KR102099500B1 - Composition of media for culturing and detecting E. coli and method for producing the same - Google Patents

Abstract

The present invention relates to a culture medium for E. coli culture and detection and a method for manufacturing the same. When the medium composition for culture and detection of E. coli of the present invention is used, E. coli and C. braakii strains that cannot be detected in a conventional medium can be effectively distinguished with the naked eye, and C. braakii strains are effectively shown by showing a different colony form from E. coli. Can be excluded. In addition, the medium of the present invention can selectively grow only E. coli and inhibit the growth of C. braakii , thereby effectively culturing E. coli, which can be usefully used in related industries.

Description

Composition and media for culturing and detecting E. coli and method for producing the same}

The present invention relates to a culture medium composition for E. coli culture and detection and a method for manufacturing the same.

E. coli ( Escherichia coli ) is classified by 1 to 136 in the O antigen, 1 to 78 in the K antigen, and 1 to 40 in the H antigen, when distinguished by antigen structure. Escherichia coli usually does not show pathogenicity in the intestine, but when it enters a part other than the intestine, it causes cystitis, pyelonephritis, peritonitis, and sepsis, and in the intestine, antigen-type E. coli, such as O 26, O 55, and O 111, sucks It is especially called pathogenic E. coli because it can cause infectious diarrhea from people to adults. E. coli is weakly resistant to heat and is sterilized when heated for 60 to about 20 minutes. Therefore, boiling and drinking water in summer can prevent diarrhea. Escherichia coli has rounded ends, 24μm in length, and 0.40.7μm in length. It does not make spores, it is a Gram-negative bacterium. Morphologically, it is difficult to distinguish it from erythrocytes, but biologically, Escherichia coli breaks down lactose and glucose to produce acids and gases, and coagulates milk to make indole. In addition, the presence or absence of E. coli is an indicator of the presence or absence of contamination by feces, and is important in hygiene as a means often applied to water quality tests.

Several mediums are used to cultivate the E. coli, and MacConkey agar, along with Eosin Methylene Blue agar, is a pathogenic E. coli O157: H7 based on the Food and Drug Administration Bacteriological Analytical Manual (FDA BAM). This medium is used for qualitative detection of the remaining pathogenic E. coli. The selectivity of these two media is due to the color change according to the pH change by the resolution of lactose in the medium of E. coli.

However, it has been reported that some bacteria in the genus Citrobacter and Enterobacter may show a typical colony of E. coli, and the bacteria have a significantly higher degree of contamination than E. coli, which is high when not selected on a selective medium. Improvement is necessary because there is room for much time and money to be confirmed and identified due to false positives.

The bacterium of the genus Citroacter belongs to the intestinal microflora, and is a Gram-negative small bacillus, usually with a main mother flagella, having mobility and no stenosis. It is a facultative anaerobic bacillus and proliferates well in agar medium. The Vogues-Proskauer (VP) reaction is characterized by producing gas by fermenting glucose using citric acid salt of Simmons.

Therefore, when using a medium for culture and detection of E. coli, it is possible to effectively distinguish E. coli from bacteria of the genus Citrobacter, and there is a need to develop a medium in which bacteria in the Citrobacter do not grow.

Korean Registered Patent 10-1109565

An object of the present invention can provide a culture medium composition for E. coli culture and detection.

To achieve the above object, the present invention provides a culture medium for E. coli culture and detection.

When the medium composition for culture and detection of E. coli of the present invention is used, E. coli and C. braakii strains that cannot be detected in a conventional medium can be effectively distinguished with the naked eye, and C. braakii strains are effectively shown by showing a different colony form from E. coli. Can be excluded. In addition, the medium of the present invention can selectively grow only E. coli and inhibit the growth of C. braakii , thereby effectively culturing E. coli, which can be usefully used in related industries.

1 is a view confirming the colony type change of Citrobacter braakii according to the culture time in this medium.

The present invention provides a medium composition for E. coli culture and detection.

In the present invention, the term "cultivation" means to grow microorganisms under environmental conditions that are artificially controlled.

The medium contains nutrients required by a microorganism to be a culture target, that is, a culture medium in order to cultivate a specific microorganism, and a material for a specific purpose may be additionally added and mixed. The medium is also referred to as a culture medium or a culture medium, and is a concept including both natural medium, synthetic medium, or selective medium.

The medium used for culture should satisfy the requirements of the target strain in an appropriate manner while controlling temperature, pH, etc. in a normal medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. As a carbon source that can be used, mixed sugars of glucose and xylose are used as the main carbon source. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soy cake or its degradation products Can be. These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate, and the corresponding sodium-containing salt as personnel. Personnel that can be used include potassium dihydrogen phosphate or dipotassium phosphate or the corresponding sodium-containing salt. Further, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above materials, essential growth materials such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium may be used. The above raw materials may be added in a batchwise, fed-batch, or continuous manner in an appropriate manner to the culture during the culture process, but are not particularly limited thereto. The pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide or ammonia or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to materialize the contents of the present invention, and the present invention is not limited thereto.

<Example 1> Preparation of medium

E. coli or Citrobacter braakii is cultured in the medium of the present invention to show colonies of the same type as E. coli in the medium used in the past, and C. braakii showing false positive as E. coli is excluded and only E. coli To correctly culture and detect, the medium of the present invention was prepared.

Specifically, MacConkey agar (Mac) used for culture and detection of E. coli was prepared according to the manufacturer's manual. In addition, the culture medium for E. coli culture and detection of the present invention is a C. braakii strain By confirming that hydrogen sulfide (H ₂ S) gas was generated, lactose concentration, which makes it difficult to confirm gas production, was minimized. In addition, ferric ammonium citrate and sodium thiosulphate, which are indicators for the generation of hydrogen sulfide gas, were added. The final composition of the medium of the present invention is shown in Table 1 below.

Configuration Mac Badge of the invention Peptone 17.0 g 10.0 g Proteose Peptone 3.0 g 5.0 g Bile Salts No.3 1.5 g 3.6 g Sodium chloride (NaCl) 5.0 g 1.5 g Agar 13.5 g 5.0 g Neutral Red 0.03 g 10 g Crystal Violet 0.001 g 6.0 g Lactose 10.0 g 2.5 g Sodium thiosulphate - 6.8 g Ferric ammonium citrate - 0.8 g Distilled water 1000 ml pH to 7.1 (+ -0.2)

<Example 2> Observation of morphological changes of colonies according to E. coli inoculation into the medium of the present invention

After inoculating E. coli with the medium of the present invention prepared in Example 1 and the conventional Mac medium, the change in colony shape according to the culture time was observed.

Specifically, the present invention is a medium and conventional Mac medium 2 types of general E. coli and 18 types of pathogenic E. coli (5 types of enterohemorrhagic Escherichia coli, 5 types of enterotoxin type E. coli, 5 types of enteropathogenic E. coli, 3 types of enteroscopic E. coli) To inoculate, each strain stored at -70 was plated on EMB (Eosin Methylene Blue agar) and cultured at 37 for 24 hours. The colonies showing green metallic luster were picked and incubated in BPW (Buffered peptone water) for 42 hours for 24 hours, followed by 10-fold dilution using PBS (Phosphate buffered saline). Plates are plated on each selected medium using dilutions of dilution multiples that can form approximately 30-300 colonies per 100 ul, and typical colonies (brick red colonies) according to the incubation time (12 hours, 24 hours, 36 hours, 48 hours). with hazy surrounding zones). The results are shown in Table 2 below.

As shown in Table 2 below, as a result of comparing the number of plates showing the colony typical colonies according to the culture time of the conventional Mac medium and the medium of the present invention for 20 species of E. coli, at 12 hours, the colony size was too small to determine whether the colonies were typical. Although it could not be determined, from 24 hours, typical E. coli colonies were found in both media. Therefore, it was confirmed that the medium of the present invention showed sufficient sensitivity to E. coli despite the composition different from that of the conventional medium.

Presumptive positive plates / tested plates (%) Incubation time (h) MacConkey agar (Mac) Badge of the invention 12 0/20 0/20 24 20/20 20/20 36 20/20 20/20 48 20/20 20/20

<Example 3> To the medium of the present invention C. braakii  Observation of colony morphology change according to inoculation

After inoculating 15 types of C. braakii into the medium of the present invention and the conventional Mac medium prepared in Example 1, the change in colony shape according to the culture time was observed using the same method as in Example 2. The results are shown in Table 3 below and FIG. 1.

As shown in Table 3 and Fig. 1, as a result of comparing the number of plates showing C. braakii typical colonies according to the culture time of the conventional Mac medium and the culture medium of the present invention for 15 types of C. braakii , colonies at 12 hours The size of was too small to determine whether a typical colony was present. However, from 24 hours, it was observed that colonies of the same type as E. coli appeared in all samples of the conventional Mac medium, but in the medium of the present invention, the center of the colonies was gray or black due to H ₂ S gas formation in most samples. It was confirmed that a color dot was formed. From 36 hours, this trend was confirmed in all samples inoculated with C. braakii , and it was confirmed that colonies turned black in some plates.

Presumptive positive plates / tested plates (%) Incubation time (h) MacConkey agar (Mac) Badge of the invention 12 0/20 0/20 24 20/20 3/20 36 20/20 0/20 48 20/20 0/20

Therefore, the medium of the present invention was able to effectively distinguish the C. braakii strain, which was impossible to distinguish in the conventional Mac medium, and also showed a different colony form from E. coli to effectively exclude it, thereby selectively selecting E. coli. It was confirmed that a discernible effect exists.

Claims (12)

Peptone 8.0-12.0 parts by weight, Proteose Peptone 3.0-7.0 parts by weight, Bile Salts 1.6-5.6 parts by weight, sodium chloride (NaCl) 0.5-3.5 parts by weight, agar 3.0 to 7.0 parts by weight, Neutral Red 8.0 to 12.0 parts by weight, Crystal Violet 4.0 to 8.0 parts by weight, Lactose 0.5 to 4.5 parts by weight, sodium thiosulphate 4.8 to 8.8 parts by weight Part and a composition for the identification of bacteria in Escherichia coli and Citroacter, containing 0.1 to 2.8 parts by weight of ferric ammonium citrate in the medium. The method of claim 1, wherein the E. coli is one type selected from the group consisting of enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxin coliform (ETEC), enteroadsorbing E. coli (EAEC) and enteroinvasive E. coli (EIEC) The above is the composition. The method of claim 1, wherein the Escherichia coli O26: H11, Escherichia coli O91, Escherichia coli O103: H2, Escherichia coli O103: H6, Escherichia coli O103: H11, Escherichia coli O111: H8, Escherichia coli O157: H7, Escherichia The composition is one or more selected from the group consisting of coli O45: H2, Escherichia coli O88, Escherichia coli O177, Escherichia coli O25, Escherichia coli O121: H9, Escherichia coli O130, Escherichia coli O145: NM and Escherichia coli O96. The composition of claim 1, wherein the E. coli is cultured under 12-48 hour conditions. delete The method of claim 1, wherein the genus Citrobacter is Citrobacter rodentium, Citrobacter braakii, Citrobacter freundii, Citrobacter youngae, Citrobacter koseri, Citrobacter sedlakii, Citrobacter murliniae, Citrobacter werkmanii, Citrobacter amaronaticus amalonaticus), is a composition of at least one strain selected from the group consisting of Citrobacter gillenii and Citrobacter farmeri. According to claim 1, The discrimination is that the colonies having a gray or black color are determined to be contaminated with bacteria in the citrobacter. delete delete delete Peptone 8.0 to 12.0 parts by weight, Proteose Peptone 3.0 to 7.0 parts by weight, Bile Salts 1.6 to 5.6 parts by weight, sodium chloride (NaCl) 0.5 to 3.5 parts by weight, agar 3.0 to 7.0 parts by weight, Neutral Red 8.0 to 12.0 parts by weight, Crystal Violet 4.0 to 8.0 parts by weight, Lactose 0.5 to 4.5 parts by weight, sodium thiosulphate 4.8 to 8.8 parts by weight Part and ferric ammonium citrate (Ferric ammonium citrate) 0.1 to 2.8 parts by weight of the medium, E. coli culture composition. Culturing E. coli in the composition of claim 1; And
Checking the color of the colonies of the E. coli, gray or black colonies comprising the step of determining as a bacterium in the genus Citrobacter (Citrobacter), E. coli and the method of determining the bacterium in the genus Citrobacter (Citrobacter).

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