CN103409495B - 一种腐生葡萄球菌选择性培养基的制备方法 - Google Patents
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Abstract
本发明公开了一种腐生葡萄球菌选择性培养基的制备方法。其特征在于，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素8~12mg；山柑子萜酮0.5g；磷酸二氢钾0.2g；蒸馏水加至1000ml。最佳优化配方制备方法为：取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾；L-半胱氨酸及硫酸铜溶于百合水煮液中，混匀，定容，灭菌后分装。本发明与现有技术相比较，具有检出可靠、腐生葡萄球菌分离率高的特点。
Description
技术领域
本发明为申请日为2013年1月13日，申请号为2013100107381，名称为《一种腐生葡萄球菌选择性培养基及其制备方法》发明的分案。本发明涉及医学检验领域，具体涉及一种腐生葡萄球菌选择性培养基的制备方法。
背景技术
腐生葡萄球菌（Staph.saparophytics）为低毒力条件致病菌，感染后症状不典型，且呈现多重耐药现象，给临床诊断和治疗带来一定困难，高效可靠的检验方法已是必然需要。
所谓微生物培养是一种用人工方法使微生物生长繁殖的技术，而选择性选择性培养基是用来促进或抑制一定类型的生物体（如细胞或细菌等）而设计的选择性培养基，利用这种选择性培养基可以将所需要的微生物从混杂的微生物中分离出来。
目前临床上普遍使用的葡萄球菌选择性培养基为甘露醇高盐琼脂（Manitol Salt Agar），用于金黄色葡萄球菌的选择性分离培养。培养基成分为：蛋白胨、鸡蛋蛋白液、D-甘露醇、氯化钠、酚红、琼脂。金黄色葡萄球菌菌落为黄色，大肠杆菌和沙门氏菌菌落为无色。但是，该种培养基并不能选择性培养腐生葡萄球菌，其原因在于在目前的选择性培养基中金黄色葡萄球菌比腐生葡萄球菌生长更快，很快覆盖了培养基，腐生葡萄球菌无法生长。
发明内容
本发明的技术任务是针对以上现有技术的不足，提供一种对腐生葡萄球菌分离率高的选择性培养基。
本发明解决其技术问题的技术方案是：一种腐生葡萄球菌选择性培养基，其特征在，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素8~12mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；硫酸铜5mg；L-半胱氨酸0.1g；浓度200g/L的百合水煮液200~300m；蒸馏水加至1000ml。
一种腐生葡萄球菌选择性培养基的制备方法，其特征在于包括以下步骤：
（1）取百合加水煮沸1小时，过滤，取滤液，调整体积到浓度为200g/L，得百合水煮液；
（2）取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾；L-半胱氨酸及硫酸铜溶于步骤1所得百合水煮液中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
其中所述的麦芽浸粉、鸡蛋蛋白液提供碳氮源和能量；枸橼酸镁和磷酸二氢钾为缓冲剂，并可以抑制杂菌生长；L-半胱氨酸属氨基酸类，为营养剂；琼脂是选择性培养基的凝固剂；假细锥甲素对于金葡菌具有特异的抑菌作用，对于腐生葡萄球菌无效；鸡蛋蛋白液含有各种多肽、脂肪、生长因子、激素等，这些物质对促进腐生葡萄球菌的繁殖和生长都是很重要的因素，此外鸡蛋蛋白液还具有维持选择性培养基pH 值恒定的作用。山柑子萜酮为腐生葡萄球菌特异性营养剂，可以特异性的促进产色葡萄球菌繁殖。铜离子属于重金属离子，在5mg/L这个浓度剂量下，沉积于金葡菌内部，导致胞壁消失或变薄，核糖体聚集成团块，最终导致细菌变形及破裂，呈现重金属源性损伤，而腐生葡萄球菌对重金属的运输能力和渗透能力低于金葡菌，也即重金属的抗性较高，故而加入硫酸铜可以有效地抑制金葡菌生长。百合为百合科植物卷丹Lilium lancifolium Thunb.、百合Lilium brownii F.E.Brown var. viridulum Baker 或细叶百合Lilium pumilum DC. 的干燥肉质鳞叶，含有蛋白质、脂肪、淀粉、还元糖、维生素B1、B2、泛酸、维生素C、β-胡萝卜素等多种成分，可以提供能量代谢、细胞发育、新陈代谢因子，有助于促进腐生葡萄球菌生长，尤其是重金属环境下菌落的生长。
本发明与现有技术相比较，具有检出可靠、腐生葡萄球菌分离率高的特点。
具体实施方式
以下结合实际情况，对本发明的具体实施方式作详细说明。
实施例1，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素8mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；蒸馏水加至1000ml。制备方法：取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾溶于蒸馏水中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
实施例2，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素12mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；硫酸铜5mg；蒸馏水加至1000ml。制备方法：取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾；硫酸铜溶于蒸馏水中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
实施例3，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素10mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；L-半胱氨酸0.1g；蒸馏水加至1000ml。制备方法：取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾；L-半胱氨酸溶于蒸馏水中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
实施例4，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素9mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；浓度200g/L的百合水煮液200ml；蒸馏水加至1000ml。制备方法：
（1）取百合加水煮沸1小时，过滤，取滤液，调整体积到浓度为200g/L，得百合水煮液；
（2）取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾溶于步骤1所得百合水煮液中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
实施例5，配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素11mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；硫酸铜5mg；L-半胱氨酸0.1g；浓度200g/L的百合水煮液300ml；蒸馏水加至1000ml。制备方法为：
（1）取百合加水煮沸1小时，过滤，取滤液，调整体积到浓度为200g/L，得百合水煮液；
（2）取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾；L-半胱氨酸及硫酸铜溶于步骤1所得百合水煮液中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
所述的百合水煮液浓度为200g/L，指的是每升百合水煮液中含有生药200g。
本发明所得腐生葡萄球菌选择性培养基具有检出可靠、腐生葡萄球菌分离率高的特点，为临床资料充分证明，有关资料如下。
1 对象与方法。
1.1 选择性培养基制备：共设3组，分别为对照组、实施例1方案组、实施例5方案组。对照组为葡萄球菌选择性琼脂110（CHAPMAN琼脂），配方为：胰酪蛋白胨、酵母浸粉、明胶、D-甘露醇、乳糖、氯化钠、磷酸氢二钾、琼脂。实施例1方案组、实施例5方案组的配方和制备方法见实施例。
1.2方法：将80份经由PCR确定腐生葡萄球菌阳性的标本中混入金色葡萄球菌，接种于培养基上，在 37℃下孵育 24 小时。
1.3统计学分析：用SPSS 13.0进行统计分析，P< 0.05表示有显著性意义。
2 结果：在80份接种标本中，细菌培养分离并鉴定为腐生葡萄球菌的，实施例1方案组为72例，实施例5方案组为76例。对照组CHAPMAN琼脂培养基菌落经鉴定最后被证实均为金葡菌，经统计学处理，本发明各实施例组与对照组比较具有明显差异（P<0.001）。结果表明，本发明实施例腐生葡萄球菌分离率明显高于现有的选择性培养基。
Claims (1)
1.一种腐生葡萄球菌选择性培养基的制备方法，其特征在于：配制1000ml选择性培养基的配方中含有：麦芽浸粉15g；鸡蛋蛋白液150ml；氯化钠5g；枸橼酸镁0.5g；琼脂15g；假细锥甲素11mg；山柑子萜酮 0.5 g；磷酸二氢钾0.2g；硫酸铜5mg；L-半胱氨酸0.1g；浓度200g/L的百合水煮液300ml；蒸馏水加至1000ml；包括以下步骤：
（1）取百合加水煮沸1小时，过滤，取滤液，调整体积到浓度为200g/L，得百合水煮液；
（2）取麦芽浸粉；鸡蛋蛋白液；氯化钠；枸橼酸镁；琼脂；假细锥甲素；山柑子萜酮；磷酸二氢钾；L-半胱氨酸及硫酸铜溶于步骤（1）所得百合水煮液中，混匀过滤后，蒸馏水定容，低温灭菌2次，每次45 min，灭菌后分装。
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