KR100868574B1 - Assessing biofilm formation onto natural soil particles - Google Patents
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- KR100868574B1 KR100868574B1 KR1020080024488A KR20080024488A KR100868574B1 KR 100868574 B1 KR100868574 B1 KR 100868574B1 KR 1020080024488 A KR1020080024488 A KR 1020080024488A KR 20080024488 A KR20080024488 A KR 20080024488A KR 100868574 B1 KR100868574 B1 KR 100868574B1
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Abstract
Description
본 발명은 토양 입자 표면에 형성된 바이오필름의 함량 측정 방법에 관한 것으로, 더욱 상세하게는 토양에 존재하는 토착 박테리아(indigenous bacteria)에 의해 자연적으로 형성되거나 또는 인위적으로 토양 입자에 형성된 바이오필름의 양을 정량적으로 측정할 수 있는 방법에 관한 것이다.The present invention relates to a method for measuring the content of a biofilm formed on the surface of the soil particles, and more particularly, the amount of biofilm naturally formed by indigenous bacteria present in the soil or artificially formed on the soil particles. It relates to a method that can be measured quantitatively.
자연상태와 같은 빈영양 상태에서 대부분의 미생물은 바이오필름을 형성하여 각각의 개체가 아닌 군집의 형태로 존재한다. 바이오필름은 미생물과 그 미생물이 분비한 세포외 중합체(EPS; extracellular polymeric substances)를 말한다. In micronutrients, such as the natural state, most microorganisms form biofilms and exist in the form of clusters instead of individual individuals. Biofilm refers to microorganisms and extracellular polymeric substances (EPS) secreted by the microorganisms.
바이오필름은 점액질 형태로서 지질학적 매질의 표면에 고착하여 발달하는 특징을 보이며, 특히 토양이나 퇴적물에서 주로 발생한다. 또한 바이오필름은 환경공학적으로 중금속을 흡착하거나 하수 슬러지에서 유해물질을 제거하는데 연구되고 있고, 미생물학적으로 식품의 보존 등에 연구되고 있다. 최근 토양 입자에 부착한 바이오필름에 관한 연구 수요가 많아지고 있는데 반해, 자연 토양층에 발달된 바이오필름을 정량하는 방법은 전무한 실정이다. Biofilm is a slime form that develops by adhering to the surface of a geological medium, especially in soils and sediments. In addition, biofilms are being researched to adsorb heavy metals in environmental engineering or to remove harmful substances from sewage sludge, and microbiologically to study food preservation. Recently, there is a growing demand for research on biofilms attached to soil particles. However, there is no method for quantifying biofilms developed in natural soil layers.
바이오필름을 정량적으로 측정하는 방법에는 크리스탈 바이올렛(crystal violet) 용액을 이용하여 염색하는 방법(O'Toole and Kolter 1998)과 미생물에서 추출되는 칼륨(K)의 양을 측정하는 방법(Sutherland et al., 1986), 세포를 염색하여 현미경으로 관찰하는 방법(Fujishige et al., 2006) 등이 있다. Methods of quantitatively measuring biofilm include staining using a crystal violet solution (O'Toole and Kolter 1998) and measuring the amount of potassium (K) extracted from microorganisms (Sutherland et al. , 1986), and staining cells and observing them under a microscope (Fujishige et al., 2006).
그중 칼륨(K)의 양을 측정하는 방법의 경우, 토양에는 다량의 칼륨이 함유 되어 있어 미생물에서 추출되는 칼륨의 양을 측정할 때 토양의 칼륨과 간섭을 일으키는 문제가 있다. 또한 현미경으로 관찰하는 방법은 그 절차가 매우 까다로울 뿐만 아니라, 토양 입자에 비해 상대적으로 미생물의 크기가 매우 작아 미생물의 정확한 측정이 어려운 단점이 있다. Among them, the method of measuring the amount of potassium (K), the soil contains a large amount of potassium, there is a problem causing interference with potassium in the soil when measuring the amount of potassium extracted from the microorganism. In addition, the method of observing under a microscope is not only very difficult, but also has a disadvantage that it is difficult to accurately measure the microorganism because the size of the microorganism is relatively small compared to soil particles.
본 발명은 상기와 같은 문제점을 해결하기 위한 것으로, 토양에 부착된 바이오필름의 양을 토양의 간섭을 배제하고 정량적으로 측정함으로써 환경공학 또는 미생물공학 등의 분야에서 토양 입자 표면에 형성된 바이오필름의 함량을 신속·간단·정확하게 측정하는 방법을 제공하는데 그 목적이 있다. The present invention is to solve the above problems, by quantitatively measuring the amount of the biofilm attached to the soil without the interference of the soil content of the biofilm formed on the surface of the soil particles in the field of environmental engineering or microbial engineering, etc. The aim is to provide a method for measuring the rate of time rapidly, easily and accurately.
전술한 바와 같은 목적을 달성하기 위한 본 발명에 의한 토양 입자 표면에 형성된 바이오필름의 함량 측정 방법은, 측정하고자 하는 토양을 멸균된 페트리 접시에 넣는 토양 채취 단계와; 상기 페트리 접시에 증류수를 투입하여 상기 토양을 침강시킴으로써 토양 입자에 형성된 바이오필름을 제외한 나머지 유동 가능한 자유유영 상태의 미생물을 씻어내 제거하는 세척 단계와; 상기 세척된 토양을 건조하는 단계와; 상기 건조 단계를 거친 건조된 토양에 크리스탈 바이올렛(crystal violet) 용액을 넣어 토양입자에 형성된 바이오필름을 염색하는 단계와; 상기 토양입자에 형성된 바이오필름에 염색되지 않고 남아있는 크리스탈 바이올렛 용액을 제거하는 단계와; 상기 바이오필름에 염색된 크리스탈 바이올렛을 탈색하는 단계와; 상기 탈색 단계를 거친 용액의 흡광도를 측정하는 단계를 포함하여 이루어진 것을 특징으로 한다.Method for measuring the content of the biofilm formed on the surface of the soil particles according to the present invention for achieving the object as described above, the soil collection step of putting the soil to be measured in a sterile petri dish; A washing step of washing and removing the remaining free-flowing microorganisms except for the biofilm formed on the soil particles by putting distilled water into the petri dish to settle the soil; Drying the washed soil; Dyeing the biofilm formed on the soil particles by putting a crystal violet solution into the dried soil after the drying step; Removing the crystal violet solution remaining in the biofilm formed on the soil particles without being dyed; Decolorizing the crystal violet stained on the biofilm; Characterized in that it comprises the step of measuring the absorbance of the solution passed through the decolorizing step.
본 발명에 의한 토양 입자 표면에 형성된 바이오필름의 함량 측정 방법에 의하면, 토양에 형성되어 있는 바이오필름의 함량을 정량적으로 확인할 수 있고, 고가의 장비가 필요없으며, 누구나 손쉽게 측정할 수 있는 염색법이라는 것에 그 장점이 있다.According to the method for measuring the content of the biofilm formed on the surface of the soil particles according to the present invention, it is possible to quantitatively check the content of the biofilm formed in the soil, and does not require expensive equipment, and anyone can easily measure the dyeing method. It has its advantages.
본 발명을 설명하는데 있어 기술되는 용어들은 본 발명에서의 기능을 고려하여 정의된 용어들로서 이는 사용자, 운용자의 의도 또는 관례 등에 따라 달라질 수 있다. 그러므로 그 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. Terms used in describing the present invention are terms defined in consideration of functions in the present invention, which may vary according to a user's or operator's intention or custom. Therefore, the definition should be made based on the contents throughout the specification.
도 1을 참조하여 본 발명에 의한 토양 입자 표면에 형성된 바이오필름의 함량 측정 방법을 설명한다.With reference to Figure 1 will be described a method of measuring the content of the biofilm formed on the surface of the soil particles according to the present invention.
(M1) 토양 채취.(M1) Soil Harvesting.
토양(미생물에 의해 바이오필름이 형성된 토양)을 채취하여 일정량, 예를 들면 3g을 정량하여 페트리 접시(petridish)에 넣는다. 이때 사용하는 상기 페트리 접시는 미리 70% 에틸 알콜(ethyl alcohol)을 이용해 세척 후, 자외선을 이용하여 살균ㆍ소독한 후 사용한다. Soil (the soil in which the biofilm is formed by microorganisms) is collected and weighed in a predetermined amount, for example, 3 g, into a petri dish. The Petri dish used at this time is used after washing with 70% ethyl alcohol in advance and sterilizing and disinfecting with ultraviolet rays.
본 발명은 박테리아에 의해 형성된 바이오필름의 양을 정량하는 방법의 특성상 토양 외부(공기 중이나 사람의 손 등)의 다른 박테리아의 간섭을 막기 위해 살균상태가 유지되는 크린 벤치(clean bench) 내부에서 진행되어야 한다. 또한 본 과정에 이용되는 모든 장비는 70% 에틸 알콜(ethyl alcohol)을 이용하여 소독 후 사용한다.The present invention has to be carried out inside a clean bench where sterilization is maintained to prevent interference of other bacteria outside the soil (such as in the air or human hands) due to the nature of the method of quantifying the amount of biofilm formed by bacteria. do. In addition, all equipment used in this process should be used after disinfection with 70% ethyl alcohol.
(M2) 토양 세척.(M2) soil washing.
고압멸균기로 멸균한 증류수를 이용하여 페트리 접시에 담겨진 토양을 3회 세척한다. 세척의 방법은 1)토양 3g이 담겨진 페트리 접시에 멸균된 증류수 20mL를 피펫을 이용하여 동일하게 넣어준다. 2)토양과 증류수가 담긴 페트리 접시를 바닥에 놓고 약 2∼3회 가볍게 흔들어 주고 토양입자를 침강시킨다. 토양을 흔들어 주는 이유는 토양 내에서 바이오필름을 형성하지 않고 자유유영 상태로 존재하는(planktonic) 미생물을 토양으로부터 분리·제거하기 위함이다. 3)페트리 접시에 담긴 증류수를 버린다. 이때 토양의 유실이 없도록 페트리 접시의 뚜껑을 닫고 한 쪽 방향으로 기울여 증류수만 제거한다. 이러한 과정을 총 3회 반복 하여 1회 세척 시 미처 제거 되지 않는 자유유영 상태의 미생물을 반복적으로 제거 한다.The soil contained in the Petri dish is washed three times using distilled water sterilized by autoclave. How to wash 1) Put the same 20mL sterilized distilled water in a petri dish containing 3g of soil using a pipette. 2) Place a Petri dish containing soil and distilled water on the bottom, shake it lightly 2 ~ 3 times and settle soil particles. The reason for shaking the soil is to separate and remove the planktonic microorganisms from the soil without forming biofilm in the soil. 3) Discard distilled water in Petri dish. At this time, close the lid of the Petri dish so that there is no loss of soil and tilted in one direction to remove only distilled water. This process is repeated three times in order to repeatedly remove free-running microorganisms that cannot be removed in one wash.
(M3) 토양 건조.(M3) soil drying.
세척이 끝난 후 상기 세척단계에서 토양간극에 남아있는 증류수를 제거하기 위하여 토양을 28 내지 32oC 온도로 25 내지 35분, 바람직하게 30oC로 30분간 건조한다. After washing, the soil is dried at a temperature of 28 to 32 ° C. for 25 to 35 minutes, preferably 30 ° C. for 30 minutes to remove distilled water remaining in the soil gap in the washing step.
(M4) 바이오필름 염색.(M4) Biofilm Staining.
상기 공정을 거쳐 건조된 토양에 1% 크리스탈 바이올렛(crystal violet) 용액 13~17mL 바람직하게 15mL를 넣어 토양과 크리스탈 바이올렛의 비율이 1:5가 되도록 한다. 또한 크리스탈 바이올렛의 농도가 높아지면 용액 자체의 색이 너무 진하여 흡광도 측정시 기계적인 신뢰 범위인 2.000을 초과하게 되므로 이를 방지하기 위해 1% 크리스탈 바이올렛을 사용하여, 하기에 나오는 흡광도 측정값의 신뢰도를 확보한다. 13 ~ 17mL of 1% crystal violet solution (15% preferably 15mL) is added to the dried soil through the above process so that the ratio of soil and crystal violet is 1: 5. In addition, when the concentration of the crystal violet becomes high, the color of the solution itself is too dark to exceed the mechanical confidence range of 2.000 when measuring the absorbance. Therefore, the 1% crystal violet is used to prevent this. Secure.
크리스탈 바이올렛(crystal violet)은 미생물의 외벽을 염색하는 시약으로 이때 주입한 용액은 토양 내 바이오필름을 형성한 미생물을 염색한다. 크리스탈 바이올렛(crystal violet)을 넣어준 후 18~22oC온도로 40~50분간, 바람직하게 20oC온도로 45분 동안 반응시켜 주입한 크리스탈 바이올렛이 토양에 형성되어 있는 바이오필름을 염색하도록 한다.Crystal violet is a reagent for staining the outer walls of microorganisms, and the injected solution dyes the microorganisms that form the biofilm in the soil. After the crystal violet is added, it is reacted for 40 to 50 minutes at 18 to 22 o C temperature, and for 45 minutes at 20 o C temperature, so that the injected crystal violet dyes the biofilm formed in the soil. .
(M5) 크리스탈 바이올렛의 제거. (M5) removal of crystal violet.
염색된 토양을 전술한 토양 세척 공정(M2)에서 증류수를 제거한 것과 동일한 방법으로 바이오필름을 염색하고 남은 크리스탈 바이올렛(crystal violet)용액을 제거한다. 이후 멸균된 증류수를 이용하여 세척해 준다. 이때 세척은 증류수로 토양내부 공극에 남아있는 크리스탈 바이올렛 용액을 제거하는 것으로 1회 세척만으로 완전히 제거되지 않기 때문에 총 3회 반복해 주며 1회 세척 시 멸균된 증류수 20mL을 사용한다. 이때 증류수 20mL를 사용하는 것은 페트리 접시의 용량에 따른 것이다. 방법은 상기 토양세척단계에서 증류수를 제거하는 것과 동일하다.The dyed soil is dyed the biofilm in the same manner as the distilled water is removed in the aforementioned soil washing process (M2), and the remaining crystal violet solution is removed. After washing with sterile distilled water. At this time, the washing is to remove the crystal violet solution remaining in the pores of the soil with distilled water. Since it is not completely removed by only one washing, the washing is repeated three times. In each washing, 20 mL of sterilized distilled water is used. The use of 20 mL of distilled water depends on the capacity of the Petri dish. The method is the same as removing the distilled water in the soil washing step.
(M6) 바이오필름에 염색된 크리스탈 바이올렛의 탈색.(M6) Bleaching of crystal violet dyed to biofilm.
총 3회의 세척이 종료된 후 95% 에틸 알콜(ethyl alcohol) 18~22mL 바람직하게 20mL을 페트리 접시에 넣어준다. 이 과정을 통하여 토양 입자에 형성된 바이오필름에 염색된 크리스탈 바이올렛(crystal violet)을 바이오필름으로부터 추출한다. Gram's iodine 등의 매염제(mordant)를 넣지 않았으므로 이 세척과정을 통하여 그램음성균 뿐만 아니라 그램양성균을 염색한 크리스탈 바이올렛(crystal violet)까지 녹아 나온다. 25~35분, 바람직하게 30분 동안 반응을 시켜 바이오필름에 염색 된 크리스탈 바이올렛(crystal violet)용액을 탈색시키고, 0.2㎛ 필터를 이용하여 여과한다. After 3 washes in total, 18-22 mL of 95% ethyl alcohol, preferably 20 mL, is placed in a Petri dish. Through this process, crystal violet (crystal violet) dyed in the biofilm formed on the soil particles is extracted from the biofilm. Since no mordant such as Gram's iodine was added, the washing process melts not only Gram-negative bacteria but also crystal violet stained Gram-positive bacteria. The reaction is carried out for 25 to 35 minutes, preferably 30 minutes to decolorize the crystal violet solution stained on the biofilm, and filtered using a 0.2 μm filter.
(M7) 흡광도 측정.(M7) Absorbance Measurement.
여과된 용액을 UV-cell에 넣어 UV-vis를 이용하여 흡광도를 측정한다. 흡광도 측정 시에는 염색된 바이오필름에서 탈색시킨 용액이 보라색을 띠기 때문에 자외선 파장 영역인 595㎚ 파장을 이용한다. Put the filtered solution in the UV-cell and measure the absorbance using UV-vis. When absorbance is measured, the solution decolored from the dyed biofilm has a violet color, so a 595 nm wavelength, which is an ultraviolet wavelength region, is used.
SAMPLE CD-OD595TSM0.473TDM0.508TSC0.158TDC0.158LSM0.898LDM0.696LSC0.122LDC0.155SAMPLE CD-OD 595 TSM0.473TDM0.508TSC0.158TDC0.158LSM0.898LDM0.696LSC0.122LDC0.155
TSM: 표토(surface soil) 내 미생물에 TSB 영양분을 공급한 후 바이오필름 TSM: Biofilm after supplying TSB nutrients to microorganisms in surface soil
측정 결과 Measurement result
TDM: 심토(subsurface soil) 내 미생물에 TSB 영양분을 공급한 후 바이오필름 TDM: Biofilm after supplying TSB nutrients to microorganisms in subsurface soil
측정 결과 Measurement result
TSC: TSM과 동일한 조건이나 미생물을 살균한 비교시료(control)TSC: Control sample sterilizing microorganisms under the same conditions as TSM
TDC: TDM과 동일한 조건이나 미생물을 살균한 비교시료(control)TDC: Control sample sterilizing microorganisms under the same conditions as TDM
LSM: TSM과 동일한 조건이나 TSB 대신 LB 영양분을 공급한 경우 LSM: Same conditions as TSM or LB nutrients instead of TSB
LDM: TDM과 동일한 조건이나 TSB 대신 LB 영양분을 공급한 경우 LDM: Same conditions as TDM or LB nutrients instead of TSB
LSC: LSM과 동일한 조건이나 미생물을 살균한 비교시료(control)LSC: Control sample sterilizing microorganisms under the same conditions as LSM
LDC: LDM과 동일한 조건이나 미생물을 살균한 비교시료(control)LDC: Control sample sterilizing microorganisms under the same conditions as LDM
위 표는 실제로 바이오필름의 양을 정량하기 위해 실시한 실험의 결과이다. TSB와 LB 배지를 토양에 공급하여 토양 토착 박테리아(indigenous bacteria)가 바이오필름을 형성하게 한 후 그 함량을 측정해 보았다. 실험결과에서도 나타나듯이, 미생물 시료가 비교시료(control)에 비하여 더 높은 흡광도 값을 보여 크리스탈 바이올렛(crystal violet)에 더 많이 염색되었음을 알 수 있다. 이를 정확한 농도로 환산하기 위해서는 각 농도별 흡광도 값을 확인하는 표준 곡선을 만들어 흡광도 측정 후, 이때 얻어진 환산식에 의해 구하는 방법을 따른다. The above table is actually the result of experiment conducted to quantify the amount of biofilm. TSB and LB medium was fed to the soil to allow the indigenous bacteria to form a biofilm and then its content was measured. As shown in the experimental results, the microbial sample showed a higher absorbance value compared to the control (control), it can be seen that more stained in the crystal violet (crystal violet). In order to convert this to the correct concentration, a standard curve for confirming the absorbance value for each concentration is made, and the absorbance is measured.
도 1 은 발명에 따른 박테리아에 의해 토양에 형성된 바이오필름의 양을 측정하는 방법을 나타낸 과정. 1 is a process showing a method for measuring the amount of biofilm formed in soil by bacteria according to the invention.
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| KR20070022023A (en) * | 2004-02-23 | 2007-02-23 | 티에리 베르나르디 | Method and device for detecting the formation and development of biofilms in a culture medium |
| KR20070038464A (en) * | 2004-05-15 | 2007-04-10 | 햅토젠 리미티드 | Methods for reducing bioflim formation in infectious bacteria |
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| KR20070022023A (en) * | 2004-02-23 | 2007-02-23 | 티에리 베르나르디 | Method and device for detecting the formation and development of biofilms in a culture medium |
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| US11135315B2 (en) | 2010-11-30 | 2021-10-05 | Convatec Technologies Inc. | Composition for detecting biofilms on viable tissues |
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