KR20020006786A - A method and medium for rapidly determining fermentation of micro-organism - Google Patents

A method and medium for rapidly determining fermentation of micro-organism Download PDF

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KR20020006786A
KR20020006786A KR1020000040205A KR20000040205A KR20020006786A KR 20020006786 A KR20020006786 A KR 20020006786A KR 1020000040205 A KR1020000040205 A KR 1020000040205A KR 20000040205 A KR20000040205 A KR 20000040205A KR 20020006786 A KR20020006786 A KR 20020006786A
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medium
fermentation
sugar
microorganisms
glucose
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양영수
김경동
김의종
최경환
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양영수
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
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    • 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
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • 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

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Abstract

PURPOSE: A medium and method for inspecting the sugar fermentation of microorganisms in a short time are provided, thereby rapidly detecting the sugar fermentatability of microorganism. CONSTITUTION: The medium for detecting the sugar fermentation of a microorganism in a short time is made by adding 10g of glucose, 2g of peptone, 0.3g of dipotassium phosphate, 0.03 to 0.05g of bromthymol blue(BTB) into 1 liter of distilled water; adjusting the hydrogen concentration to pH 7.1; and further adding 1 to 2g of yeast extract and 3 to 4g of beef extract into the medium. The method comprises the steps of: inserting the medium into each tube; inoculating a microorganism into the medium; coating mineral oil on the medium and cultivating it at 37 deg.C for 4 hours; and analyzing the color of the medium to determine the microorganism's sugar fermentatability.

Description

미생물의 당발효여부의 단시간 검사를 위한 배지와 그 검사법 {A method and medium for rapidly determining fermentation of micro-organism}Medium and method for rapidly determining fermentation of micro-organisms for microbial sugar fermentation {A method and medium for rapidly determining fermentation of micro-organism}

장내세균과 비장내그람음성균 등의 미생물의 생화학적 동정에서, 첫단계는 포도당을 발효하는지의 여부를 결정하는 것이다. 즉, 당 발효 대사 기능이 있는 미생물과 상기 기능이 없는 미생물간에 추가 검사의 방법을 달리함에 따라, 상기 당 발효 여부를 확인하는 단계를 최우선으로 실시한다. 그러나 상기 과정은 보통 포도당이 포함되어 있는 당발효 배지에 감별하고자 하는 미생물을 접종하여 18∼24시간 배양한 뒤에 미생물의 당 발효여부 결과를 알 수 있고 그 결과에 따라 추가적인 검사를 진행하여 검사미생물의 동정을 실시하므로 상기 기존의 당발효검사를 통할 경우 최종 미생물 동정의 완료는 최소 48시간후에 결과를 알 수 있어서 미생물의 신속한 동정을 진행할 수 없다.In the biochemical identification of microorganisms such as enterobacteria and spleen gram-negative bacteria, the first step is to determine whether or not to ferment glucose. That is, according to the method of additional testing between the microorganisms having a sugar fermentation metabolism function and the microorganisms without the function, the step of confirming whether the sugar fermentation is carried out as a top priority. However, the above process is usually inoculated with the microorganisms to be discriminated in the glucose fermentation medium containing glucose and cultured for 18 to 24 hours, and the result of microbial sugar fermentation can be determined. Since the identification is carried out through the existing sugar fermentation test, the completion of the final microorganism identification can be found at least 48 hours later, and therefore, the rapid identification of the microorganisms cannot proceed.

이에 대한 예로서, 현재 가장 널리 쓰이고 있는 프랑스 바이오메리으 (BioMeriuex)의 오에프 배지(OF medium)는 균 접종을 한 뒤 24시간 배양을 한 후 색깔 변화를 판독하여 당발효여부를 결정하고, 상기 결과에 따라 미생물 동정 키트를 선택하여 균을 접종한 뒤에 미생물의 동정을 실시한다. 따라서 상기 당발효여부 확인부터 최종 미생물동정 확인까지의 과정은 최소 48시간이 소요되므로 신속히 치료해야 될 환자이거나 식품유통업체 등에서는 균동정 업무의 소요시간에 따라 치료 시간 또는 처리시간의 지연으로 많은 피해가 발생할 수 있다.As an example of this, the most widely used bio medium of BioMeriuex of France (OF medium) is inoculated with the bacteria after incubation for 24 hours and then read the color change to determine whether the sugar fermentation, Based on the results, microbial identification kits are selected and the microorganisms are identified after inoculation. Therefore, the process from checking whether the sugar fermentation is confirmed to the final microorganism identification takes at least 48 hours, so that patients who need to be treated quickly or food distribution companies, etc., suffer a lot of damage due to the delay of treatment time or processing time depending on the time required for the bacteriological work. May occur.

본 발명의 목적은 상기 문제점을 해결하기 위하여 단시간내에 미생물의 당발효여부를 확인할 수 있는 검사시약을 제공하는 것이다.It is an object of the present invention to provide a test reagent which can confirm the fermentation of microorganisms within a short time to solve the above problems.

본 발명의 다른 목적은 상기 검사시약을 이용한 단시간내에 미생물의 당발효여부를 확인하는 검사방법을 제공하는 것이다.Another object of the present invention is to provide a test method for checking whether the microorganisms are fermented within a short time using the test reagent.

도1은 본 발명의 검사법에 대한 전체 순서를 나타낸 개략도이다.1 is a schematic diagram showing the overall procedure for the inspection method of the present invention.

본 발명은 미생물 동정을 위한 검사 방법의 선택에서, 가장 큰 기준이 되는 검사대상인 미생물(검체균)의 당 발효대사과정의 존재여부를 단시간에 확인하는 지시약을 포함한 배지 및 상기 배지를 이용한 검사법에 관한 것이다. 즉, 검체균에 대한 상기 당 발효대사과정의 존재여부 확인결과를 4시간만에 알아, 상기 당발효여부의 확인결과에 따라 선택된 동정 키트를 이용한 검체균 동정이 24시간내에 이루어질 수 있도록 한 것이다.The present invention relates to a medium containing an indicator for confirming the presence of a sugar fermentation metabolism process of a microorganism (sample bacterium), which is the largest standard, in the selection of a test method for microorganism identification, and a test method using the medium. will be. In other words, the result of confirming the presence of the sugar fermentation metabolism process for the sample bacteria in 4 hours, so that the identification of the specimen bacteria using the identification kit selected according to the result of the sugar fermentation can be made within 24 hours.

본 발명은 기존의 당발효 배지에 미생물이 필요로 하는 영양물질(이스트 추출물 : Yeast Extract, 소고기 추출물 : Beef Extract)을 첨가하여 당분해속도를 증가시키는 것으로서 일반적인 당발효배지는 증류수 1000㎖에 포도당(Glucose) 10g, 펩톤(Peptone) 2g, 제2인산칼륨(Dipotassium Phosphate) 0.3g, BTB(Bromthymol Blue) 0.08g을 첨가한 후에 pH를 6.6∼7.0으로 조정하여 제조되는 것에 비하여, 본 발명은 상기 일반적인 당 발효배지중에 BTB첨가량을 감소하여 0.03∼0.05g을 첨가하고 pH=7.1 또는 중성으로 하였으며 미생물 생장에 유용한 영양물질인 이스트 추출물(Yeast Extract) 1∼2g, 소고기 추출물(Beef Extract) 3∼4g을 상기 용액에 첨가하여 당발효대사가 최대 4시간내에 신속히 일어나도록 하였다.The present invention is to increase the rate of glycolysis by the addition of nutrients (yeast extract: Yeast Extract, Beef Extract: Beef Extract) that microorganisms need to the existing sugar fermentation medium. Compared to 10 g of Glucose, 2 g of Peptone, 0.3 g of Dipotassium Phosphate, and 0.08 g of BTB (Bromthymol Blue), the pH is adjusted to 6.6 to 7.0. The amount of BTB added in the fermentation broth was reduced to 0.03 to 0.05 g, pH = 7.1 or neutral, and 1 to 2 g of yeast extract and 3 to 4 g of beef extract were used for microbial growth. The solution was added to allow the fermentation metabolism to occur rapidly within a maximum of 4 hours.

상기 배지를 이용한 검사법은 도1에 나타내었다. 즉, 상기 방법으로 제조한 배지를 뚜껑이 있는 튜브에 약 4∼5㎖정도로 분주한 후 맥파랜드 네페로미터 기준(McFarland Nephelometer Standard) 탁도 1 (Mcf.=1)에 맞추어 균을 접종하고 발효대사는 공기를 이용하지 않는 과정이므로 균접종한 배지위에 미네랄 오일을 1cm두께로 덮은 다음 37℃에서 4시간동안 배양한다. BTB는 산성에서는 노란색, 염기성에서는 푸른색을 띄는 지시제이므로 결과 판독시 접종한 균이 포도당(Glucose)을 발효하면 강산을 생성해 pH를 낮추므로 배지내 지시약인 BTB의 색깔이 노란색으로 변해 양성으로 판단하고 음성은 이와 반대로 포도당(Glucose)을 분해하지 못하므로 푸른색으로 배지의 색깔 변화가 없다.The test method using the medium is shown in FIG. In other words, the medium prepared by the above method was dispensed in a tube with a lid of about 4 to 5 ml, and then inoculated with bacteria in accordance with the McFarland Nephelometer Standard turbidity 1 (Mcf. = 1) and fermented metabolism. Since the process does not use air, cover the mineral oil with a 1cm thickness on the inoculated medium and incubate for 4 hours at 37 ℃. BTB is yellow in acid and blue in basic. Therefore, when inoculating bacteria in fermented glucose, GTB produces strong acid and lowers pH. Judgment and negative, on the contrary, do not break down glucose, so there is no change in the color of the medium.

상기 검사법을 통하여 검체균의 당발효대사 가능 여부를 판단하고 상기 결과에 따라 양성의 결과를 나타낸 경우는 포도당 발효 미생물 동정 키트를 이용하여 검체균의 동정을 실시하고, 상기 결과가 음성으로 나타난 경우에는 포도당 비발효 미생물 동정 키트를 이용하여 검체균의 동정을 실시한다.In the case of judging whether the fermentation metabolism of the specimen bacteria is possible through the above test method, and if the result was positive according to the above results, the identification of the specimen bacteria was performed using a glucose fermentation microorganism identification kit. Specimen bacteria are identified using a glucose non-fermentation microorganism identification kit.

상기 신속한 당발효를 위한 배지를 활용하여 비발효균주 3종 및 발효균주 3종에 대한 4시간 배양후의 결과를 아래 표에 나타내었다.The results after 4 hours of incubation for three non-fermented strains and three fermented strains using the medium for rapid fermentation are shown in the table below.

신속 당발효 배지(Rapid Fermentation Medium)Rapid Fermentation Medium 대상균주Target strain 4시간 배양후의 판독결과Reading result after 4 hours incubation 비발효균주Unfermented strain 슈도모나스 에루지노사(Pseudomonas aeruginosa)Pseudomonas aeruginosa 음성(초록)Voice (green) 프라보박테리움 메니고셉티컴(Flavobacterium meningosepticum) Flavobacterium meningosepticum 음성(초록)Voice (green) 아시네토박터 바우마니(Acinetobacter baumanii) Acinetobacter baumanii 음성(초록)Voice (green) 발효균주Fermented strain 크레브실라 뉴모니애(Klebsiella pneumoniae) Klebsiella pneumoniae 양성(노랑)Positive (yellow) 프로테우스 미라빌리스(Proteus mirabillis) Proteus mirabillis 양성(노랑)Positive (yellow) 시트로박터 프론디이(Citrobactor freundii) Citrobactor freundii 양성(노랑)Positive (yellow)

상기 결과에서 본 발명의 신속한 당발효배지를 이용할 경우에 4시간의 배양시간이 당발효여부를 평가하는데 있어서 충분한 시간임을 확인할 수 있다.In the above results, it can be confirmed that when using the fast sugar fermentation medium of the present invention, the culture time of 4 hours is sufficient time to evaluate whether the sugar fermentation is effective.

검체균의 당발효여부를 4시간에 검사할 수 있음에 따라 세균의 동정에 걸리는 전체 시간을 20시간정도 단축할 수 있고, 이에 따라 세균의 동정에 의한 병원에서 환자에 대한 정확하고 신속한 치료가 가능하고, 식품 유통업체에서는 결과를 대기하는 시간의 단축을 통해 빠른 유통제품에 따른 신선도 유지가 가능하고 유통제품의 폐기율을 낮출 수 있다.By checking whether the bacteria are fermented in 4 hours, the total time required to identify bacteria can be reduced by about 20 hours, thus enabling accurate and rapid treatment of patients in the hospital by identifying bacteria. In addition, the food retailer can maintain the freshness according to the rapid distribution products and reduce the disposal rate of the distribution products by reducing the time to wait for the result.

Claims (4)

미생물의 당발효 여부를 확인하는 당발효배지에 영양물질을 첨가함을 특징으로 하는 미생물의 당발효여부의 단시간 검사를 위한 배지Medium for short-term test of sugar fermentation of microorganisms, characterized by adding nutrients to the sugar fermentation medium to check the sugar fermentation of microorganisms 제1항에서, 상기 영양물질이 이스트 추출물과 소고기 추출물임을 특징으로 하는 미생물의 당발효여부의 단시간 검사를 위한 배지[Claim 2] The medium for short-term test of fermentation of sugars of microorganisms according to claim 1, wherein the nutrients are yeast extract and beef extract. 제2항에서, 상기 배지가The method of claim 2, wherein the medium is 증류수 1000㎖에 포도당(Glucose) 10g, 펩톤(Peptone) 2g, 제2인산칼륨 (Dipotassium Phosphate) 0.3g, BTB(Bromthymol Blue) 0.03∼0.05g을 첨가하고;10 g of glucose, 2 g of peptone, 0.3 g of dipotassium phosphate, and 0.03 to 0.05 g of BTB (Bromthymol Blue) were added to 1000 ml of distilled water; pH를 7.1로 조정하고; 그리고,adjust pH to 7.1; And, 이스트 추출물(Yeast Extract) 1∼2g, 소고기 추출물(Beef Extract) 3∼4g을 첨가하여 제조되는 것을 특징으로 하는 미생물의 당발효여부의 단시간 검사를 위한 배지Yeast Extract 1 ~ 2g, Beef Extract (Beef Extract) Medium for a short time test of sugar fermentation of microorganisms, characterized in that it is prepared by adding 제1항 내지 제3항중의 어느 한 항의 배지를 뚜껑이 있는 튜브에 약 4∼5㎖정도로 분주하는 단계;Dispensing the medium of any one of claims 1 to 3 in a tube with a lid of about 4 to 5 ml; 맥파랜드 네페로미터 기준(McFarland Nephelometer Standard) 탁도 1 (Mcf.=1)에 맞추어 균을 접종하는 단계;Inoculating the bacterium according to the McFarland Nephelometer Standard turbidity 1 (Mcf. = 1); 균접종한 배지위에 미네랄 오일을 1cm두께로 덮은 다음 37℃에서 4시간동안 배양하는 단계; 및,Covering the mineral oil with a 1 cm thickness on the inoculated medium and incubating at 37 ° C. for 4 hours; And, 배지의 색깔이 노란색으로 변하면 양성으로 접종한 균이 포도당(Glucose)을 발효하는 것으로, 배지의 색깔이 푸른색 또는 초록색이면 음성으로 접종한 균이 포도당(Glucose)을 발효하지 못하는 것으로 판단하는 단계;Determining that the positively inoculated bacteria ferment glucose when the color of the medium changes to yellow, and that the negatively inoculated bacteria do not ferment the glucose when the color of the medium is blue or green; 를 포함하는 것을 특징으로 하는 미생물의 당발효여부의 단시간 검사법Short-term test method of the fermentation of sugar of the microorganism comprising a
KR1020000040205A 2000-07-13 2000-07-13 A method and medium for rapidly determining fermentation of micro-organism KR20020006786A (en)

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KR20030096986A (en) * 2002-06-18 2003-12-31 주식회사 코메드 Identification composition for non-fermentative gram negative bacilli and identification kit containing the same

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