本文關鍵詞：結核休眠菌模型構建及復蘇探索　出處：《重慶醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 缺鉀培養(yǎng) 結核休眠菌 分枝桿菌噬菌體 復蘇

【摘要】：目的構建結核休眠菌缺鉀模型,并探索結核菌培養(yǎng)上清液(supernatant,SN)、分枝桿菌噬菌體TM4、分枝桿菌噬菌體Guo1對模型菌的復蘇作用。方法1.構建結核休眠菌缺鉀模型采用缺鉀Sauton培養(yǎng)基培養(yǎng)對數(shù)生長期結核菌,誘導其進入休眠狀態(tài)。采用菌落計數(shù)、刃天青試驗和最大或然數(shù)法(MPN)檢測結核菌存活率及休眠菌比例;用透射電子顯微鏡(transmission electron microscope,TEM)觀察結核菌細胞壁的厚度改變;采用金胺O-尼羅紅雙熒光染色法檢測結核菌是否失去抗酸染色特性和發(fā)生脂質聚集;藥敏測試檢測結核菌對異煙肼及利福平的耐藥情況。2.復蘇結核休眠菌將對數(shù)期結核菌的培養(yǎng)上清液、分枝桿菌噬菌體TM4、分枝桿菌噬菌體Guo1分別與缺鉀建模的結核休眠菌混合培養(yǎng),采用菌落計數(shù)法檢測復蘇情況。結果1.構建結核休眠菌缺鉀模型缺鉀培養(yǎng)30天后,結核菌從桿狀變?yōu)榍蛐?細胞壁增厚,失去抗酸染色特性,發(fā)生脂質聚集,并對抗結核藥物耐藥。2.復蘇結核休眠菌混合培養(yǎng)第1日時,空白對照組、上清組、TM4組、Guo1組菌落計數(shù)分別為(2.67±0.58)×102CFU/mL、(3.00±1.00)×102CFU/mL、(2.67±0.58)×102CFU/mL、(2.33±0.58)×102CFU/mL;培養(yǎng)第15日時,各組菌落計數(shù)分別為(7.33±1.53)×104CFU/mL、(6.67±2.08)×1010CFU/mL、(3.00±1.00)×1010CFU/mL、(3.33±1.53)×1010CFU/mL。結論缺鉀培養(yǎng)可誘導對數(shù)期結核菌進入休眠狀態(tài)。對數(shù)期結核菌培養(yǎng)上清液、分枝桿菌噬菌體TM4、分枝桿菌噬菌體Guo1均能夠復蘇結核休眠菌。其中以上清液復蘇能力最強,分枝桿菌噬菌體TM4和Guo1復蘇能力無明顯差異。
[Abstract]:Objective to establish the potassium deficiency model of Mycobacterium tuberculosis dormant bacteria and to explore the supernatant TM4 of Mycobacterium tuberculosis culture supernatant. The resuscitation of Mycobacterium bacteriophage Guo1 on the model bacteria. 1. The model of Mycobacterium tuberculosis dormancy bacteria was established and cultured in the logarithmic growth phase using Sauton medium. 2. The survival rate of tuberculous bacteria and the proportion of dormant bacteria were detected by colony count, edge azurol test and maximum probability method (MPN). The thickness of the cell wall of tuberculous bacilli was observed by transmission electron microscope (TEM) and transmission electron microscopesimetry (TM). Whether the mycobacterium tuberculosis lost its acid fast staining characteristics and the accumulation of lipid was detected by the double fluorescence staining method of Amidine O- Nile red. Drug sensitivity test to detect the drug resistance of Mycobacterium tuberculosis to isoniazid and rifampicin. 2. Resuscitation tuberculosis dormancy bacteria culture supernatant of logarithmic phase tuberculous bacteria, Mycobacterium bacteriophage TM4. Mycobacterium bacteriophage Guo1 was mixed with Mycobacterium tuberculosis dormancy bacteria which was modeled by potassium deficiency, and the resuscitation was detected by colony counting method. Results 1. After 30 days of potassium deficiency culture, the model of Mycobacterium tuberculosis dormant bacteria was cultured. 2. Tuberculous bacteria from rod-shaped to spherical, cell wall thickening, loss of acid-fast staining characteristics, lipid accumulation, and anti-tuberculosis drug resistance .2. resuscitation tuberculosis dormancy bacteria mixed culture on 1st, blank control group. The colony count of Guo1 group was 2.67 鹵0.58 脳 10 ~ 2 CFU 路m ~ (-1) 脳 10 ~ (2) CFU / mL in supernatant group (TM4) and 3.00 鹵1.00 脳 10 ~ (2) CFU / mL respectively. 2.67 鹵0.58) 脳 10 ~ (2) CFU / m ~ (-1) (2.33 鹵0.58) 脳 10 ~ (2) CFU / m ~ (-1); On 15th, the colony count of each group was 7.33 鹵1.53 脳 10 ~ 4 CFU / m ~ (-1) (6.67 鹵2.08) 脳 10 ~ (10) CFU / mL. 3.00 鹵1.00) 脳 10 ~ (10) CFU / mL. Conclusion potassium deficiency culture can induce mycobacterium tuberculosis into dormancy state in logarithmic phase. The supernatant of logarithmic culture of Mycobacterium tuberculosis, Mycobacterium phage TM4. Mycobacterium bacteriophage Guo1 was able to resuscitate the dormancy bacteria of tuberculosis, among which the supernatant was the strongest, but the TM4 and Guo1 of Mycobacterium bacteriophages had no significant difference.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R378

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