CN117286213A - 肠杆菌科细菌阳性血培养直接药敏试验处理方法 - Google Patents
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Abstract
本方案属于细菌药敏试验领域，具体涉及一种肠杆菌科细菌阳性血培养直接药敏试验处理方法，包括以下步骤：步骤S10：进行涂片；步骤S20：待其自然干燥后进行革兰染色的初染，滴加革兰染色A液于涂布细菌处，染色15s后用细流水冲洗甩干；步骤S30：进行媒染，滴加革兰染色B液数滴，染色15s后用细流水冲洗，甩干；步骤S40：进行脱水处理，滴加革兰染液C液数滴，轻轻晃动玻片至无紫色脱出为止，然后用流水冲洗，甩干；步骤S50：复染，滴加革兰染色D液数滴，染色15s，用流水冲洗；步骤S60：待标本片自然干燥或用吸水纸吸干后在涂菌处滴加一滴香柏油油镜观察、观察是否为革兰阴性杆菌；若为革兰阴性杆菌则进行分离操作；步骤S70：分离；步骤S70：用上述分离的细菌悬液，按全自动鉴定和药敏分析仪说明书调制0.5 McF的ID肉汤和AST肉汤进行鉴定和药敏试验。与传统的试验方法相比，本方案大幅度地缩短了整个过程的时间。
Description
技术领域
本方案属于细菌药敏试验领域，具体涉及一种肠杆菌科细菌阳性血培养直接药敏试验处理方法。
背景技术
细菌培养及药敏试验是诊治感染性疾病(如肺炎、脑膜炎、泌尿道感染、结核、伤害、霍乱、败血症，以及疖、痈)全过程的关键。我们不妨把这个过程当作一次军事行动，首先侦辑元凶，接着制定方案，最后克敌制胜。
细菌培养——侦缉元凶。我们知道，自然界存在着较多的微生物，其中能引起人体患病的细菌侵犯人体后，可引起各种感染性疾病。同一种细菌侵犯人体不同的部位，可引起不同疾病的发生；而同一种疾病又可由不同细菌的侵犯而引起。为了明确诊断，只有从病人的血、尿或其他分泌物中取样进行细菌培养，快速、准确地“侦缉”到病原菌。
药敏试验——制定方案。由于各种细菌对抗菌药物的敏感性不同，即使同一种细菌的不同菌株对各种抗菌药物的敏感性也有差别；在治疗过程中，细菌对药物的敏感性也会发生变化；随着各种抗菌药物的广泛应用，细菌的抗药性也随之增多。只有对病原菌进行多种抗菌药物的敏感度试验，才能选择到有效的抗菌药物及浓度。
血流感染是一种严重的全身感染性疾病，发病率和死亡率较高。血培养是诊断血流感染的金标准,快速、准确的提供病原菌鉴定及药敏结果，是临床进行抗感染的关键。更早的进行正确的抗感染治疗可以明显提高患者的生存率。
专利号为CN202211395997.6的专利，公开了一种革兰氏阴性菌的药敏鉴定方法，包括以下步骤：S1：抗生素、氘水孵育细菌：向抗生素溶液中接种革兰氏阴性菌，配制成浓度为103？5×103CFU/ml的悬浮菌液，再于96孔板的各孔中加入20ul的该悬浮菌液和20ul氘水，37℃恒温孵育30min；S2：药敏样品自动化层流洗涤：将含S1孵育后悬浮菌液的96孔板置于层流洗涤系统中，利用重力和流体力学的层流效应进行自动化洗涤；S3：拉曼光谱检测；S4：药敏性结果分析：观察拉曼位移在2200波数处是否出现氘水拉曼峰，以鉴定革兰氏阴性菌对抗生素是否敏感。本发明药敏鉴定方法将抗生素、氘水孵育一体化，结合自动化层流洗涤，无需15h-7d长周期培养、染色即可检测出革兰氏阴性菌的拉曼光谱图，通过所建数据库和智能识别鉴定。然而该方法需要拉曼光谱仪，不太适合基层实验室。
而现有的肠杆菌科细菌阳性血培养的药敏试验方法是：血培养仪阳性报警后按传统流程处理，涂片革兰染色、显微镜观察、转种血或巧克力平板在厌氧或需氧条件下培养24-48h获得纯菌落后，再使用现有的商品化仪器如Vitek 2Compact、BD PhoenixTM-100和MicroScan WalkAway 96Plus等进行鉴定和药敏试验。
但是目前这样的方法需要将阳性血培养液转种至培养基上，培养孵育至肉眼可见的单个纯菌落后，再挑选单个纯菌落通过后续的方法进行鉴定和药敏试验，整个过程耗时较长(约24h-48h)。
发明内容
本方案提供一种肠杆菌科细菌阳性血培养直接药敏试验处理方法，有效避免了现有技术中整个过程耗时长的缺陷。
为了达到上述目的，本方案提供一种肠杆菌科细菌阳性血培养直接药敏试验处理方法，包括以下步骤：
步骤S10：用无菌注射器抽取1ml血培养阳性液滴两滴于干净的载玻片上，进行涂片；
步骤S20：待其自然干燥后进行革兰染色的初染，滴加革兰染色A液于涂布细菌处，染色15s后用细流水冲洗甩干；
步骤S30：进行媒染，滴加革兰染色B液数滴，染色15s后用细流水冲洗，甩干；
步骤S40：进行脱水处理，滴加革兰染液C液数滴，轻轻晃动玻片至无紫色脱出为止，然后用流水冲洗，甩干；
步骤S50：复染，滴加革兰染色D液数滴，染色15s，用流水冲洗；
步骤S60：待标本片自然干燥或用吸水纸吸干后在涂菌处滴加一滴香柏油油镜观察、观察是否为革兰阴性杆菌；若为革兰阴性杆菌则进行分离操作；
步骤S70：分离，用无菌注射器抽取阳性瓶内血标本8ml，分别等量注入两只血清分离胶促凝管；3000r/min离心10min,弃上层血清,分别用无菌吸管吸取1ML无菌生理盐水小心吹打灰白色细菌层后吸取该菌液置于同一只血清分离胶促凝管；在装有菌液的血清分离胶促凝管中加入适量无菌生理盐水混匀,3000r/min离心10min,弃上清备用，用无菌吸管吸取1ML无菌生理盐水小心吹打灰白色细菌层后吸取该菌液置于一只无菌试管，在装有菌液的无菌试管中加入适量无菌生理盐水混匀,3000r/min离心10min,弃上清备用，用于调制接种菌液；
步骤S70：用上述分离的细菌悬液，按全自动鉴定和药敏分析仪说明书调制0.5McF的ID肉汤和AST肉汤进行鉴定和药敏试验。
进一步，所述滴加革兰染色A液为结晶紫染液。
进一步，所述革兰染色B液为碘液。
进一步，所述革兰染液C液为95％的乙醇液。
进一步，所述革兰染色D液为稀释沙黄。
具体实施方式
下面通过具体实施方式进一步详细的说明：
实施例
本实施例提供一种肠杆菌科细菌阳性血培养直接药敏试验处理方法，具体步骤如下：
1、用无菌注射器抽取1ml血培养阳性液滴两滴于干净的载玻片上，进行涂片，
2、待其自然干燥后进行革兰染色。初染滴加结晶紫染液(革兰染色A液)于涂布细菌处，染色15s后用细流水冲洗，甩干。媒染滴加碘液(革兰染色B液)数滴，染色15s后用细流水冲洗，甩干。脱色滴加95％的乙醇液(革兰染液C液)数滴，轻轻晃动玻片至无紫色脱出为止，大约3-5s左右(灵活掌握时间)，用流水冲洗，甩干。复染滴加稀释沙黄(革兰染色D液)数滴，染色15s，用流水冲洗。
3、待标本片自然干燥或用吸水纸吸干后在涂菌处滴加一滴香柏油油镜观察、观察是否为革兰阴性杆菌。若为革兰阴性杆菌则进行下面操作，
4、用无菌注射器抽取阳性瓶内血标本8ml，分别等量注入两只血清分离胶促凝管(5ML)。3000r/min离心10min,弃上层血清,分别用无菌吸管吸取1ML无菌生理盐水小心吹打灰白色细菌层后吸取该菌液置于同一只血清分离胶促凝管(5ML)。在装有菌液的血清分离胶促凝管中加入适量无菌生理盐水混匀(用于稀释可溶性血红蛋白),3000r/min离心10min,弃上清备用，用无菌吸管吸取1ML无菌生理盐水小心吹打灰白色细菌层后吸取该菌液置于一只无菌试管(5ML)，在装有菌液的无菌试管中加入适量无菌生理盐水混匀(用于稀释可溶性血红蛋白),3000r/min离心10min,弃上清备用，用于调制接种菌液。用上述分离的细菌悬液，按全自动鉴定和药敏分析仪说明书调制0.5McF的ID肉汤和AST肉汤进行鉴定和药敏试验。
以上所述的仅是本发明的实施例，方案中公知的具体结构及特性等常识在此未作过多描述。应当指出，对于本领域的技术人员来说，在不脱离本发明结构的前提下，还可以作出若干变形和改进，这些也应该视为本发明的保护范围，这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准，说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (5)
1.肠杆菌科细菌阳性血培养直接药敏试验处理方法，其特征在于：包括以下步骤：
步骤S10：用无菌注射器抽取1ml血培养阳性液滴两滴于干净的载玻片上，进行涂片；
步骤S20：待其自然干燥后进行革兰染色的初染，滴加革兰染色A液于涂布细菌处，染色15s 后用细流水冲洗甩干；
步骤S30：进行媒染，滴加革兰染色B液数滴，染色15s后用细流水冲洗，甩干；
步骤S40：进行脱水处理，滴加革兰染液C液数滴，轻轻晃动玻片至无紫色脱出为止，然后用流水冲洗，甩干；
步骤S50：复染，滴加革兰染色D液数滴，染色15s，用流水冲洗；
步骤S60：待标本片自然干燥或用吸水纸吸干后在涂菌处滴加一滴香柏油油镜观察、观察是否为革兰阴性杆菌；若为革兰阴性杆菌则进行分离操作；
步骤S70：分离，用无菌注射器抽取阳性瓶内血标本8ml，分别等量注入两只血清分离胶促凝管；3000r／min 离心10 min,弃上层血清,分别用无菌吸管吸取1ML无菌生理盐水小心吹打灰白色细菌层后吸取该菌液置于同一只血清分离胶促凝管；在装有菌液的血清分离胶促凝管中加入适量无菌生理盐水混匀,3000r／min 离心10min,弃上清备用，用无菌吸管吸取1ML无菌生理盐水小心吹打灰白色细菌层后吸取该菌液置于一只无菌试管，在装有菌液的无菌试管中加入适量无菌生理盐水混匀,3000r／min 离心10min,弃上清备用，用于调制接种菌液；
步骤S70：用上述分离的细菌悬液，按全自动鉴定和药敏分析仪说明书调制0.5McF的ID肉汤和AST肉汤进行鉴定和药敏试验。
2.根据权利要求1所述的肠杆菌科细菌阳性血培养直接药敏试验处理方法，其特征在于：所述革兰染色A液为结晶紫染液。
3.根据权利要求1所述的肠杆菌科细菌阳性血培养直接药敏试验处理方法，其特征在于：所述革兰染色B液数滴。
4.根据权利要求1所述的肠杆菌科细菌阳性血培养直接药敏试验处理方法，其特征在于：所述革兰染液C液为95%的乙醇液。
5.根据权利要求1所述的肠杆菌科细菌阳性血培养直接药敏试验处理方法，其特征在于：所述革兰染色D液为稀释沙黄。
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