小鼠的共性共性隔离和识别
1Department of Medicine, Division of Infectious Diseases, Brigham and Women’s Hospital,Harvard Medical School, 2Massachusetts Host-Microbiome Center, Department of Pathology, Brigham and Women’s Hospital,Harvard Medical School, 3Clinical Microbiology Laboratory, Department of Pathology, Brigham and Women’s Hospital,Harvard Medical School
Summary
这里介绍的是一个协议，用于分离和放大有氧和体性厌氧小鼠结膜共体细菌使用独特的眼拭子和基于培养的丰富步骤，随后通过微生物为基础的方法和MALDI-TOF质谱识别。
Abstract
眼表面曾经被认为是免疫特权和非生物，但最近看来，有一个小的，但持久的共生存在。眼粘膜细菌物种的鉴定和监测一直具有挑战性，因为其丰度低，而且缺乏适当的共生和识别方法。有两种标准方法：基于文化或DNA测序方法。第一种方法是有问题的，由于有限的可回收细菌和第二种方法识别活细菌和死细菌导致异常的眼部空间表示。我们利用标准的微生物培养技术，开发出一种坚固灵敏的细菌分离方法。这是一种基于拭子的技术，使用”实验室内”制成的薄拭子，瞄准下结膜，然后是有氧和足部厌氧基因的放大步骤。该协议使我们能够分离和识别结膜物种，如 科琳细菌、凝固负葡萄球菌、链球菌等。该方法适用于定义不同疾病条件下小鼠的同体多样性。
Introduction
本议定书的目的是加强从眼结膜中分离出可行和罕见的有氧和足性厌氧微生物的具体隔离，以特性眼微生物群。广泛的研究已经剖析了皮肤、肠道、呼吸道和生殖器上的粘膜群落，并表明这些社区影响免疫系统的发展和反应1，2，3。眼科共生社区已被证明在某些疾病病理学中发生改变，如干眼病4、Sjogren综合征5和糖尿病6。然而，与其他粘膜部位6、7、8相比，其丰度相对较低，妨碍了定义典型眼表面共体群落的能力。这引发了一场关于是否有常驻眼微生物群以及是否存在、它是否与皮肤微生物群不同以及其对与生俱来的免疫系统发育和反应的局部影响的争议。此协议可以帮助解决此问题。
一般来说，定义眼部共融利基的方法是基于测序和基于文化的技术4，7，9。16 S rDNA测序和BRISK分析7显示，与基于文化的技术相比，其多样性更为广泛，但无法区分活微生物和死微生物。由于眼部表面由于撕裂膜的抗微生物特性而对许多微生物怀有敌意，因此基于DNA的方法将检测这些文物，这些文物可能会使数据偏向于将死细菌识别为常驻共产物而不是污染物。这导致异常的共体识别和描述的眼部空间是较高的微生物丰度和多样性10。这使得很难通过基于DNA的方法定义常驻眼微生物群。然而，基于文化的标准技术无法检测共体，因为负载太低11。我们的方法改进了标准做法，使用薄拭子，可以瞄准结膜，从而避免来自邻近皮肤的污染，以及可行的生物体可以通过营养密集介质的短暂培养来丰富的概念，目的是恢复可行但不可耕种的微生物，以及丰富稀有可行的微生物。
结果，每个眼拭子的眼部共生体相对丰度，是结膜常驻微生物群的特征，对比较目的很重要。我们的数据显示，皮肤和结膜微生物之间有差异，随着年龄的增长和性别的丰度差异，多样性也越来越大。此外，这种方法在淘汰小鼠12中重新发现了共性差异。此协议可用于描述可能因细胞实践、地理或疾病状态而变化的眼微生物群，以及共生代谢物和产品对免疫系统发育和反应的局部影响。
Protocol
所有涉及小鼠的程序均遵循机构动物护理和使用委员会的准则。在与微生物和潜在受污染材料合作时，请遵循实验室安全指南（由您的机构环境健康和安全部门指导）。在处置可能受到生物危害的受污染材料之前，使用适当的废物贮器和净化程序。 1. 眼拭子准备、工作现场设置、鼠标眼拭子和样品浓缩 准备无菌眼拭子注：无菌眼拭子是薄涂层的木制牙…
Representative Results
显示不同电镀方法的眼拭板的代表性结果见 图 3A， 显示与 C57BL/6 鼠标的形态多样性隔离。对于每个不同的隔离，殖民地被计算在条和相对丰度，独特的殖民地形成单位（CFU）每个眼拭子，计算和绘制的比较目的。对于微生物特征，细菌从单个鼠标眼拭子板中采摘，以产生主 TSA 板（通过从每个鼠眼拭子板中采摘三元形的形态分明分离物创建）。从主板块，在第二天或当增长…
Discussion
由于眼表面的低菌态，许多实验室难以分离眼科共生体7、20，导致生长、丰度低、多样性低的样品数量少。这种方法通过增加充实步骤以及MALDI-TOF MS重新设计的眼拭子和识别，显著改进了标准文化实践4，21。浓缩步骤通过放大细菌负荷来解决低可回收性问题。可回收细菌从不到100个CFU显著增…
Declarações
The authors have nothing to disclose.
Acknowledgements
来自P30 DK034854的资金支持VY，LB和马萨诸塞州主机微生物中心的研究，以及NIH/NEI R01 EY022054支持MG的资金。
Materials
| 0.1 to 10 µl pipet tip | USA Scientific | 1110-300 | autoclave before use |
| 0.5 to 10 µl Eppendorf pipet | Fisher Scientific | 13-690-026 | |
| 1 ml syringe | Fisher Scientific | BD309623 | 1 syringe for each eye swab group |
| 1.5 ml Eppendorf tubes | USA Scientific | 1615-5500 | autoclave before use |
| 1000 µ ml pipet tip | USA Scientific | 1111-2021 | autoclave before use |
| 200 to 1000µl Gilson pipetman (P1000) | Fisher Scientific | F123602G | |
| 25 G needle | Fisher Scientific | 14-826AA | 1 needle per eye swab group |
| 3 % Hydrogen Peroxide | Fisher Scientific | S25359 | |
| 37 ° C Incubator | Lab equipment | ||
| 70 % Isopropanol | Fisher Scientific | PX1840-4 | |
| Ana-Sed Injection (Xylazine 100 mg/ml) | Santa Cruz Animal Health | SC-362949Rx | |
| BD BBL Gram Stain kit | Fisher Scientific | B12539 | |
| Bunsen Burner | Lab equipment | ||
| Clean paper towels | Lab equipment | ||
| Cotton Batting/Sterile rolled cotton | CVS | ||
| Disposable 1 ml Pipets | Fisher Scientific | 13-711-9AM | for Gram stain and catalase tests |
| E.coli | ATTC | ATCC 8739 | |
| Glass slides | Fisher Scientific | 12-550-A3 | for Gram stain and catalase tests |
| Ketamine (100mg/ml) | Henry Schein | 9950001 | |
| Mac Conkey Agar Plates | Fisher Scientific | 4321270 | store at 4 °C until ready to use |
| Mannitol Salt Agar | Carolina Biological Supply | 784641 | Prepare plates according to mfr's instructions, store at 4 °C for 1 week |
| Mice | Jackson Labs | C57/BL6J | |
| Petri Dishes | Fisher Scientific | 08-757-12 | for Mannitol Salt agar plates |
| RPI Brain Heart Infusion Media | Fisher Scientific | 50-488525 | prepare according to directions and autoclave |
| SteriFlip (0.22 µm pore size polyester sulfone) | EMD/Millipore, Fisher Scientifc | SCGP00525 | to sterilize anesthesia |
| Sterile Corning Centrifuge Tube | Fisher Scientific | 430829 | anesthesia preparation |
| Sterile mouse cage | Lab equipment | ||
| Tooth picks (round bamboo) | Kitchen Essentials | autoclave before use and swab preparation | |
| Trypticase Soy Agar II with 5% Sheep's Blood Plates | Fisher Scientific | 4321261 | store at 4 °C until ready to use |
| Vitek target slide | BioMerieux Inc. Durham,NC | ||
| Vitek-MS | BioMerieux Inc. Durham,NC | ||
| Vitek-MS CHCA matrix solution | BioMerieux Inc. Durham, NC | 411071 | |
| Single use eye drops | CVS Pharmacy | Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each |
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Smith-Page, K., Kugadas, A., Lin, T., Delaney, M., Bry, L., Gadjeva, M. Conjunctival Commensal Isolation and Identification in Mice. J. Vis. Exp. (171), e61672, doi:10.3791/61672 (2021).