培养和维护<em>难辨梭状芽孢杆菌</em>在厌氧环境
Summary
艰难梭菌是一种致病细菌是严格厌氧并导致抗生素相关性腹泻（AAD）。这里，用于分离，培养和维持C.方法艰难梭菌营养细胞和孢子的描述。这些技术的必要厌氧室，这就需要定期保养，以确保最佳的C。适当的条件难辨栽培。
Abstract
艰难梭菌是一种革兰氏阳性，厌氧，产孢子的细菌，主要负责抗生素相关性腹泻（AAD）和一个显著院内病原体。C.艰难是出了名的难以分离和培养，并是即使是低氧气的环境水平极为敏感。这里，对于隔离C.方法从难辨粪便样品，随后C.培养难辨用于制备甘油股票长期贮存介绍。编制和列举孢子股票在实验室用于各种下游应用，包括显微镜和动物实验技术也有所说明。这些技术的必要厌氧室，它保持一致的厌氧环境，以确保最佳的C。适当的条件艰难成长。我们提供的协议在室的进出转印材料未经CA使用显著氧污染以及用于维持适当的厌氧环境，高效的和一致的要求下定期保养建议难辨栽培。
Introduction
艰难梭菌是一种革兰氏阳性，形成孢子的细菌是专性厌氧菌以及人类和动物的一种潜在的致命胃肠道病原体。在1935年最初描述为粪便样本中发现从新生儿1，C的共生有机体难辨后来被证明是伪膜性肠炎的病原体使用抗生素治疗2有关。C.艰难梭菌感染（CDI），通常先通过抗生素治疗而导致的正常结肠菌群的破坏，形成一个利基C。难辨茁壮成长2。C.难辨传输为通过粪-口途径休眠孢子，并随后在胃肠道内发芽，生产营养细胞能够产生多种毒素，引起严重的疾病，结肠炎3。 CDI往往是难治常规治疗而这些fections经常经常性的4。因此，课程发展处负责可达至48十亿在医疗保健费用在美国5-7。
艰难梭菌是即使低氧气的环境中的电平非常敏感。对于C。难辨坚持环境和有效地从主机到主机的传输，一个代谢不活跃孢子的形成是至关重要的8。因为C的实验室维护和操作难辨需要控制，无氧的环境中，这些技术需要使用在厌氧室。利用厌氧室已导致增加的回收和专性厌氧菌9-11的隔离，并允许一些要在厌氧气氛中进行的分子技术。
除了C。难辨 ，厌氧室使用和维护这里描述适用其它专性厌氧菌如其他梭菌物种（ 如产气荚膜梭菌 ），其它胃肠物种（ 如拟杆菌属种12）和牙周病原体（ 如消化链球菌属 13种）。
Protocol
注：C。艰难梭菌是一种人类和动物病原体，可引起胃肠道疾病。涉及C.实验难辨必须以适当的生物安全防范措施（BSL-2）进行。 1。厌氧室使用与维护 艰难梭菌是一种严格厌氧菌和是即使是低浓度的氧的大气中极为敏感。因此，受控的，厌氧环境是必要的，它的成功操作。利用厌氧室中（ 图1A）的提供了最稳定的环?…
Representative Results
C.示例艰难梭菌生长在BHIS和哥伦比亚厌氧绵羊血琼脂培养基上可以看出，在图2中 。C.难辨形成不规则的菌落是平的，拥有一个毛玻璃的外观，这是显而易见的两种培养基上。这里，C的红霉素敏感型临床分离难辨 ，630E 30，生长在BHIS琼脂，一个富集，非选择性培养基中，放置24小时，在37℃（ 图2A）。哥伦比亚厌氧羊血琼脂上菌落…
Discussion
这里描述的方法允许简单和快速C的恢复从各种粪便样品，包括人类，小鼠和仓鼠，以及在长期贮存下的难辨难辨甘油或孢子的股票。C.难辨可以是一个困难的有机体培养，但细心保养的厌氧环境和无菌技术的应用可以提供强劲的增长和污染的减少。
厌氧室：注意事项和维护
有两种类型的厌氧室：刚性室或乙烯基室。刚性腔通常由?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢大队实验室的好心提供厌氧室的照片。这项工作是由卫生部授予DK087763（SMM）和一个STEP /霍华德休斯医学研究所课程发展奖学金（ANE）国家机构的支持。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| Proteose Peptone no. 2 | BD | 212120 | |
| Na2HPO4 | Fisher | S373 | |
| KH2PO4 | Fisher | BP362 | |
| NaCl | Fisher | S27 | |
| MgSO4 (anhydrous) | Fisher | M65 | |
| ᴅ-Fructose | Fisher | L96 | |
| Sodium taurocholate | Sigma | T4009 | |
| ᴅ-cycloserine | Sigma | C6880 | |
| Cefoxitin | Fluka | C4786 | |
| Brain heart infusion medium | BD | 237300 | |
| Proteose Peptone | BD | 211684 | |
| (NH4)2SO4 | Sigma | A5132 | |
| Tris base | Fisher | BP152 | |
| Agar | BD | 214010 | |
| L-cysteine | Sigma | C7755 | |
| BactoPeptone | BD | 211684 | |
| Columbian sheep blood agar | Fisher | L21928 | |
| NaCl | Fisher | S27 | |
| KCl | Fisher | P217 | |
| Glycerol | Fisher | BP2291 | |
| Sterile inoculating loops | Fisher | 22363596 | |
| Sterile swabs | Fisher | 1495990 | |
| Coy Vinyl Anaerobic Chamber and Accessories | Coy Laboratory Products, Inc | Customer Specified | These items are custom ordered per laboratory needs |
| Materials | |||
| TCCFA agar Proteose peptone no. 2 (Difco) 40 g Na2HPO4 5 g KH2PO4 1 g NaCl 2 g MgSO4 (anhydrous) 0.1 g Fructose 6 g Agar 20 g Bring to 1 L with deionized water and autoclave at 121 °C for 20 min to sterilize. After autoclaving, add: 10 ml of 10% (w/v) sodium taurocholate, filter-sterilized (dissolve in water; final concentration, 0.1%) 25 ml of 10 mg/ml ᴅ-cycloserine, filter-sterilized (dissolve in water; final concentration, 250 μg/ml) 1.6 ml of 10 mg/ml cefoxitin, filter-sterilized (dissolve in water; final concentration, 16 μg/ml) BHIS Medium Brain heart infusion 37 g Yeast extract 5 g For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 °C for 20 min to sterilize. Optional (add after autoclaving): 3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 10 ml of 10% (w/v) sodium taurocholate (dissolve in water; final concentration, 0.1%) SMC Sporulation Medium BactoPeptone 90 g Protease peptone 5 g (NH4)2SO4 1 g Tris base 1.5 g Agar 15 g Bring to 1 L with deionized water and autoclave at 121 °C for 20 min to sterilize. Optional (add after autoclaving): 3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 70:30 Medium BactoPeptone 63 g Protease peptone 3.5 g Brain heart infusion 11.1 g Yeast extract 1.5 g (NH4)2SO4 0.7 g Tris base 1.06 g For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 °C for 20 min to sterilize. After autoclaving, add 3 ml of 10% (w/v) L-cysteine (final concentration, 0.03%). Blood agar The use of premade Columbia anaerobic sheep blood agar plates (Fisher Scientific, L21928)35 is recommended. 1X Phosphate buffered saline (PBS) NaCl 8.01 g KCl 0.2 g Na2HPO4 1.44 g KH2PO4 0.27 g Bring to 1 L with deionized water and adjust pH to 7.4 with HCl. Filter sterilize before use. |
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Citer Cet Article
Edwards, A. N., Suárez, J. M., McBride, S. M. Culturing and Maintaining Clostridium difficile in an Anaerobic Environment. J. Vis. Exp. (79), e50787, doi:10.3791/50787 (2013).