CN110241171A - 一种不含动植物源性的伊红美蓝纯化学成分琼脂培养基及配制方法 - Google Patents
一种不含动植物源性的伊红美蓝纯化学成分琼脂培养基及配制方法 Download PDFInfo
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Abstract
本发明专利公开一种伊红美蓝无动植物源性纯化学成分的琼脂培养基及配制方法,其目的是提高实验结果的准确性,消除现市售伊红美蓝培养基中因蛋白胨营养成分不明确对菌落生长带来的干扰,提高结果重现率。用营养成分明确的多种氨基酸和无机盐代替现市售该种培养基中的蛋白胨成分。主要用于革兰氏阴性肠杆菌的分离与鉴定,特别是大肠杆菌的分离与鉴定。
Description
技术领域
本发明涉及专利公开一种伊红美蓝无动植物源性纯化学成分的琼脂培养基及配制方法,旨在提供一种无动植物源性,低成本,可用于医药、食品、医院等不同领域革兰氏阴性肠杆菌的分离与鉴定,特别是大肠杆菌的分离与鉴定。
背景技术
伊红美蓝培养基(eosin-methylene blue medium,简称EMB medium),一般用于检测大肠杆菌。伊红属于酸性染料,美蓝属于碱性染料。伊红美蓝琼脂培养基又名曙红亚甲蓝琼脂培养基。当大肠杆菌分解乳糖产酸时细菌带正电荷被染成红色,再与美蓝结合形成紫黑色菌落,并带有金属光泽。在碱性环境中不分解乳糖产酸的细菌不着色,伊红和美蓝不能结合,故沙门氏菌等为无色或琥珀色半透明菌落。
培养基根据原料的来源可分为合成培养基、天然培养基和半合成培养基。天然培养基和半合成培养基中包括一些动植物源性成分如蛋白胨、牛肉膏等,是目前食品微生物检验常用到的两种成分,为微生物的生长提供碳源、氮源、维生素、生长因子等。但是动植物源性成分制作过程复杂,且存在引入外源性病毒的安全隐患,可变因素多,质量难以控制,进而影响实验结果的准确性。而合成培养基是用已知的纯化学试剂配制的培养基,这种培养基成分精确,重复性强,质量可控。现如今市售伊红美蓝培养基中均含有复杂成分蛋白胨,该培养基中因蛋白胨营养成分不明确,对菌落生长带来许多干扰,并且难以人为控制,因此研发无动植物源性纯化学成分的该种培养基迫在眉睫,且可以为参考培养基体系的建立提供数据及实物支撑。
发明内容
本发明公开一种伊红美蓝无动植物源性纯化学成分的琼脂培养基及制备方法。通过解析蛋白胨中氨基酸的种类和含量,配制模拟蛋白胨营养成分的纯化学合成物质,替代培养基中动物源性成分—蛋白胨。
本发明所述的一种伊红美蓝无动植物源性纯化学成分的琼脂培养基,只含纯化学物质,包括多种氨基酸、无机盐、糖类、染色剂;其中用于替代蛋白胨的氨基酸组分和无机盐组分如表1:
在一定浓度范围内甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸、天冬氨酸、谷氨酸对大肠埃希杆菌的生长具有促进作用,丝氨酸、苏氨酸、精氨酸、赖氨酸、组氨酸、天冬氨酸、谷氨酸、半胱氨酸、甲硫氨酸、甘氨酸在高浓度时会对大肠杆菌的生长有抑制作用。采用如下对比试验分别培养大肠埃希杆菌ATCC8739:配方1称取上述表中氨基酸组分8.08g用于代替蛋白胨;配方2称取氨基酸组分16.16g,无机盐组分3.84g用于代替蛋白胨;配方3称取氨基酸组分8.08g,无机盐组分1.92g用于代替蛋白胨。其中大肠埃希杆菌ATCC8739在配方3培养基中的金属光泽最明显,菌落形态一致,说明本申请所述的氨基酸组分与无机盐组分对大肠埃希氏杆菌的生长缺一不可,且浓度适当效果更优。
进一步的,用于替代蛋白胨的氨基酸组分和无机盐组分如表2:
本发明所述的伊红美蓝无动植物源性纯化学成分的琼脂培养基的制备方法:先将准确称量的20种氨基酸在涡旋仪上混合30分钟以上,再加入5种无机盐,总量为100g,再次在涡旋仪上混匀30分钟以上,以确保充分混匀,制备成蛋白胨替代物;按照伊红美蓝培养基配方称取混匀后的蛋白胨替代物10g,乳糖10g,磷酸氢二钾2.0g,伊红0.4g,美蓝0.065g,琼脂15g溶于1000mL蒸馏水中,加热煮沸至完全溶解,调节pH至7.1±0.2,121℃高压灭菌15min,冷却至55℃倾注平板备用。
伊红美蓝无动植物源性纯化学成分的琼脂培养基的应用,所述步骤如下:
(1)平板的制备与保存:根据GB4789.28-2013选择性分离和计数固体培养基的测试方法中平板的制备与保存要求,倾注融化的培养基到平皿中,形成一个至少3mm厚的琼脂层,倾注后将平板放置于水平平面,使琼脂冷却凝固。凝固后的培养基应立即使用或存放在暗处或2℃~8℃冰箱的密封袋中,在有效期内保存。使用前应对琼脂表面进行干燥。
(2)工作菌悬液的制备:将大肠埃希氏杆菌ATCC 25922、ATCC8739、鼠伤寒沙门氏菌ATCC14028、金黄色葡萄球菌ATCC6538、ATCC25923、粪肠球菌ATCC29212、肺炎克雷伯菌CICC 46117、肺炎克雷伯CICC 24165、痢疾志贺氏菌CMCC51105、福氏志贺氏菌CMCC51571、蜡样芽孢杆菌ATCC11778、产气肠杆菌ATCC13048、奇异变形杆菌 CMCC49005、铜绿假单胞菌ATCC27853、白色念珠菌CMCC98001接种到脑心浸出液肉汤或胰酪大豆胨琼脂平板培养过夜,将过夜菌稀释至20~200CFU/100uL。
(3)接种:取稀释好的菌悬液100uL,均匀涂布接种于待测平板和参比平板,平行接种2个平板,36℃培养18~24h ;取一环过夜培养的菌在平板上划线,36℃培养18~24h。
(4)计算:对2个平板进行计数后,计算其平均值按下列公式进行计算生长率:
PR=NS/NO
PR---生长率
NS---待测培养基平板上获得的菌落总数
NO---参比培养基平板上获得的菌落总数(菌落数≥100CFU)
参比培养基为TSA
(5)结果解释:目标菌PR值应不小于0.7,非目标菌应不大于0.5
平板划线菌落特征应符合要求。
目前,食品微生物纯化学培养基在全球范围内整体较为匮乏,本发明所述培养基组分以及制备方法为我国食品微生物检验培养基相关参考体系的建立提供实验数据;消除现市售伊红美蓝培养基中因蛋白胨营养成分不明确对菌落生长带来的干扰,提高结果重现率;提升整体检验水平,为我国培养基生产与检定提供稳定的自主标尺。
具体实施方式
下面结合表格对发明进行详细说明
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明公开一种伊红美蓝无动植物源性纯化学成分的琼脂培养基及配制方法。称取氨基酸组分和无机盐组分10g,乳糖10g,2.0g磷酸氢二钾,0.4g伊红,0.065g美蓝,15g琼脂溶于1000mL蒸馏水中,加热煮沸至完全溶解,调节pH至7.1±0.2,121℃高压灭菌15min,冷却至55℃倾注平板备用。
1、材料与方法
1.1菌株
大肠埃希氏杆菌ATCC 25922、ATCC8739、鼠伤寒沙门氏菌ATCC14028、金黄色葡萄球菌ATCC6538、ATCC25923、粪肠球菌ATCC29212、肺炎克雷伯菌CICC 46117、肺炎克雷伯CICC24165、痢疾志贺氏菌CMCC51105、福氏志贺氏菌CMCC51571、蜡样芽孢杆菌ATCC11778、产气肠杆菌ATCC13048、奇异变形杆菌 CMCC49005、铜绿假单胞菌 ATCC27853、白色念珠菌CMCC98001
1.2工作菌悬液的制备
将大肠埃希氏杆菌ATCC 25922、ATCC8739、鼠伤寒沙门氏菌ATCC14028、金黄色葡萄球菌ATCC6538、ATCC25923、粪肠球菌ATCC29212、肺炎克雷伯菌CICC 46117、肺炎克雷伯CICC24165、痢疾志贺氏菌CMCC51105、福氏志贺氏菌CMCC51571、蜡样芽孢杆菌ATCC11778、产气肠杆菌ATCC13048、奇异变形杆菌 CMCC49005、铜绿假单胞菌 ATCC27853、白色念珠菌CMCC98001接种到脑心浸出液肉汤或胰酪大豆胨琼脂平板培养过夜。
1.3接种
将过夜菌稀释至20~200CFU/100uL。取稀释好的菌悬液100uL,均匀涂布接种于待测平板和参比平板,平行接种2个平板,36℃培养18~24h ;取一环过夜培养的菌在平板上划线,36℃培养18~24h。
2、实验结果
2.1菌落形态
2.2生长率测定结果
2.3鼠伤寒沙门氏菌和金黄色葡萄球菌特异性和选择性结果
3、参考国外EMB培养基验证用菌种试验结果
各菌种在纯化学合成EMB、市售EMB平板上均生长良好,菌落特征基本一致,有些革兰氏阳性菌在本发明培养基上不生长而在市售培养基上生长,说明其抑制作用优于某些现市售EMB。
伊红美蓝无动植物源性纯化学成分的琼脂培养基验证符合食品安全国家标准-食品微生物学检验-培养基和试剂(GB4789.28-2013)的质量要求,且参考国外此培养基的厂家检测菌种,其在无动植物源性纯化学成分的琼脂培养基的菌落形态与厂家描述结果基本一致,甚至有些优于市售培养基。
Claims (6)
1.一种伊红美蓝无动植物源性纯化学成分的琼脂培养基,其特征在于,所述琼脂培养基只含纯化学物质,包括多种氨基酸、无机盐、糖类、染色剂;其中用于替代蛋白胨的氨基酸组分和无机盐组分如下:
氨基酸组分
名称 g/100g
L-丙氨酸 4.0~8.0
L-精氨酸 2.5~6.0
L-天冬氨酸 5.5~12.0
L-谷氨酸 16.0~33.0
甘氨酸 2.0~4.5
L-组氨酸 2.0~4.0
L-异亮氨酸 5.5~12.0
L-亮氨酸 7.5~15.0
L-赖氨酸水合物 6.5~14.0
L-蛋氨酸 1.5~4.0
L-苯基丙氨酸 5.5~11.0
L-脯氨酸 6.0~13.0
L-丝氨酸 2.0~4.5
L-苏氨酸 1.5~4.0
L-酪氨酸 1.0~3.0
L-缬氨酸 6.0~12.5
L-天冬酰胺(游离) 0.5~2.0
L-半胱氨酸(游离) 0.3~0.6
L-谷氨酰胺(游离) 0.3~0.6
L-色氨酸(游离) 0.5~1.5
无机盐组分
氯化钙 0.0~0.15
硫酸镁 0.0~0.4
磷酸二氢钾 0.0~15.0
氯化钠 0.0~9.0
尿素 0.0~15.0
氨基酸组合和无机盐组分的质量和为100g。
2.如权利要求1所述的一种伊红美蓝无动植物源性纯化学琼脂培养基,其特征在于,氨基酸组分及无机盐组分如下所示:
氨基酸组分
名称 g/100g
L-丙氨酸 4.2
L-精氨酸 2.9
L-天冬氨酸 5.9
L-谷氨酸 16.1
甘氨酸 2.2
L-组氨酸 2.0
L-异亮氨酸 5.8
L-亮氨酸 7.7
L-赖氨酸水合物 6.6
L-蛋氨酸 1.9
L-苯基丙氨酸 5.5
L-脯氨酸 6.2
L-丝氨酸 2.2
L-苏氨酸 1.9
L-酪氨酸 1.4
L-缬氨酸 6.1
L-天冬酰胺(游离) 0.9
L-半胱氨酸(游离) 0.3
L-谷氨酰胺(游离) 0.3
L-色氨酸(游离) 0.7
无机盐组分
氯化钙 0.1
硫酸镁 0.2
磷酸二氢钾 7.4
氯化钠 4.4
尿素 7.1
氨基酸组合和无机盐组分的质量和为100g。
3.一种伊红美蓝无动植物源性纯化学成分的琼脂培养基的配制方法,其特征在于,采用如权利要求1或2所述的琼脂培养基组分:先将准确称量的20种氨基酸在涡旋仪上混合30分钟以上,再加入5种无机盐,总量为100g,再次在涡旋仪上混匀30分钟以上,以确保充分混匀,制备成蛋白胨替代物;按照伊红美蓝培养基配方称取混匀后的蛋白胨替代物10g,乳糖10g,磷酸氢二钾2.0g,伊红0.4g,美蓝0.065g,琼脂15g溶于1000mL蒸馏水中,加热煮沸至完全溶解,调节pH至7.1±0.2,121℃高压灭菌15min,冷却至55℃倾注平板备用。
4.一种伊红美蓝无动植物源性纯化学成分的琼脂培养基的应用,其特征在于,包括工作菌悬液的制备:将大肠埃希氏杆菌ATCC 25922、ATCC8739、鼠伤寒沙门氏菌ATCC14028、金黄色葡萄球菌ATCC6538、ATCC25923、粪肠球菌ATCC29212、肺炎克雷伯菌CICC 46117、肺炎克雷伯CICC 24165、痢疾志贺氏菌CMCC51105、福氏志贺氏菌CMCC51571、蜡样芽孢杆菌ATCC11778、产气肠杆菌ATCC13048、奇异变形杆菌 CMCC49005、铜绿假单胞菌 ATCC27853、白色念珠菌CMCC98001接种到脑心浸出液肉汤或胰酪大豆胨琼脂平板培养过夜,将过夜菌稀释至20~200CFU/100uL;取稀释好的菌悬液100uL,均匀涂布接种于待测平板和参比平板,平行接种2个平板,36℃培养18~24h;取一环过夜培养的菌在平板上划线,36℃培养18~24h;所述待测平板为权利要求3所得到的平板。
5.如权利要求4所述的一种伊红美蓝无动植物源性纯化学成分的琼脂培养基的应用,结果计算:对2个平板进行计数后,计算其平均值按下列公式进行计算生长率:
PR=NS/NO
PR---生长率
NS---待测培养基平板上获得的菌落总数
NO---参比培养基平板上获得的菌落总数(菌落数≥100CFU)
参比培养基为TSA培养基。
6.如权利要求5所述的一种伊红美蓝无动植物源性纯化学成分的琼脂培养基的应用,结果解释:目标菌PR值应不小于0.7,非目标菌应不大于0.5;平板划线菌落特征应符合要求。
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