Effect Of Simulating Soil Environment On Soil Bacterial Diversity By PCR-RFLP Analysis

At present, due to insufficient level of awareness of human and technical means to limit, making more than 99% of the microorganisms in nature is difficult to be cultured and isolated by traditional pure culture technique.However,isolating pure culture stains is the base of the microbial ecology research. Therefore, how to improve the culturability of microorganisms, development and utilization of the 99% of uncultured microorganisms has become a hot topic at home and abroad.In this research, the bacteria in three types of soil samples was cultured on four different types of media and sub-period count. Bacterial universal primers were used to amplify 16S rDNA fragments; PCR products digested by restriction endonucleases Hha I and Rsa I and their fingerprints analyzed for influence of medium type on soil microbial diversity and culturability.At last,we summed up a training method which can more the number of bacterial culture and the diversity of bacterial.According to the experimental results of the analysis, we have the following conclusions:First, extend the incubation time can increase the number of bacteria culture, but for different types of media,the effect of icreasing is somewhat different.in the vegetable soil sample,after 144 hours in culture,the bacteria on the LB mediem increased by 1.46 times than after 48 hours.while the same time,the bactera on the soil particles LB medium increased by 2.78 times, the bactera on the WSA medium increased by 6.33 times and the bactera on the soil particles WSA medium increased by 8.69 times. The other two soil samples also showed the same trend, namely:On the LB medium, bacterial colonies appeared earlier, but growth is slow, almost 96 hours to stop growth trends; the soil particles WSA is the fastest growing medium,a large number of new colonies remain appeared after 96 hours.Second, reduce the nutrient medium can increase the cultivation of microorganisms.In this experiment, the bacteria in three types of soil samples was cultured on four different types of media.After the PCR-RFLP analysis we got Shannon-Wiener index, Simpson index and Margalef index.We found that low nutrition culture medium can obtain a higher bacterial diversity.Third, soil particles water instead of conventioal water in culture medium, not only can get more number of bacteria,and can get more types of them.The vegetable soil sample,for example,we have 8.23×10'CFUs on the soil particles LB medium after 144 hours,compared to the LB medum,the number is 2.46 times. we have 6.01×107 CFUs on the soil particles WSA medium after 144 hours,compared to the WSA medum,the number is 2.11 times.According to the Shannon-Wiener index,Simpson index and Margalef's index which gained from the PCR-RFLP analysis,the medium with soil particles can be used to get more types of bacteria.This is also can be found in the phytotype richness curves of soil bacteria cultured in four culturing mediaThe Jaccard index was calculated through the PCR-RFLP analysis. the Jaccard index,which between four types of medium,of the vegetable soil sample is 24.31%,the Jaccard index of the wood soil sample is 12.50%, the. Jaccard index of the rice field sample is 9.14%.The smaller the Jaccard index is, the little similarity of bacterial community is, suggesting that only a very small number of soil bacteria can grow under different environmental conditions.The culture of bacteria needs suitable medium,therefore,the medium of choice is very important.