3种致病性弧菌选择性鉴别培养基的研制
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摘要
水产品是人类赖以生存的基础物质和重要蛋白来源,深得消费者的喜爱,消费量也在日益提高。致病性弧菌是水产品中引发人体疾病的主要致病菌,会引发人的季节性传播流行性的肠道疾病,导致人类发生严重的腹泻等消化道疾病或创伤感染病变、创伤感染或发生败血症,甚至死亡。研究和建立致病性弧菌快速检测的新技术和新方法受到各国研究机构的重视。用选择性鉴别培养基即可满足抽检率,更真实的反应水产品的污染状况,又能克服标准方法的费时、繁琐和成本较高等许多不足。
本论文主要围绕以下几个方面开展了一些工作：
1霍利斯弧菌选择性鉴别培养基的研制
基于霍利斯弧菌的基础营养和特殊生态环境需求,优化了其增菌培养基及培养条件。通过单因素试验结合响应面组合试验得到如下结果：培养基组成成分为混合蛋白胨2.11%(胰蛋白胨：鱼蛋白胨为1：1)、酵母浸膏0.210%、NaCl2.13%、pH7.5；培养温度为36土1℃。经过8h增菌后,该培养基的菌液浓度是GB/T4789.7-2008中规定方法的碱性蛋白胨水(APW)增菌菌液浓度的1.39倍,增菌效果明显。
以霍利斯弧菌特有的酶系统及其代谢特点和对某些特定抑菌成分的抵抗力为基础,设计研制了霍利斯弧菌选择性鉴别培养基(VhDM)。试验中对VhDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：VhDM对霍利斯弧菌具有较好的选择性和特异性；用VhDM在36±1℃培养16h-24h,霍利斯弧菌菌落为黄色；其它各种弧菌及非弧菌属致病菌菌落呈红色或未形成有效的可见菌落；VhDM对霍利斯弧菌的出菌效率与嗜盐琼脂相比为(90.65±1.72)%；检出限为101cfu/mL:因此,VhDM对海产品中霍利斯弧菌分离鉴别与检测是可行的,且具有操作简便、经济、结果易于观察等优点。
2河弧菌选择性鉴别培养基的研制
基于河弧菌的基础营养和特殊生态环境需求,优化了其增菌培养基及培养条件。通过单因素试验结合响应面组合试验得到如下结果：培养基组成成分为混合蛋白胨2.24%(胰蛋白胨：鱼蛋白胨为1：3)、酵母浸膏0.204%、NaCl1.81pH8.0；培养温度为36±1℃。经过8h增菌后,该培养基的菌液浓度是GB/T4789.7-2008中规定方法的碱性蛋白胨水(APW)增菌菌液浓度的1.59倍,增菌效果明显。
以河弧菌特有的酶系统及其代谢特点和对某些特定抑菌成分的抵抗力为基础,设计研制了河弧菌选择性鉴别培养基(VfDM)。试验中对VfDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：VfDM对河弧菌具有较好的选择性和特异性；用VfDM在36±1℃培养18h-24h,河弧菌菌落为深褐色；其它各种弧菌及非弧菌属致病菌菌落呈红色或未形成有效的可见菌落；VfDM对河弧菌的出菌效率与嗜盐琼脂相比为(92.80±2.88)%；检出限为101cfu/mL;因此,VfDM对海产品中河弧菌分离鉴别与检测是可行的,且具有操作简便、经济、结果易于观察等优点。
3弗尼斯弧菌选择性鉴别培养基的研制
基于弗尼斯弧菌的基础营养和特殊生态环境需求,优化了其增菌培养基及培养条件。通过单因素试验结合响应面组合试验得到如下结果：培养基组成成分为混合蛋白胨2.08%(胰蛋白胨：鱼蛋白胨为1：3)、酵母浸膏0.221%、NaCl1.94%、pH8.5；培养温度为36±1℃。经过8h增菌后,该培养基的菌液浓度是GB/T4789.7-2008中规定方法的碱性蛋白胨水(APW)增菌菌液浓度的1.48倍,增菌效果明显。
以弗尼斯弧菌特有的酶系统及其代谢特点和对某些特定抑菌成分的抵抗力为基础,设计研制了弗尼斯弧菌选择性鉴别培养基(VfuDM)。试验中对VfuDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：VfuDM对弗尼斯弧菌具有较好的选择性和特异性；用VhDM在36±1℃培养18h-24h,弗尼斯弧菌菌落为黄色；其它各种弧菌及非弧菌属致病菌菌落呈蓝色或未形成有效的可见菌落；VfuDM对弗尼斯弧菌的出菌效率与嗜盐琼脂相比为(92.98±2.58)%;检出限为101cfu/mL;因此, VfuDM对海产品中弗尼斯弧菌分离鉴别与检测是可行的,且具有操作简便、经济、结果易于观察等优点。
通过增菌培养基与选择性鉴别培养基结合检测3种致病性弧菌的方法,与常规方法(TCBS结合法国梅里埃细菌鉴定系统API20E试剂条或弧菌科生化鉴定管)相比,提高了目的菌的检出率,检测结果直观,易于观察,灵敏度高、特异性强,且降低检测成本,操作简便,减少工作量,具有更高的检测效率和鉴别能力,为构建同时快速筛检12种致病性弧菌的增菌和选择性鉴别培养基系统做了部分工作。
Aquatic products are the survival foundation material and important protein source of human. It is loved by the consumers with a continuous increase of consumption. Pathogenic Vibrio in the aquatic products was the main pathogenic bacteria which causes human diseases. It can cause the intestinal diseases which spread seasonally and epidemically, such as severe diarrhea, wound infection and even septicemia. Research and setting up new techniques and methods of rapid detection of Pathogenic Vibrio were taken seriously by research institutions of various countries. The selective differential medium can not only meet the need of the rate of inspection, but also reflect the contamination status of the aquatic products. Moreover, it can overcome the weak points of the standard method, which was time-consuming, cockamamie and high-cost.
The researches carried out in this paper would be described as follows:
1. Study on Vibrio hollisae Differential Medium(VhDM)
To save enrichment culture time and enhance the detection efficiency, the enrichment mediums of Vibrio hollisae(Vh) were optimized based on nutrient requirements and living environment demands. The results showed as follow:Vh enrichment medium compositions for mixture peptone2.11%(tryptone:fish peptone=l:l), yeast extract0.210%, NaCl2.13%, pH7.5; temperature of culturing was36±1℃. After8hours culturing, the bacteria liquid concentration of Vh enrichment medium was1.39times than that of Vh cultured on APW (GB/T4789.7-2008) under the same condition, it was obvious.
Based on the specific enzyme systems and the corresponding metabolisms of Vh, according to the resistibility to different antibacterial ingredients, VhDM was designed for the rapid separation, identification or detection of Vh. The tests of color effects, plating efficiency, sensitivity and specificity were done to evaluate the efficiency of VhDM. The results showed that:VhDM has good selectivity and specificity for Vh. After incubation for16-24h at36±1℃, The Vh formed yellow colonies on VhDM. The other strains did not form colonies or formed red colonies.VhDM showed a mean plating efficiency of Vh cells of (90.65±1.72)%and its detection limit was101cfu/mL. Accordingly, VhDM is suitable for kinds of inspection agencies to detect Vh, which is simple operation, lower cost, convenient for observation and so on.
2. Study on Vibrio flurialis Differential Medium(VfDM)
To save enrichment culture time and enhance the detection efficiency, the enrichment mediums of Vibrio flurialis(Vf) were optimized based on nutrient requirements and living environment demands. The results showed as follow:Vf enrichment medium compositions for mixture peptone2.24%(tryptone:fish peptone=l:3), yeast extract0.204%, NaCl1.81%, pH8.0; temperature of culturing was36±1℃. After8hours culturing, the bacteria liquid concentration of Vf enrichment medium was1.59times than that of Vfcultured on APW (GB/T4789.7-2008) under the same condition, it was obvious.
Based on the specific enzyme systems and the corresponding metabolisms of Vf according to the resistibility to different antibacterial ingredients, VfDM was designed for the rapid separation, identification or detection of Vf. The tests of color effects, plating efficiency, sensitivity and specificity were done to evaluate the efficiency of VfDM. The results showed that:VhDM has good selectivity and specificity for Vf. After incubation for18-24h at36±1℃, The Vf formed yellow colonies on VfDM. The other strains did not form colonies or formed red colonies.VfDM showed a mean plating efficiency of Vf cells of (92.80±2.88)%and its detection limit was101cfu/mL. Accordingly, VfDM is suitable for kinds of inspection agencies to detect Vf, which is simple operation, lower cost, convenient for observation and so on.
3. Study on Vibrio furnissii Differential Medium(VfuDM)
To save enrichment culture time and enhance the detection efficiency, the enrichment mediums of Vibrio furnissii(Vfu) were optimized based on nutrient requirements and living environment demands. The results showed as follow:Vfu enrichment medium compositions for mixture peptone2.08%(tryptone:fish peptone=l:3), yeast extract0.211%, NaCl1.94%, pH8.5; temperature of culturing was36±1℃. After8hours culturing, the bacteria liquid concentration of Vfu enrichment medium was1.48times than that of Vh cultured on APW (GB/T4789.7-2008) under the same condition, it was obvious.
Based on the specific enzyme systems and the corresponding metabolisms of Vfu, according to the resistibility to different antibacterial ingredients, VfuDM was designed for the rapid separation, identification or detection of Vfu. The tests of color effects, plating efficiency, sensitivity and specificity were done to evaluate the efficiency of VfuDM. The results showed that:VfuDM has good selectivity and specificity for Vfu. After incubation for16-24h at36±1℃, The Vfu formed yellow colonies on VfuDM. The other strains did not form colonies or formed red colonies.VfuDM showed a mean plating efficiency of Vfu cells of (90.65±1.72)%and its detection limit was101cfu/mL. Accordingly, VhDM is suitable for kinds of inspection agencies to detect Vfu, which is simple operation, lower cost, convenient for observation and so on.
We have successfully established an isolation and identification method for Vibrio hollisae, Vibrio flurialis, and Vibrio furnissii. As compared with the conventional methods (TCBS combined with the API20E system or biochemistry assessor of Vibrionaceae), vibrio enrichment mediums combined with three Selective Differential medias respectively, used for isolation and detection of Pathogenic Vibrio, possess the advantages of simple operation, economic, convenient for observation, high sensitivity and specificity, which could lay the foundation for the further study.
本论文主要围绕以下几个方面开展了一些工作：
1霍利斯弧菌选择性鉴别培养基的研制
基于霍利斯弧菌的基础营养和特殊生态环境需求,优化了其增菌培养基及培养条件。通过单因素试验结合响应面组合试验得到如下结果：培养基组成成分为混合蛋白胨2.11%(胰蛋白胨：鱼蛋白胨为1：1)、酵母浸膏0.210%、NaCl2.13%、pH7.5；培养温度为36土1℃。经过8h增菌后,该培养基的菌液浓度是GB/T4789.7-2008中规定方法的碱性蛋白胨水(APW)增菌菌液浓度的1.39倍,增菌效果明显。
以霍利斯弧菌特有的酶系统及其代谢特点和对某些特定抑菌成分的抵抗力为基础,设计研制了霍利斯弧菌选择性鉴别培养基(VhDM)。试验中对VhDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：VhDM对霍利斯弧菌具有较好的选择性和特异性；用VhDM在36±1℃培养16h-24h,霍利斯弧菌菌落为黄色；其它各种弧菌及非弧菌属致病菌菌落呈红色或未形成有效的可见菌落；VhDM对霍利斯弧菌的出菌效率与嗜盐琼脂相比为(90.65±1.72)%；检出限为101cfu/mL:因此,VhDM对海产品中霍利斯弧菌分离鉴别与检测是可行的,且具有操作简便、经济、结果易于观察等优点。
2河弧菌选择性鉴别培养基的研制
基于河弧菌的基础营养和特殊生态环境需求,优化了其增菌培养基及培养条件。通过单因素试验结合响应面组合试验得到如下结果：培养基组成成分为混合蛋白胨2.24%(胰蛋白胨：鱼蛋白胨为1：3)、酵母浸膏0.204%、NaCl1.81pH8.0；培养温度为36±1℃。经过8h增菌后,该培养基的菌液浓度是GB/T4789.7-2008中规定方法的碱性蛋白胨水(APW)增菌菌液浓度的1.59倍,增菌效果明显。
以河弧菌特有的酶系统及其代谢特点和对某些特定抑菌成分的抵抗力为基础,设计研制了河弧菌选择性鉴别培养基(VfDM)。试验中对VfDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：VfDM对河弧菌具有较好的选择性和特异性；用VfDM在36±1℃培养18h-24h,河弧菌菌落为深褐色；其它各种弧菌及非弧菌属致病菌菌落呈红色或未形成有效的可见菌落；VfDM对河弧菌的出菌效率与嗜盐琼脂相比为(92.80±2.88)%；检出限为101cfu/mL;因此,VfDM对海产品中河弧菌分离鉴别与检测是可行的,且具有操作简便、经济、结果易于观察等优点。
3弗尼斯弧菌选择性鉴别培养基的研制
基于弗尼斯弧菌的基础营养和特殊生态环境需求,优化了其增菌培养基及培养条件。通过单因素试验结合响应面组合试验得到如下结果：培养基组成成分为混合蛋白胨2.08%(胰蛋白胨：鱼蛋白胨为1：3)、酵母浸膏0.221%、NaCl1.94%、pH8.5；培养温度为36±1℃。经过8h增菌后,该培养基的菌液浓度是GB/T4789.7-2008中规定方法的碱性蛋白胨水(APW)增菌菌液浓度的1.48倍,增菌效果明显。
以弗尼斯弧菌特有的酶系统及其代谢特点和对某些特定抑菌成分的抵抗力为基础,设计研制了弗尼斯弧菌选择性鉴别培养基(VfuDM)。试验中对VfuDM的显色效果、平板效率、灵敏度和特异性进行评价。结果显示：VfuDM对弗尼斯弧菌具有较好的选择性和特异性；用VhDM在36±1℃培养18h-24h,弗尼斯弧菌菌落为黄色；其它各种弧菌及非弧菌属致病菌菌落呈蓝色或未形成有效的可见菌落；VfuDM对弗尼斯弧菌的出菌效率与嗜盐琼脂相比为(92.98±2.58)%;检出限为101cfu/mL;因此, VfuDM对海产品中弗尼斯弧菌分离鉴别与检测是可行的,且具有操作简便、经济、结果易于观察等优点。
通过增菌培养基与选择性鉴别培养基结合检测3种致病性弧菌的方法,与常规方法(TCBS结合法国梅里埃细菌鉴定系统API20E试剂条或弧菌科生化鉴定管)相比,提高了目的菌的检出率,检测结果直观,易于观察,灵敏度高、特异性强,且降低检测成本,操作简便,减少工作量,具有更高的检测效率和鉴别能力,为构建同时快速筛检12种致病性弧菌的增菌和选择性鉴别培养基系统做了部分工作。
Aquatic products are the survival foundation material and important protein source of human. It is loved by the consumers with a continuous increase of consumption. Pathogenic Vibrio in the aquatic products was the main pathogenic bacteria which causes human diseases. It can cause the intestinal diseases which spread seasonally and epidemically, such as severe diarrhea, wound infection and even septicemia. Research and setting up new techniques and methods of rapid detection of Pathogenic Vibrio were taken seriously by research institutions of various countries. The selective differential medium can not only meet the need of the rate of inspection, but also reflect the contamination status of the aquatic products. Moreover, it can overcome the weak points of the standard method, which was time-consuming, cockamamie and high-cost.
The researches carried out in this paper would be described as follows:
1. Study on Vibrio hollisae Differential Medium(VhDM)
To save enrichment culture time and enhance the detection efficiency, the enrichment mediums of Vibrio hollisae(Vh) were optimized based on nutrient requirements and living environment demands. The results showed as follow:Vh enrichment medium compositions for mixture peptone2.11%(tryptone:fish peptone=l:l), yeast extract0.210%, NaCl2.13%, pH7.5; temperature of culturing was36±1℃. After8hours culturing, the bacteria liquid concentration of Vh enrichment medium was1.39times than that of Vh cultured on APW (GB/T4789.7-2008) under the same condition, it was obvious.
Based on the specific enzyme systems and the corresponding metabolisms of Vh, according to the resistibility to different antibacterial ingredients, VhDM was designed for the rapid separation, identification or detection of Vh. The tests of color effects, plating efficiency, sensitivity and specificity were done to evaluate the efficiency of VhDM. The results showed that:VhDM has good selectivity and specificity for Vh. After incubation for16-24h at36±1℃, The Vh formed yellow colonies on VhDM. The other strains did not form colonies or formed red colonies.VhDM showed a mean plating efficiency of Vh cells of (90.65±1.72)%and its detection limit was101cfu/mL. Accordingly, VhDM is suitable for kinds of inspection agencies to detect Vh, which is simple operation, lower cost, convenient for observation and so on.
2. Study on Vibrio flurialis Differential Medium(VfDM)
To save enrichment culture time and enhance the detection efficiency, the enrichment mediums of Vibrio flurialis(Vf) were optimized based on nutrient requirements and living environment demands. The results showed as follow:Vf enrichment medium compositions for mixture peptone2.24%(tryptone:fish peptone=l:3), yeast extract0.204%, NaCl1.81%, pH8.0; temperature of culturing was36±1℃. After8hours culturing, the bacteria liquid concentration of Vf enrichment medium was1.59times than that of Vfcultured on APW (GB/T4789.7-2008) under the same condition, it was obvious.
Based on the specific enzyme systems and the corresponding metabolisms of Vf according to the resistibility to different antibacterial ingredients, VfDM was designed for the rapid separation, identification or detection of Vf. The tests of color effects, plating efficiency, sensitivity and specificity were done to evaluate the efficiency of VfDM. The results showed that:VhDM has good selectivity and specificity for Vf. After incubation for18-24h at36±1℃, The Vf formed yellow colonies on VfDM. The other strains did not form colonies or formed red colonies.VfDM showed a mean plating efficiency of Vf cells of (92.80±2.88)%and its detection limit was101cfu/mL. Accordingly, VfDM is suitable for kinds of inspection agencies to detect Vf, which is simple operation, lower cost, convenient for observation and so on.
3. Study on Vibrio furnissii Differential Medium(VfuDM)
To save enrichment culture time and enhance the detection efficiency, the enrichment mediums of Vibrio furnissii(Vfu) were optimized based on nutrient requirements and living environment demands. The results showed as follow:Vfu enrichment medium compositions for mixture peptone2.08%(tryptone:fish peptone=l:3), yeast extract0.211%, NaCl1.94%, pH8.5; temperature of culturing was36±1℃. After8hours culturing, the bacteria liquid concentration of Vfu enrichment medium was1.48times than that of Vh cultured on APW (GB/T4789.7-2008) under the same condition, it was obvious.
Based on the specific enzyme systems and the corresponding metabolisms of Vfu, according to the resistibility to different antibacterial ingredients, VfuDM was designed for the rapid separation, identification or detection of Vfu. The tests of color effects, plating efficiency, sensitivity and specificity were done to evaluate the efficiency of VfuDM. The results showed that:VfuDM has good selectivity and specificity for Vfu. After incubation for16-24h at36±1℃, The Vfu formed yellow colonies on VfuDM. The other strains did not form colonies or formed red colonies.VfuDM showed a mean plating efficiency of Vfu cells of (90.65±1.72)%and its detection limit was101cfu/mL. Accordingly, VhDM is suitable for kinds of inspection agencies to detect Vfu, which is simple operation, lower cost, convenient for observation and so on.
We have successfully established an isolation and identification method for Vibrio hollisae, Vibrio flurialis, and Vibrio furnissii. As compared with the conventional methods (TCBS combined with the API20E system or biochemistry assessor of Vibrionaceae), vibrio enrichment mediums combined with three Selective Differential medias respectively, used for isolation and detection of Pathogenic Vibrio, possess the advantages of simple operation, economic, convenient for observation, high sensitivity and specificity, which could lay the foundation for the further study.
引文
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