本文選題：鰻弧菌 + 生物膜��； 參考：《上海海洋大學》2017年碩士論文

【摘要】：采用激光共聚焦顯微鏡技術觀察鰻弧菌生物膜形成過程,體外構建法構建鰻弧菌生物膜模型,并分析結構特征。本實驗以致病性鰻弧菌BYK0638為研究對象,使用蓋玻片生物膜培養(yǎng)法體外構建鰻弧菌的生物膜,并在生物膜形成過程中,采用CCK-8法定量檢測生物膜的形成量;在培養(yǎng)不同時間后,用免疫熒光劑碘化吡啶(PI)染色,用激光共聚焦顯微鏡(Confocal laser scanning microscope,CLSM)觀察鰻弧菌生物膜的形成過程和結構特征。結果顯示,鰻弧菌能夠在玻片上形成較致密的生物膜,鰻弧菌生物膜的形成量在培養(yǎng)前24 h升高較快,60 h后趨于穩(wěn)定。獲得了生物膜形成過程不同時間節(jié)點的CLSM圖像,觀察到鰻弧菌一般在24 h后開始逐漸形成生物膜,在72 h形成穩(wěn)定的生物膜。探討致病性鰻弧菌(V.anguillarum)BYK0638生物膜的形成特性,為進一步研究鰻弧菌生物膜的形成機制和致病機理提供參考。采用改良的微孔板法研究靜置培養(yǎng)條件下鰻弧菌(V.anguillarum)BYK0638在96孔酶標板上的成膜情況,CCK-8法(Cell Counting Kit-8)定量檢測生物膜中鰻弧菌的活力。鰻弧菌BYK0638能夠在聚苯乙烯酶標板上形成穩(wěn)定而明顯的生物膜,其生物膜的OD450值在24h達到峰值,60h后趨于穩(wěn)定;在107-108CFU/mL范圍內(nèi),鰻弧菌生物膜的OD450值顯著高于其他試驗組(P0.05);25℃時的生物膜OD450值顯著高于其他溫度生物膜的形成量(P0.05);在pH4-11范圍內(nèi),當pH值為7時鰻弧菌形成的生物膜量最高,在pH值為3和12時,鰻弧菌幾乎不形成生物膜;在TSB培養(yǎng)基中加入0.03-2.00mmol/L CaCl2,鰻弧菌生物膜形成量與未添加CaCl2對照組無顯著性差異;在TSB培養(yǎng)基中加入0.03mmol/L MgCl_2,可促進鰻弧菌生物膜形成;NaCl濃度為5%時,形成的生物膜OD450值最高;鰻弧菌在大黃魚表皮黏液、肝臟、前腸、后腸組織提取液包被的96孔酶標板上形成的生物膜顯著高于其他黏液和組織提取液包被組(P0.05)。致病性鰻弧菌BYK0638能形成穩(wěn)定而明顯的生物膜,其生物膜形成與外界環(huán)境因素變化有密切的關系,培養(yǎng)時間、溫度、初始菌濃度、pH、鹽度、Mg~(2+)、魚體黏液及組織等各種環(huán)境因素均能顯著影響鰻弧菌生物膜的形成。為探索大黃、穿心蓮與五倍子對鰻弧菌及其生物膜活性的影響。采用微量二倍稀釋法分別測定大黃、穿心蓮及五倍子對鰻弧菌游離菌的最小抑菌濃度(MIC)、最小殺菌濃度(MBC)。用1/8、1/4、1/2、1、2、4、8倍MIC的大黃、穿心蓮與五倍子分別作用于鰻弧菌生物膜,CCK-8法測定藥物干預后生物膜活力變化。結果表明,五倍子能較好地干預鰻弧菌生物膜形成,在2倍MIC濃度時,在培養(yǎng)4 h時即可完全抑制BYK0638菌株生物膜的生長,且其SMIC50濃度為2MIC。推薦生產(chǎn)防治用量為6.25mg/mL。
[Abstract]:The formation process of Vibrio anguilis biofilm was observed by laser confocal microscopy. The model of Vibrio eel biofilm was constructed in vitro, and the structure characteristics were analyzed. This experiment was made to study the BYK0638 of Vibrio Anguilla. The biofilm of Vibrio Anguilla was constructed in vitro by means of cover glass biofilm culture, and C was used in the process of biofilm formation. CK-8 method was used to quantify the formation of biofilm. After different times of culture, the formation and structural characteristics of the biofilm of Vibrio Anguilla were observed with the immunofluorescent iodipyridine (PI) staining and the Confocal laser scanning microscope (CLSM). The results showed that Vibrio Anguilla was able to form a dense biofilm on the slide. The formation of Vibrio anguilis biofilm increased rapidly at 24 h before culture and stabilized after 60 h. The CLSM image of different time nodes in the biofilm formation process was obtained. It was observed that Vibrio Anguilla formed a biofilm gradually after 24 h and formed a stable biofilm at 72 h. The formation of pathogenic Vibrio anguilis (V.anguillarum) BYK0638 biofilm was formed. In order to provide reference for further study on the formation mechanism and pathogenesis of Vibrio anguilis biofilm, the membrane formation of Vibrio Anguilla (V.anguillarum) BYK0638 on the 96 hole enzyme labeled plate was studied by modified microplate method. The activity of Vibrio Anguilla in the biofilm was quantified by CCK-8 (Cell Counting Kit-8). The BYK0638 energy of Vibrio Anguilla was determined by the CCK-8 method. A stable and obvious biofilm was formed on the polystyrene label plate. The OD450 value of the biofilm reached its peak value at 24h and then stabilized after 60H; in the 107-108CFU/mL range, the OD450 value of the biofilm of Vibrio Anguilla was significantly higher than that of the other experimental groups (P0.05), and the OD450 value of the biofilm at 25 C was significantly higher than that of the other temperature biofilms (P0.05); In the range of pH4-11, when the value of pH was 7, the biofilm formed by Vibrio anguilis was the highest. When the value of pH was 3 and 12, Vibrio eel almost did not form a biofilm. The formation of Vibrio anguilis biofilm in the TSB medium was not significantly different from that of the non CaCl2 control group, and the addition of 0.03mmol/L MgCl_2 to the TSB medium could promote the eel arc. Bacterial biofilm was formed; when the concentration of NaCl was 5%, the OD450 value of the biofilm was highest, and the biofilm formed on the 96 orifice of the epidermal mucus, the liver, the foregut and the posterior intestinal tissue was significantly higher than that of the other mucus and tissue extracts (P0.05). The pathogenic Vibrio anguilis BYK0638 could form a stable and obvious birth. Membrane formation has a close relationship with the changes of external environmental factors. The culture time, temperature, initial bacteria concentration, pH, salinity, Mg~ (2+), fish body mucus and tissue can significantly affect the formation of the biofilm of Vibrio Anguilla. The effects of rhubarb on the activity of Vibrio Anguilla and its biofilm are explored. The minimum bacteriostasis concentration (MIC) and minimum bactericidal concentration (MBC) of rhubarb, andrographolic and Galla chinensis were measured by two times dilution method respectively. Using 1/8,1/4,1/2,1,2,4,8 times MIC of rhubarb, Andrographis paniculata and Galla chinensis were respectively acted on the biofilm of Vibrio Anguilla, and the activity of biofilm after drug intervention was determined by CCK-8 method. The results showed that the Chinese gall was the Chinese gall. It can interfere with the formation of the biofilm of Vibrio anguilis. At the concentration of 2 times MIC, the growth of the biofilm of the BYK0638 strain can be completely suppressed when the culture of 4 h is cultured, and the concentration of SMIC50 is 2MIC. recommended for the production of 6.25mg/mL..

【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S941.4;S948

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