KR100374358B1 - A Glucoside and Cellobiose mediums and test methods for replacing Voges-Proskauer and Citrate Test in identification of microorganism - Google Patents

A Glucoside and Cellobiose mediums and test methods for replacing Voges-Proskauer and Citrate Test in identification of microorganism Download PDF

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KR100374358B1
KR100374358B1 KR10-2000-0040204A KR20000040204A KR100374358B1 KR 100374358 B1 KR100374358 B1 KR 100374358B1 KR 20000040204 A KR20000040204 A KR 20000040204A KR 100374358 B1 KR100374358 B1 KR 100374358B1
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glucoside
cellobiose
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kit
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KR20020006785A (en
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양영수
김경동
김의종
최경환
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주식회사 코메드
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/20—Redox indicators

Abstract

본 발명은 일반적으로 장내 세균을 동정하는데 사용하던 수입산 동정용 키트(Kit)의 생화학적 성상반응 검사방법중 번거로움과 재현성이 문제가 되던 VP(Voges-Proskauer)검사법과 구연산염(Citrate)검사법을 글루코시드(Glucoside)와 셀로비오스(Cellobiose)의 분해검사법으로 대체하는 것에 관한 것이다.The present invention is a glucosate test (Voges-Proskauer) test and citrate test that has been troublesome and reproducible problem of the biochemical reaction test of the imported kit (Kit) used to identify intestinal bacteria in general It is related to the replacement by seed digestion (Glucoside) and Cellobiose (Cellobiose).

즉, 증류수 1000㎖에 펩톤(Peptone) 10.0g, 염화나트륨(NaCl) 0.5g, 페놀레드(Phenol Red) 0.108g을 첨가한 뒤 pH를 7.2로 조정하고, 121℃, 15psi 가압조건에서 15분간 멸균하고, 40∼45℃로 냉각한 후 글루코시드 45∼50g을 0.45㎛ 여과멸균을 통해 첨가하여 글루코시드 배지와 상기 글루코시드 대신에 셀로비오스를 45∼50g첨가하여 셀로비오스 배지를 제조한다.That is, 10.0 g of peptone, 0.5 g of sodium chloride, and 0.108 g of phenol red were added to 1000 ml of distilled water, and then the pH was adjusted to 7.2, and sterilized for 15 minutes at 121 ° C. and 15 psi pressure. After cooling to 40-45 ° C., 45-50 g of glucoside was added through 0.45 μm filtration sterilization to prepare cellobiose medium by adding 45-50 g of glucoside medium and cellobiose instead of the glucoside.

또한 상기 과정을 통해 제조된 배지를 동정용 키트에 50㎕씩 분주하여 상온에서 건조한 후 균을 접종하고 37℃에서 18∼24시간동안 배양하고, 사용자는 접종한 균이 글루코시드와 셀로비오스의 분해여부를 색깔 변화만으로 판독할 수 있다.In addition, 50 μl of the medium prepared by the above procedure is dispensed in a kit for identification, dried at room temperature, inoculated with the bacteria, and incubated at 37 ° C. for 18 to 24 hours, and the user inoculates the inoculated bacteria to degrade glucoside and cellobiose. Whether it can be read by color change alone.

따라서 장내세균의 동정에서, 본 발명의 글루코시드와 셀로비오스 분해검사법이 상기 종래의 두 검사법(VP검사법 및 구연산염 검사법)을 대체함으로써 종래와 동일한 반응결과를 보이면서도, 추가시약이 필요하지 않으며, 색깔의 변화가 명확하여 판독이 용이하고, 재현성이 뛰어난 효과를 나타낸다.Therefore, in the identification of intestinal bacteria, the glucoside and cellobiose digestion assays of the present invention replace the two conventional assays (VP assay and citrate assay) with the same reaction results as before, but do not require additional reagents. The change of is clear, and it is easy to read and shows the effect which was excellent in reproducibility.

Description

미생물 동정을 위한 비피검사법과 구연산염 검사법을 대체하는 글루코시드 분해검사배지와 셀로비오스 분해검사배지 및 그 검사법 {A Glucoside and Cellobiose mediums and test methods for replacing Voges-Proskauer and Citrate Test in identification of microorganism}Glucoside and Cellobiose medium and test methods for replacing Voges-Proskauer and Citrate Test in identification of microorganism}

일반적인 장내 세균의 동정에서, 기존의 미생물 동정 키트(Kit)는 VP(Voges-Proskauer)검사법와 구연산염(Citrate)검사법을 포함하는데, VP검사법은 균접종을 하고, 18∼24시간 균배양을 한 후 2개의 추가시약을 차례로 떨어뜨려 10∼15분 이상 경과한 후 색깔변화를 읽어야 한다. 그러나 대량의 검체를 동정해야하는 병원이나 검사기관 등에서는 추가시약 적하후 10여분 이상 기다린 후 또 다시 색깔 변화를 읽어야하므로 번거롭고, 시간적 손실과 추가시약 사용에 의한 경제적 부담을 발생시킨다. 또한 추가 시약의 성분이 대개 몸에 해로운 유기 화합물로 이루어져 있어 각별히 주의를 요하는 문제점이 있다.In the identification of common intestinal bacteria, the existing microbial identification kit (Kit) includes the VP (Progester-Proskauer) test and the citrate test. The VP test is performed after inoculation, 18 to 24 hours of incubation, and 2 After two to ten minutes have passed, the additional reagents must be dropped and the color change read. However, hospitals and inspection institutions that need to identify large samples need to wait for more than 10 minutes after loading additional reagents and read the color change again, which is cumbersome, causing time-consuming and economic burdens from using additional reagents. In addition, the components of the additional reagents are usually composed of organic compounds harmful to the body, there is a problem that requires special attention.

또한 구연산염 검사법은 배양후 색깔 변화가 애매한 결과를 나타내므로 색깔 판독이 명확치 않아 정확성을 감소시키며, 키트 사용자의 주관적 판단이 개입될 수있으며 재현성도 좋지 않은 문제점을 가지고 있다.In addition, the citrate test results in an ambiguous color change after incubation, so the accuracy of the color reading is not clear, thereby reducing the accuracy, the subjective judgment of the kit user may be involved, and the reproducibility is poor.

상기 VP검사법과 구연산염 반응의 검사법은 매우 복잡한 단계에 의해 설명되므로 본 발명의 설명에는 기술하지 않고 진 에프 맥패딘의 의학상 박테리아의 생화학적 동정법을 참조토록 한다. (Jean F. Mac faddin, Biochemical Tests for Identification of Medical Bacteria, 1980, Williams Wilkins, USA, pp59-63, pp308-320)The above VP test and citrate reaction test are described by a very complicated step, and therefore, the description of the present invention is referred to the biochemical identification of gin F. McFadin's medical bacteria. (Jean F. Mac faddin, Biochemical Tests for Identification of Medical Bacteria, 1980, Williams Wilkins, USA, pp59-63, pp308-320)

본 발명의 목적은 상기 문제점을 해결하기 위하여 추가 시약을 사용하지 않으며 확연한 검사결과를 나타내도록 동일 미생물에 대해서 상기 VP검사법 및 구연산염 검사법과 동일한 결과를 제공하는 페놀 레드 브로스 베이스에 글루코시드를 첨가한 글루코시드 분해검사 배지 및 셀로비오스를 첨가한 셀로비오스 분해검사 배지를 제공하는 것이다.An object of the present invention is to add glucoside to a phenol red broth base which provides the same results as the VP test and the citrate test for the same microorganism without using additional reagents to solve the above problems and showing a clear test result. It is to provide a seed digestion medium and cellobiose digestion medium added with cellobiose.

본 발명의 또 다른 목적은 상기 글루코시드 분해검사 배지와 셀로비오스 분해검사 배지를 이용한 장내 세균의 동정법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a method for identifying intestinal bacteria using the glucoside digestion medium and cellobiose digestion medium.

본 발명은 일반적으로 장내 세균을 동정하는데 사용하던 수입산 동정용 키트(Kit)의 생화학적 성상반응 검사방법중 번거로움과 재현성이 문제가 되던 VP(Voges-Proskauer)검사법과 구연산염(Citrate)검사법을 글루코시드(Glucoside)와셀로비오스(Cellobiose)의 분해검사법으로 대체하는 것에 관한 것이다.The present invention is a glucosate test (Voges-Proskauer) test and citrate test that has been troublesome and reproducible problem of the biochemical reaction test of the imported kit (Kit) used to identify intestinal bacteria in general It is related to the replacement by seed digestion (Glucoside) and Cellobiose (Cellobiose).

상기 글루코시드 분해 검사법에서 클루코시드의 정확한 시약명은 메틸 알파-디-클루코피라노시드(Methyl α-D-Glucopyranoside) 혹은 메틸 알파-디-글루코시드 (Methyl α-D-Glucoside) (C7H14O6, FW194.2 SIGMA사)이며 줄여서 글루코시드라고 칭하도록 한다.The glucoside decomposition test inclusive nose seed accurate reagent in said alpha-methyl-D-Inclusion nose pyrano seed (Methyl α- D -Glucopyranoside) or alpha-methyl-D-glucoside (Methyl α- D -Glucoside) (C 7 H 14 O 6 , FW194.2 SIGMA), abbreviated as glucoside.

먼저, 검사 원리는 장내 세균은 당을 이용해 산화 및 발효를 활발하게 하는 세균들이므로 pH수치에 따른 지시제인 페놀 레드(Phenol Red)를 첨가한 페놀 레드 브로스 베이스(Phenol Red Broth Base)에 당의 일종인 글루코시드를 첨가하면, 상기 페놀 레드 브로스 베이스내의 글루코시드를 균이 분해하는 경우는 이산화탄소를 생성함으로써 배지의 pH수치를 감소시켜 지시제인 페놀 레드의 색깔을 노란색으로 변화시키고 글루코시드를 분해하지 않으면 페놀 레드 브로스 베이스의 색깔은 변화하지 않는 것이다.First of all, the test principle is that enterobacteria are bacteria that actively oxidize and ferment using sugars. Therefore, the phenol red broth base added with phenol red, an indicator according to pH value, is a type of sugar. When glucoside is added, when bacteria decompose the glucoside in the phenol red broth base, carbon dioxide is generated to reduce the pH value of the medium, thereby changing the color of phenol red, which is an indicator, to yellow, and not decomposing glucoside. The color of the red broth base does not change.

당의 산화, 발효 검사에 이용되는 일반적인 페놀 레드 브로스 베이스(Phenol Red Broth Base)는 증류수 1000㎖에 펩톤(Peptone) 10.0g, 염화나트륨(NaCl) 0.5g, 페놀레드(Phenol Red) 0.018g을 첨가한 뒤 pH를 7.4로 조정해서 제조할 수 있고, 이미 시약들이 제조된 상태로 시중에 판매되고 있는 것을 이용할 수도 있다. 그리고 그 용액에 원하는 당 10g(1%)를 첨가하면 당배지가 완성된다.Phenol Red Broth Base, which is used for sugar oxidation and fermentation, is added to 1000 ml of distilled water after adding 10.0 g of peptone, 0.5 g of sodium chloride (NaCl), and 0.018 g of phenol red. It can be prepared by adjusting the pH to 7.4, and it is also possible to use a commercially available with reagents already prepared. The sugar medium is completed by adding 10 g (1%) of the desired sugar to the solution.

본 발명에서는 페놀레드 브로스 베이스를 제조할 때 펩톤과 염화 나트륨의 첨가량은 원래의 제법과 동일하게 하고 페놀 레드를 기존의 0.018g보다 6배인0.108g을 첨가하고 pH는 7.2로 조정했으며 멸균조건은 원법과 동일한 조건인 121℃, 15psi의 가압조건에서 15분간 멸균하여 제조하였다. 상기 멸균한 페놀 레드 브로스 베이스를 40∼45℃로 냉각한 후 글루코시드 45∼50g(4.5∼5%)을 0.45㎛ 여과멸균을 통해 첨가하여 글루코시드 분해검사법에 사용하는 검사용 배지를 제조한다.In the present invention, when the phenol red broth base is prepared, the addition amount of peptone and sodium chloride is the same as the original method, and phenol red is added to 0.108 g, which is 6 times higher than the existing 0.018 g, and the pH is adjusted to 7.2. It was prepared by sterilizing for 15 minutes under the same conditions as 121 ℃, 15psi pressure conditions. After cooling the sterilized phenol red broth base to 40 ~ 45 ℃ 45 to 50g (4.5 to 5%) of glucoside is added through 0.45㎛ filtration sterilization to prepare a test medium for the glucoside decomposition test.

셀로비오스 분해 검사법을 위한 검사용 배지의 경우도 상기 제조방법과 동일한 방법으로 제조된 페놀레드 브로스 베이스에 글루코시드와 마찬가지로 당의 일종인 셀로비오스(Cellobiose 또는D-(+)-Cellobiose : C12H22O11, FW 342.3 SIGMA사)를 상기 서술한 과정과 동일하게 여과 멸균을 통해 45∼50g(4.5∼5%)을 첨가하여 제조한다.In the case of the test medium for the cellobiose digestion assay, the phenol red broth base prepared in the same manner as in the above-described manufacturing method, like glucoside, is a celloose (Cellobiose or D -(+)-Cellobiose: C 12 H 22). O 11 , FW 342.3 SIGMA) was prepared by adding 45-50 g (4.5-5%) through filtration sterilization in the same manner as described above.

상기 배지들을 이용한 동정 방법으로, 상기 과정을 통해 제조된 배지를 동정용 키트에 50㎕씩 분주하여 상온에서 건조한 후 균을 접종하고, 균이 접종된 키트를 37℃에서 18∼24시간동안 배양한다. 사용자는 접종한 균이 글루코시드와 셀로비오스를 분해했는지 안했는지를 확인해야 하는데 이는 배양후 색깔 변화만으로 판독할 수 있다.In the identification method using the medium, 50μl of the medium prepared by the above procedure was dispensed in the kit for identification, dried at room temperature, inoculated with the bacteria, and the inoculated kit was incubated at 37 ° C for 18-24 hours. . The user should check whether the inoculated bacteria have degraded glucosides and cellobiose, which can be read only by color change after incubation.

VP검사와 구연산염검사의 결과가 당의 일종인 글루코시드와 셀로비오스 분해 실험의 결과와 대부분 일치하며 재현성을 확인한 결과를 아래의 표에 나타내었다. 즉, 대장균(Escherichia coli)과 크렙시엘라(Klebsiella), 엔테로박터 (Enterobacter)를 감별하는데 전통적으로 사용되어져 왔던 VP검사 및 구연산염 검사와 본 발명의 글루코시드 분해검사와 셀로비오스 분해검사의 상기대장균(Escherichia coli)과 크렙시엘라(Klebsiella), 엔테로박터(Enterobacter)에 대한 검사결과가 동일한 양상을 나타내었다.The results of VP test and citrate test were mostly consistent with those of glucoside and cellobiose decomposition experiments, which are sugars. That is, E. coli (Escherichia coli) and keurep when Ella (Klebsiella), Enterobacter the E. coli glucoside overhaul and cellobiose overhaul of the VP test and the citrate test with the invention to discriminate (Enterobacter) came been traditionally used ( the Escherichia coli) and test results for keurep when Ella (Klebsiella), Enterobacter (Enterobacter) showed the same pattern.

VPVP 글루코시드Glucoside 구연산염citrate 셀로비오스Cellobiose 에쉐리키아 코라이(Escherichia coli)살모넬라 티파이(Salmonella typhi)쉬겔라(Shigella) Escherichia coli Salmonella typhi Shigella 음성voice 음성voice 음성voice 음성voice 크렙시엘라 코라이(Klebsiella coli)엔테로박터 크로케(Enterobacter cloacae)엔테로박터 에로게네스(Enterobacter aerogenes) Klebsiella coli Enterobacter cloacae Enterobacter aerogenes 양성positivity 양성positivity 양성positivity 양성positivity

또한 각 균들의 글루코시드와 셀로비오스에 대한 예상반응치와 실제 본 발명의 글루코시드 분해검사와 셀로비오스 분해검사의 결과가 아래 표에 나타낸 바와 같이 서로 일치하였다.In addition, the expected response of each bacterium to glucoside and cellobiose and the results of the glucoside and cellobiose digestion tests of the present invention were in agreement with each other as shown in the following table.

접종균명Inoculation 글루코시드Glucoside 셀로비오스Cellobiose 예상반응Expected Response 결과result 예상반응Expected Response 결과result 에쉐리키아 코라이(Escherichia coli) Escherichia coli 0%0% 음성voice 2%2% 음성voice 살모넬라 티파이(Salmonella typhi) Salmonella typhi 0%0% 음성voice 0%0% 음성voice 쉬겔라 프렉스네리(Shigella flexneri) Shigella flexneri 0%0% 음성voice 0%0% 음성voice 쉬겔라 보이디이(Shigella boydii) Shigella boydii 음성voice 0%0% 음성voice 크렙시엘라 코라이(Klebsiella coli) Klebsiella coli 90%90% 양성positivity 98%98% 양성positivity 엔테로박터 크로케(Enterobacter cloacae) Enterobacter cloacae 85%85% 양성positivity 99%99% 양성positivity 엔테로박터 에로게네스(Enterobacter aerogenes) Enterobacter aerogenes 100%100% 양성positivity 100%100% 양성positivity

미생물 동정시, VP검사법은 균의 배양후에 추가로 검사시약을 적하해야 하고, 양성 반응은 10∼15분정도 뒤에 판독하고 음성반응은 30분이상 관찰해야 하는 번거로움이 있으며, 구연산염 검사법은 균 배양후 색깔 판독의 애매함과 재현성 문제를 나타내므로 이를 글루코시드 분해검사와 셀로비오스 분해검사로 대체하면 배양후 즉시 색깔 변화만 읽어주면 되는 간편함과 추가시약을 사용하지 않음으로써 원가절감효과 및 가격경쟁력도 갖출 수 있을 뿐만 아니라 색깔 판독의 애매함에 의한 주관적 판단을 배제할 수 있음에 따라 균동정의 신뢰성을 높일 수 있으며 재현성도 향상되는 효과가 있다.When identifying microorganisms, the VP test should be added dropwise after the incubation of the bacteria, the positive reaction should be read after 10 to 15 minutes, and the negative reaction should be observed for 30 minutes or more. Since it shows ambiguity and reproducibility problems of color reading after substitution, it is easy to read color change immediately after incubation and glucoside digestion test, and cost saving effect and price competitiveness can be achieved by not using additional reagents. In addition, the subjective judgment due to the ambiguity of the color reading can be excluded, thereby increasing the reliability of the identification and improving the reproducibility.

Claims (4)

증류수 1000㎖에 펩톤(Peptone) 10.0g, 염화나트륨(NaCl) 0.5g, 페놀레드(Phenol Red) 0.108g을 첨가한 뒤 pH를 7.2로 조정하고;10.0 g of peptone, 0.5 g of sodium chloride (NaCl), and 0.108 g of phenol red were added to 1000 ml of distilled water, and then the pH was adjusted to 7.2; 121℃, 15psi의 가압조건에서 15분간 멸균한 후에, 상기 멸균한 페놀 레드 브로스 베이스(Phenol Red Broth Base)를 40∼45℃로 냉각하고; 그리고,After sterilization for 15 minutes at a pressure of 121 ° C. and 15 psi, the sterilized Phenol Red Broth Base was cooled to 40 to 45 ° C .; And, 상기 냉각된 페놀 레드 브로스 베이스에 글루코시드(Glucoside)를 0.45㎛ 여과멸균을 통해 45∼50g을 첨가하여;Adding 45-50 g of glucoside to the cooled phenol red broth base through 0.45 μm filtration sterilization; 제조되는 글루코시드 분해검사용 배지Glucoside digestion medium prepared 증류수 1000㎖에 펩톤(Peptone) 10.0g, 염화나트륨(NaCl) 0.5g, 페놀레드(Phenol Red) 0.108g을 첨가한 뒤 pH를 7.2로 조정하고;10.0 g of peptone, 0.5 g of sodium chloride (NaCl), and 0.108 g of phenol red were added to 1000 ml of distilled water, and then the pH was adjusted to 7.2; 121℃, 15psi의 가압조건에서 15분간 멸균한 후에, 상기 멸균한 페놀 레드 브로스 베이스를 40∼45℃로 냉각하고; 그리고,After sterilization for 15 minutes at a pressure of 121 ° C. and 15 psi, the sterilized phenol red broth base was cooled to 40 to 45 ° C .; And, 상기 냉각된 페놀 레드 브로스 베이스에 셀로비오스(Cellobiose)를 0.45㎛ 여과멸균을 통해 45∼50g을 첨가하여;Adding 45 to 50 g of Cellobiose to the cooled phenol red broth base through 0.45 μm filtration sterilization; 제조되는 셀로비오스 분해검사용 배지Cellobiose digestion medium prepared 장내 세균의 동정에서,In the identification of intestinal bacteria, VP(Voges-Proskauer)검사배지와 구연산염(Citrate)검사배지를 대체하여, 제1항의 글루코시드 분해검사용 배지와 제2항의 셀로비오스 분해검사용 배지를 포함하는 것을 특징으로 하는 미생물 동정키트Microorganism identification kit comprising a medium for glucoside digestion test of claim 1 and cellobiose digestion test medium of claim 2 in place of VP (Voges-Proskauer) test medium and citrate test medium. 장내 세균의 동정에서,In the identification of intestinal bacteria, 제1항의 글루코시드 분해검사용 배지와 제2항의 셀로비오스 분해검사용 배지를 VP(Voges-Proskauer)검사배지와 구연산염(Citrate)검사배지 대신에 미생물 동정용 키드에 분주하고;Dispensing the glucoside digestion medium of claim 1 and the cellobiose digestion medium of claim 2 into a microorganism identification kit instead of VP (Progester-Proskauer) and Citrate assay media; 상기 키트를 상온에서 건조하여 미생물 동정용 키트를 준비하고;Drying the kit at room temperature to prepare a kit for identifying microorganisms; 상기 미생물 동정용 키트에 균을 접종하고; 그리고,Inoculating bacteria to the microorganism identification kit; And, 37℃에서 18∼24시간동안 배양한 후 색깔 변화를 통하여 글루코시드와 셀로비오스의 당분해 여부를 판단하는 단계를 포함하는 것을 특징으로 하는 미생물 동정법After culturing for 18 to 24 hours at 37 ℃ microorganism identification method comprising the step of determining the glycosylation of glucoside and cellobiose through color change
KR10-2000-0040204A 2000-07-13 2000-07-13 A Glucoside and Cellobiose mediums and test methods for replacing Voges-Proskauer and Citrate Test in identification of microorganism KR100374358B1 (en)

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