미생물 접합 실험과 항생제 내성 선별

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1. Serial Dilution (희석계대법)

Serial dilution은 미생물 수, DNA, 단백질, 화학물질 등의 농도가 너무 높아 정확한 측정이 어려울 때 사용되는 기본적인 실험 방법이다. 일정한 비율로 연속적으로 희석하여 농도를 점차 낮추는 기법으로, 본 실험에서는 항생제 농도를 최적화하기 위해 사용되었다. 각 항생제의 최저 유효 농도를 결정하는 데 중요한 역할을 한다.
2. Bacterial Conjugation (박테리아 접합)

박테리아 접합은 두 균주 간의 유전자 전달 과정이다. 본 실험에서는 donor cell인 A. baumannii 17978 △ompA - nptI ? pHKD01 in S17-1 lambda pir로부터 recipient cell인 A. baumannii 17978으로 kanamycin 내성 유전자를 전달하는 과정을 확인한다. 접합을 통해 유전자 재조합이 이루어지며, 이는 기본적인 유전자 조작 기술의 예시이다.
3. Antibiotic Resistance Selection (항생제 내성 선별)

항생제 내성은 특정 항생제에 대한 미생물의 저항성을 의미한다. 본 실험에서는 kanamycin과 ampicillin 내성을 이용하여 유전자 전달이 성공한 균주를 선별한다. Amp30 Km50 배지(ampicillin 30μl/ml, kanamycin 50μl/ml)를 사용하여 두 항생제 모두에 내성인 재조합 균주만 생존하도록 하여 실험 결과를 확인한다.
4. Spreading Method (도말 배양법)

Spreading은 액체 배양액을 고체 배지 표면에 균일하게 펼치는 배양 기법이다. 본 실험에서는 멸균된 spreader를 사용하여 균 배양액을 agar 배지에 도말한다. Spreader는 알코올램프 불꽃에 멸균한 후 식혀서 사용하며, 이는 오염을 방지하고 정확한 실험 결과를 얻기 위한 중요한 절차이다.

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1. Serial Dilution (희석계대법)

Serial dilution is a fundamental microbiological technique that enables accurate quantification of microbial populations by systematically reducing sample concentration through successive dilutions. This method is essential for obtaining countable colonies on culture plates, typically in the range of 30-300 colonies. The logarithmic reduction at each step (usually 10-fold) provides a practical approach to handle extremely high cell densities that would otherwise be impossible to enumerate. Serial dilution's reliability depends on proper mixing and accurate pipetting at each stage. While the technique is straightforward and cost-effective, it requires careful attention to detail to avoid errors that could compound through successive dilutions. The method remains indispensable in microbiology laboratories for viable cell counting, antimicrobial susceptibility testing, and quality control in pharmaceutical and food industries.
2. Bacterial Conjugation (박테리아 접합)

Bacterial conjugation represents a remarkable mechanism of horizontal gene transfer that fundamentally shapes bacterial evolution and adaptation. This direct cell-to-cell contact method allows transfer of plasmids and chromosomal DNA, particularly important for spreading antibiotic resistance genes among bacterial populations. The process involves complex pili formation and mating bridge establishment, demonstrating sophisticated bacterial communication systems. Conjugation's significance extends beyond laboratory settings, as it plays a critical role in the dissemination of virulence factors and resistance determinants in clinical environments. Understanding conjugation mechanisms is crucial for predicting and controlling the spread of pathogenic traits in bacterial communities. However, the efficiency of conjugation varies considerably depending on bacterial species, environmental conditions, and plasmid characteristics, making it a complex phenomenon worthy of continued research.
3. Antibiotic Resistance Selection (항생제 내성 선별)

Antibiotic resistance selection is a critical process that demonstrates the power of natural selection at the microbial level, where resistant mutants survive and proliferate in antibiotic-containing environments. This selective pressure mechanism is both a valuable laboratory tool for isolating specific bacterial strains and a serious public health concern when occurring in clinical settings. The technique effectively eliminates susceptible organisms while allowing resistant populations to dominate, making it essential for genetic engineering and strain development. However, the widespread use of antibiotics in clinical practice and agriculture has inadvertently created selection pressures that accelerate the emergence of multidrug-resistant pathogens. Understanding resistance selection mechanisms is vital for developing strategies to combat antibiotic resistance. The technique's dual nature—useful in research but problematic in uncontrolled environments—highlights the importance of judicious antibiotic use and continued surveillance of resistance patterns.
4. Spreading Method (도말 배양법)

The spreading method, or spread plate technique, is a simple yet highly effective microbiological procedure for isolating and enumerating individual bacterial colonies on solid media. Using a sterile spreader to distribute inoculum evenly across the agar surface creates isolated colonies that can be easily picked for further analysis or pure culture development. This technique is superior to pour plating in many applications because it avoids heat exposure that might damage temperature-sensitive organisms. The method's success depends on proper aseptic technique, appropriate inoculum volume, and spreader sterilization between applications. While the spreading method is economical and requires minimal equipment, it demands operator skill and experience for consistent results. The technique remains widely used in clinical microbiology, food testing, and research laboratories for isolation of pure cultures and colony counting. Its simplicity and effectiveness make it an indispensable tool in microbiology despite the availability of more automated alternatives.

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