Effect of Simulating Soil Environment on Soil Bacterial Diversity by PCR-RFLP Analysis

Author :DanZuo
Tutor:CaoHui
School :Nanjing Agricultural College
CLC :S154.3
TYPE :Masters thesis
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Year:2011
Abstract:
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 8.2310CFUs on the soil particles LB medium after 144
hours,compared to the LB medum,the number is 2.46 times. we have 6.01107 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 Margalefs 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.