false positive and false negative occur, detected result is accurate.
Embodiment
Embodiment 1: preparation qualitative detection coliform and colibacillary substratum.
(1) take by weighing peptone 3.5g, yeast extract 2.0g, lactose 0.6g, 4-methyl umbelliferone-β-D-galactoside 0.05g, sodium-chlor 6.0g, dipotassium hydrogen phosphate 2.5g, potassium primary phosphate 0.7g, Sodium Lauryl Sulphate BP/USP 0.1g, adding distil water is to the 1000mL heating for dissolving;
(2) treat the cooling of above solution after, supply this solution to 1000mL with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add preparation just and respectively the 25mL concentration of filtration sterilization be that 15mg/mL indolol-β-D-glucoside solution and 5.0mL concentration are 1.1mg/mL cefsulodin solution, the substratum that is not promptly contained agar, but fast qualitative detects coliform and intestinal bacteria in tap water, ambient water or the food.
Embodiment 2: preparation qualitative detection coliform and colibacillary substratum.
(1) take by weighing peptone 5.0g, yeast extract 3.0g, lactose 1.2g, 4-methyl umbelliferone-β-D-galactoside 0.25g, sodium-chlor 7.5g, dipotassium hydrogen phosphate 3.4g, potassium primary phosphate 1.2g, Sodium Lauryl Sulphate BP/USP 0.2g, adding distil water is to the 1000mL heating for dissolving;
(2) treat the cooling of above solution after, supply this solution to 1000mL with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add preparation just and the 25mL concentration 32mg/mL indolol-β-D-glucoside solution and the 4.5mL concentration of filtration sterilization are 0.3mg/mL cefsulodin solution respectively, the substratum that is not promptly contained agar, but fast qualitative detects coliform and intestinal bacteria in tap water, ambient water or the food.
Embodiment 3: preparation qualitative detection coliform and colibacillary substratum.
1) take by weighing peptone 4g, yeast extract 2.5g, lactose 0.9g, 4-methyl umbelliferone-β-D-galactoside 0.15g, sodium-chlor 6.5g, dipotassium hydrogen phosphate 3g, potassium primary phosphate 1g, Sodium Lauryl Sulphate BP/USP 0.15g, adding distil water is to the 1000mL heating for dissolving;
(2) treat the cooling of above solution after, supply this solution to 1000mL with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add preparation just and respectively the 20mL concentration of filtration sterilization be that 25mg/mL indolol-β-D-glucoside solution and 5mL concentration are 0.7mg/mL cefsulodin solution, the substratum that is not promptly contained agar, but fast qualitative detects coliform and intestinal bacteria in tap water, ambient water or the food.
Embodiment 4: preparation detects the substratum of coliform and intestinal bacteria maximum possible value (MPN value).
(1) take by weighing peptone 3.5g, yeast extract 2.0g, lactose 0.6g, 4-methyl umbelliferone-β-D-galactoside 0.05g, sodium-chlor 6.0g, dipotassium hydrogen phosphate 2.5g, potassium primary phosphate 0.7g, Sodium Lauryl Sulphate BP/USP 0.1g, adding distil water is to the 1000mL heating for dissolving;
(2) treat the cooling of above solution after, supply this solution to 1000mL with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add preparation just and respectively the 25mL concentration of filtration sterilization be the indolol-β-D-glucoside solution of 15mg/mL and the cefsulodin solution that 5.0mL concentration is 1.1mg/mL, but coliform in the substratum rapid detection tap water, ambient water or the food that obtain and colibacillary maximum possible value.
Embodiment 5: preparation detection by quantitative coliform and colibacillary substratum.
(1) take by weighing peptone 5.0g, yeast extract 3.0g, lactose 1.2g, 4-methyl umbelliferone-β-D-galactoside 0.25g, sodium-chlor 7.5g, dipotassium hydrogen phosphate 3.4g, potassium primary phosphate 1.2g, Sodium Lauryl Sulphate BP/USP 0.2g, agar 12g, adding distil water is to the 1000mL heating for dissolving;
(2) treat the cooling of above solution after, supply this solution to 1000mL with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add preparation just and respectively the 15mL concentration of filtration sterilization be the indolol-β-D-glucoside solution of 32mg/mL and the cefsulodin solution that 4.5mL concentration is 0.3mg/mL, but the substratum fast quantification that obtains detects coliform and intestinal bacteria in tap water, ambient water or the food.
Embodiment 6: preparation detection by quantitative coliform and colibacillary substratum.
1) take by weighing peptone 4g, yeast extract 2.5g, lactose 0.9g, 4-methyl umbelliferone-β-D-galactoside 0.15g, sodium-chlor 6.5g, dipotassium hydrogen phosphate 3g, potassium primary phosphate 1g, Sodium Lauryl Sulphate BP/USP 0.15g, agar 15g, adding distil water is to the 1000mL heating for dissolving;
(2) treat the cooling of above solution after, supply this solution to 1000mL with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add preparation just and respectively the 20mL concentration of filtration sterilization be the indolol-β-D-glucoside solution of 25mg/mL and the cefsulodin solution that 5mL concentration is 0.7mg/mL, the substratum that obtains, but fast quantification detects coliform and intestinal bacteria in tap water, ambient water or the food.
Claims (3)
1, rapid detection coliform and colibacillary substratum, mixed by the 1000mL distilled water solution that contains peptone 3.5-5.0g, yeast extract 2.0-3.0g, lactose 0.6-1.2g, 4-methyl umbelliferone-β-D-galactoside 0.05-0.25g, sodium-chlor 6.0-7.5g, dipotassium hydrogen phosphate 2.5-3.4g, potassium primary phosphate 0.7-1.2g, Sodium Lauryl Sulphate BP/USP 0.1-0.2g, agar 0-15.0g and 15-25mL indolol-β-D-glucoside solution and 4.5-5.0mL cefsulodin solution and to form, the final pH value is 6.9 ± 0.2.
2, rapid detection coliform according to claim 1 and colibacillary substratum is characterized in that, the concentration of indolol-β wherein-D-glucoside solution is 15-32mg/mL, and the cefsulodin strength of solution is 0.3-1.1mg/mL.
3, the preparation method of rapid detection coliform as claimed in claim 1 and colibacillary substratum, its step is as follows:
(1) component content by above-mentioned culture medium solution takes by weighing peptone, yeast extract, lactose, 4-methyl umbelliferone-β-D-galactoside, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, Sodium Lauryl Sulphate BP/USP and agar, adds the distilled water heating for dissolving;
Perhaps the component content by above-mentioned culture medium solution takes by weighing peptone, yeast extract, lactose, 4-methyl umbelliferone-β-D-galactoside, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, Sodium Lauryl Sulphate BP/USP, adds in the distilled water to dissolve;
(2) treat the cooling of above solution after, supply this solution after heat front volume with distilled water, regulate its pH value to 6.9 ± 0.2, put into the autoclaving still, be heated to 121 ℃ ± 1 ℃, keep sterilizing in 15-20 minute, then, naturally cooling;
(3) when above solution is cooled to 50 ℃ ± 5 ℃, add 15-25mL indolol-β-D-glucoside solution and 4.5-5.0mL cefsulodin solution preparation just and filtration sterilization respectively.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914613A (en) * | 2010-08-12 | 2010-12-15 | 上海市疾病预防控制中心 | Kit for screening four enteric pathogenic bacteria by using biochemical and enzyme reaction test sieve and screening method |
| CN102311990A (en) * | 2011-09-16 | 2012-01-11 | 广州绿洲生化科技有限公司 | Chromogenic medium of coliform group and quick detection card thereof |
| CN103184172A (en) * | 2011-12-30 | 2013-07-03 | 广州拜迪生物医药有限公司 | Culture medium used in Escherichia coli high-density culturing |
| CN104293667A (en) * | 2014-09-29 | 2015-01-21 | 青岛康合伟业商贸有限公司 | Lauryl sulfate tryptose MUG medium |
| CN104388524A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Selective chromogenic medium for coliform and test paper with selective chromogenic medium |
| CN111073950A (en) * | 2019-12-31 | 2020-04-28 | 中国科学院长春应用化学研究所 | Fluorescence signal enhancement method for detecting escherichia coli by enzyme substrate method |
| CN113832212A (en) * | 2021-09-10 | 2021-12-24 | 奎泰斯特(上海)科技有限公司 | Aquatic enterococcus enzyme substrate method determination agent and preparation method thereof |
-
2004
- 2004-12-28 CN CN 200410081655 patent/CN1796568A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914613A (en) * | 2010-08-12 | 2010-12-15 | 上海市疾病预防控制中心 | Kit for screening four enteric pathogenic bacteria by using biochemical and enzyme reaction test sieve and screening method |
| CN101914613B (en) * | 2010-08-12 | 2012-08-08 | 上海市疾病预防控制中心 | Kit for screening four enteric pathogenic bacteria by using biochemical and enzyme reaction test sieve and screening method |
| CN102311990A (en) * | 2011-09-16 | 2012-01-11 | 广州绿洲生化科技有限公司 | Chromogenic medium of coliform group and quick detection card thereof |
| CN103184172A (en) * | 2011-12-30 | 2013-07-03 | 广州拜迪生物医药有限公司 | Culture medium used in Escherichia coli high-density culturing |
| CN104293667A (en) * | 2014-09-29 | 2015-01-21 | 青岛康合伟业商贸有限公司 | Lauryl sulfate tryptose MUG medium |
| CN104388524A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Selective chromogenic medium for coliform and test paper with selective chromogenic medium |
| CN111073950A (en) * | 2019-12-31 | 2020-04-28 | 中国科学院长春应用化学研究所 | Fluorescence signal enhancement method for detecting escherichia coli by enzyme substrate method |
| CN113832212A (en) * | 2021-09-10 | 2021-12-24 | 奎泰斯特(上海)科技有限公司 | Aquatic enterococcus enzyme substrate method determination agent and preparation method thereof |
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