CN107843475A - 一种改良分支杆菌染色液及染色方法 - Google Patents
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Abstract
本发明公开了一种改良分支杆菌染色液,所述染色液由氧化液、初染液、脱色液和复染液组成;所述氧化液采用H2O2溶液或H5IO6溶液,所述初染液采用碱性品红溶液。本发明为非荧光染色,成本较低,操作简单,对人体无害,经氧化液氧化后进行染色,从而使碱性品红与抗酸杆菌结合更加牢固,染出的片子不但颜色鲜艳,而且可以长期保存。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种改良分支杆菌染色液及染色方法。
背景技术
近年来由于获得性免疫缺陷综合征(AIDS)的流行,结核分枝杆菌耐药菌株的出现,免疫抑制剂的应用,吸毒、贫困及人口流动等因素,结核病的流行在世界有卷土重来的趋势。结核病又成为了全球范围内重大的公共卫生问题,对病变组织进行抗酸染色,查找抗酸染色阳性结核分枝杆菌仍然是诊断结核病有效和准确的重要方法之一,但传统的Ziehi-Neelsen抗酸染色法在不同医院操作差异较大,阳性率较低,对诊断造成了一定的困难。十九世纪八十年代Ziehi与Neelsen共同发明了分枝杆菌抗酸染色法,在以后的一百多年来国内来诸多学者对该方法进行了诸多改良,其中以荧光染色法(即金胺“O”法)敏感性好,阳性率高,但由于:其一,荧光法需配备荧光显微镜,荧光显微镜价格较高,目前大多数医院及疾控尚未配备该设备;其二,荧光显微镜需使用高压汞灯(或氙灯)作为激发光源,使用时产生大量摄像对人体有一定损伤;其三,染片后需要及时观察,不能保存涂片,故一直未能很好的推广。
分枝杆菌是一种抗酸菌,抗酸菌是一类细胞壁中含有大量分枝菌酸等蜡质的特殊革兰氏阳性菌,一般不易着色,但是若经加温或延长染色时间,被碱性品红染色后就不易再被盐酸乙醇溶液脱色。传统的Ziehi-Neelsen抗酸染色法正是因为抗酸菌的蜡质屏障,即使加热或者延长时间仍然不能良好牢固着色,造成阳性率偏低。
发明内容
本发明针对上述技术问题,目的在于提供一种改良分支杆菌染色液及染色方法,为非荧光染色,成本较低,操作简单,对人体无害,经氧化液氧化后进行染色,从而使碱性品红与抗酸杆菌结合更加牢固,染出的片子不但颜色鲜艳,而且可以长期保存。
本发明通过下述技术方案实现:
一种改良分支杆菌染色液,所述染色液由氧化液、初染液、脱色液和复染液组成;所述氧化液采用H2O2溶液或H5IO6溶液,所述初染液采用碱性品红溶液。
优选地,所述H2O2溶液为:体积百分含量为3%~10%的H2O2水溶液。
优选地,所述H5IO6溶液为质量百分含量为5%~20%的H5IO6水溶液。
优选地,所述初染液的组成为:碱性品红、乙醇、蒸馏水和渗透剂,所述渗透剂采用高分子量嵌段共聚物溶液。
优选地,所述高分子量嵌段共聚物溶液包括DISPERBYK-2200或DISPERBYK-180。
优选地,所述初染液组成配比为:碱性品红2.0g~5.0g,乙醇200~400mL,蒸馏水600~800mL,DISPERBYK-2200或DISPERBYK-180 20~100mL。
优选地,所述脱色液采用3%~5%的盐酸乙醇溶液。
优选地,所述复染液采用次甲基蓝溶液,组成为:次甲基蓝3.0~6.0g、乙醇200~400mL、蒸馏水600~800mL、KOH 0.05~0.20g。
基于上述一种改良分支杆菌染色液的染色方法,包括以下步骤:
步骤1,涂片经火焰固定,切片脱蜡至水;
步骤2,滴加氧化液盖满玻片,氧化后倾去氧化液;
步骤3,滴加初染液盖满玻片,染色;
步骤4,水洗并沥干水分;
步骤5,滴加脱色液,轻轻摇动玻片,脱色,然后水洗;水洗后可见残余红色,再滴加脱色液,摇动玻片直至涂片无红色脱出或略呈粉红色为止,水洗;
步骤6,滴加复染液,复染,水洗;干后镜检。
优选地,所述步骤2中氧化时间为5~20min,所述步骤3中染色时间为5~15min。
本发明与现有技术相比,具有如下的优点和有益效果:
1、本发明一种改良分支杆菌染色液及染色方法,先使用氧化液将抗酸杆菌细胞壁中的分枝菌酸上的两相邻羟基氧化成醛基,然后碱性品红在渗透剂的作用下能与醛基发生反应,产生抗酸性的紫红色基团,从而使碱性品红与抗酸杆菌结合更加牢固。并且不会破坏抗酸菌的蜡质屏障,复染不能上色;
2、本发明一种改良分支杆菌染色液及染色方法,采用的H2O2溶液作为氧化液,H2O2溶液为常用药品,成本较低,配制简单,在本发明中具有较高的应用附加值;此外H2O2(过氧化氢)的相对于H5IO6(正高碘酸)的稳定性更好,不仅不会破坏抗酸菌的蜡质屏障,且使用较少量就可以达到良好的效果,产品更易商品化;
3、本发明一种改良分支杆菌染色液及染色方法,经氧化液氧化后,染出的片子不但颜色鲜艳,而且可以长期保存,现有染色液保存时间均为1周左右,而本发明提供的染色液能够保存1个月以上;
4、本发明一种改良分支杆菌染色液及染色方法,用带有颜料亲和基团的高分子量嵌段共聚物溶液替代苯酚作为渗透剂,解决苯酚的毒性、刺激性及挥发性对人体吸收造成的健康危害;
5、本发明一种改良分支杆菌染色液及染色方法,改良的渗透剂无毒、无味,并且是一种良好的湿润分散剂,能够增加碱性品红的溶解性和稳定性(抗絮凝、抗结晶析出);
6、本发明一种改良分支杆菌染色液及染色方法,同时该渗透剂还是一种良好的表面活性剂,能显著减低表面张力,无需加热即可帮助碱性品红穿透抗酸菌细胞壁的蜡质屏障。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
实施例1
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:3%H2O2溶液
②初染液:碱性品红5.0g
乙醇400mL
蒸馏水600mL
DISPERBYK-2200 20mL
③脱色液:5%盐酸乙醇(95%)溶液
④复染液:次甲基蓝6.0g
乙醇200mL
蒸馏水800mL
KOH 0.05g。
实施例2
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:5%H2O2溶液
②初染液:碱性品红3.0g
乙醇250mL
蒸馏水700mL
DISPERBYK-2200 50mL
③脱色液:3%盐酸乙醇(95%)溶液
④复染液:次甲基蓝4.0g
乙醇300mL
蒸馏水700mL
KOH 0.10g。
实施例3
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:5%H2O2溶液
②初染液:碱性品红3.0g
乙醇250mL
蒸馏水700mL
DISPERBYK-180 50mL
③脱色液:3%盐酸乙醇(95%)溶液
④复染液:次甲基蓝4.0g
乙醇300mL
蒸馏水700mL
KOH 0.10g。
实施例4
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:8%H2O2溶液
②初染液:碱性品红5.0g
乙醇300mL
蒸馏水650mL
DISPERBYK-180 50mL
③脱色液:5%盐酸乙醇(95%)溶液
④复染液:次甲基蓝5.0g
乙醇300mL
蒸馏水700mL
KOH 0.12g。
实施例5
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:10%H2O2溶液
②出染液:碱性品红2.0g
乙醇200mL
蒸馏水800mL
DISPERBYK-180 100mL
③脱色液:5%盐酸乙醇(95%)溶液
④复染液:次甲基蓝3.0g
乙醇400mL
蒸馏水600mL
KOH 0.20g。
实施例6
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:10%H5IO6溶液
②初染液:碱性品红5.0g
乙醇300mL
蒸馏水650mL
DISPERBYK-2200 50mL
③脱色液:5%盐酸乙醇(95%)溶液
④复染液:次甲基蓝5.0g
乙醇300mL
蒸馏水700mL
KOH 0.12g。
实施例7
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:10%H5IO6溶液
②初染液:碱性品红5.0g
乙醇300mL
蒸馏水650mL
DISPERBYK-180 50mL
③脱色液:5%盐酸乙醇(95%)溶液
④复染液:次甲基蓝5.0g
乙醇300mL
蒸馏水700mL
KOH 0.12g。
实施例8
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:5%H5IO6溶液
②初染液:碱性品红2.0g
乙醇000mL
蒸馏水800mL
DISPERBYK-180 20mL
②脱色液:3%盐酸乙醇(95%)溶液
③复染液:次甲基蓝3.0g
乙醇200mL
蒸馏水800mL
KOH 0.05g。
实施例9
本发明提供了一种改良分支杆菌染色液,按如下配比进行配置:
①氧化液:20%H5IO6溶液
②初染液:碱性品红5.0g
乙醇400mL
蒸馏水600mL
DISPERBYK-180 100mL
②脱色液:5%盐酸乙醇(95%)溶液
③复染液:次甲基蓝6.0g
乙醇400mL
蒸馏水600mL
KOH 0.20g。
上述实施例1~9中配制的染色液,均按以下方法进行染色:
步骤1,涂片经火焰固定,切片脱蜡至水;
步骤2,滴加氧化液盖满玻片,氧化5~20min后,倾去氧化液;
步骤3,滴加初染液盖满玻片,染色5~15min;实际操作中可依据涂片厚度、环境温度适当增加染色时间;
步骤4,水洗并沥干水分;
步骤5,滴加脱色液,轻轻摇动玻片,脱色1~2min,然后水洗;水洗后可见残余红色,再滴加脱色液,摇动玻片直至涂片无红色脱出或略呈粉红色为止,水洗;
步骤6,滴加复染液,复染0.5~1min,水洗;干后镜检。
对比例1
与实施例4提供的分支杆菌染色液配置相同、染色方法相同,区别在于初染液采用石碳酸品红。
对比例2
与实施例9提供的分支杆菌染色液配置相同、染色方法相同,区别在于初染液采用石碳酸品红。
对比例3
与实施例4提供的分支杆菌染色液配置相同、染色方法相同,区别在于:
初染液的组成为:碱性品红5.0g,乙醇300mL,蒸馏水650mL,不添加DISPERBYK-180。
对比例4
与实施例9提供的分支杆菌染色液配置相同、染色方法相同,区别在于:
初染液的组成为:碱性品红5.0g,乙醇300mL,蒸馏水650mL,不添加DISPERBYK-180。
性能测试:收集疑似结核菌感染的病人痰标本200例,分别采用传统的Ziehi-Neelsen抗酸染色法、对比例1~4、及实施例1~9对比染色。传统的Ziehi-Neelsen抗酸染色液的配制、染色方法及所有染色结果判定根据《结核病防治规划痰涂片镜检标准化操作及质量保证手册》实施,实施例染色方法见以上具体实施方式。同时以非抗酸菌金黄色葡萄球菌(ATCC25923)和大肠埃希氏菌(ATCC25922)涂片抗酸染色,每组10片,统计结果如表1所示:
表1性能测试结果
从以上测试可知,改良后的染色法与传统的Ziehi-Neelsen抗酸染色法相比,可大大提高阳性检出率,其中以实施例4和实施例9最优。
金黄色葡萄球菌(ATCC25923)和大肠埃希氏菌(ATCC25922)涂片抗酸染色,10组(每组10片)镜检均为阴性,证明不会产生假阳性的结果。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种改良分支杆菌染色液,其特征在于,所述染色液由氧化液、初染液、脱色液和复染液组成;所述氧化液采用H2O2溶液或H5IO6溶液,所述初染液采用碱性品红溶液。
2.根据权利要求1所述的一种改良分支杆菌染色液,其特征在于,所述H2O2溶液为:体积百分含量为3%~10%的H2O2水溶液。
3.根据权利要求1所述的一种改良分支杆菌染色液,其特征在于,所述H5IO6溶液为质量百分含量为5%~20%的H5IO6水溶液。
4.根据权利要求1所述的一种改良分支杆菌染色液,其特征在于,所述初染液的组成为:碱性品红、乙醇、蒸馏水和渗透剂,所述渗透剂采用高分子量嵌段共聚物溶液。
5.根据权利要求4所述的一种改良分支杆菌染色液,其特征在于,所述高分子量嵌段共聚物溶液包括DISPERBYK-2200或DISPERBYK-180。
6.根据权利要求5所述的一种改良分支杆菌染色液,其特征在于,所述初染液组成配比为:碱性品红2.0g~5.0g,乙醇200~400mL,蒸馏水600~800mL,DISPERBYK-2200或DISPERBYK-180 20~100mL。
7.根据权利要求1所述的一种改良分支杆菌染色液,其特征在于,所述脱色液采用3%~5%的盐酸乙醇溶液。
8.根据权利要求1所述的一种改良分支杆菌染色液,其特征在于,所述复染液采用次甲基蓝溶液,组成为:次甲基蓝3.0~6.0g、乙醇200~400mL、蒸馏水600~800mL、KOH 0.05~0.20g。
9.基于权利要求1至8任一项所述的一种改良分支杆菌染色液的染色方法,其特征在于,包括以下步骤:
步骤1,涂片经火焰固定,切片脱蜡至水;
步骤2,滴加氧化液盖满玻片,氧化后倾去氧化液;
步骤3,滴加初染液盖满玻片,染色;
步骤4,水洗并沥干水分;
步骤5,滴加脱色液,轻轻摇动玻片,脱色,然后水洗;水洗后可见残余红色,再滴加脱色液,摇动玻片直至涂片无红色脱出或略呈粉红色为止,水洗;
步骤6,滴加复染液,复染,水洗;干后镜检。
10.根据权利要求9所述的染色方法,其特征在于,所述步骤2中氧化时间为5~20min,所述步骤3中染色时间为5~15min。
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