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Culture and Detection of Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (BCG)
结核分枝杆菌(MTB)和牛分枝杆菌(BCG)的培养和鉴定   

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Abstract

Mycobacterium tuberculosis (MTB) is the bacterial pathogen responsible for tuberculosis, a human pulmonary infectious disease. Mycobacterium bovis (BCG) is the causative agent of tuberculosis in cattle, and is often used as the vaccine stain in humans. Specific recipes and methods for culture of MTB and BCG are described in this protocol.

Keywords: Mycobacterium tuberculosis(结核分枝杆菌), Mycobacterium bovis BCG(卡介苗), Culture(文化)

Materials and Reagents

  1. Middlebrook 7H9 broth base powder (Sigma-Aldrich, catalog number: M0178 )
  2. Middlebrook 7H11 agar base powder (Sigma-Aldrich, catalog number: M0428 )
  3. Middlebrook OADC growth supplement (Sigma-Aldrich, catalog number: M0678 )
  4. BD TB quick stain kit (BD Biosciences, catalog number: 212522 )
  5. General chemicals (Sigma-Aldrich)
  6. ADNaCl (see Recipes)
  7. 7H9 liquid medium (see Recipes)
  8. 7H11 agar plates (see Recipes)

Equipment

  1. T75 tissue culture flasks (Sigma-Aldrich, catalog number: Z707546 )
  2. Standard glass microscope slides
  3. Light microscope
  4. Bunsen burner or fixed flame source
  5. Standard sterile petri dishes

Procedure

  1. Culture
    1. Preparing 7H9 liquid medium
    2. Day 1: Resuspend 1 frozen vial of 1 ml (450 million) MTB or BCG (stored in 15% glycerol and 85% 7H9 liquid medium) in 20 ml 7H9 liquid medium in a T75 flask.
    3. Culture horizontally in an incubator humidified at 37 °C without CO2 for 5 - 14 days.
    4. Measure the OD600 every 5 days, till OD600 reaches 2.0.
    5. Viable MTB/BCG colonies can be counted by plating bacterial suspensions at different dilutions on Middlebrook 7H11 agar plates supplemented with OADC, and then counting colonies after two weeks.
    6. Harvest the bacteria through spin-down at room temperature, 3,000 x g for 7 min.

  2. Detection: Ziehl-Neelsen acid-fast staining detection procedure
    1. Pipet 10 μl MTB/BCG culture on a glass microscope slide and heat on top of a Bunsen flame until it is completely dry to fix the bacteria.
    2. Flood the slide with carbol fuchsin stain (BD kit reagent A).
    3. Heat the slide gently until it steams (5 min).
    4. Pour off the carbol fuchsin.
    5. Wash slide thoroughly with tap water (5 min).
    6. Decolorize with acid-alcohol (5 min).
    7. Wash slide thoroughly with tap water (5 min).
    8. Flood slide with methylene blue (BD kit reagent B) counterstain (1 min).
    9. Wash with tap water.
    10. Blot excess water and dry in hand over Bunsen flame.
    11. The slide is now ready to observe under a standard light microscope.

Recipes

  1. ADNaCl
    15 g BSA
    6 g Dextrose
    2.55 g NaCl
    Dissolve in 300 ml ddH2O
    Note: BSA will take some time to go into solution. Filter to sterilize the supplement and store covered in foil at 4 °C. Do not store for more than one month.
  2. 7H9 liquid medium
    Dissolve 4.7 g of 7H9 powder in 900 ml dH2O
    Add 2 ml glycerol and mix well
    Autoclave and let cool completely
    Store medium at room temperature in the dark
    Note: Do not use medium that has been stored for longer than a month.
    Right before use, add 2.5 ml 20% Tween 80 (filter sterilized) and 10% ADNaCl supplement. At this point the media can be filter sterilized or used directly. Media to which the supplement and Tween have been added should be stored at 4 °C and used within a few weeks.
  3. 7H11 agar plates
    Dissolve 21 g of 7H11 powder in 900 ml dH2O
    Add 5 ml glycerol and swirl to obtain a smooth suspension
    Note: Boil if necessary to completely dissolve the powder.
    Autoclave at 121 °C for 15 min
    Add 100 ml Middlebrook OADC Enrichment and 2.5 ml 20% Tween 80 (filter sterilized) to the medium when cooled to 50-55 °C
    Mix well and pour to make agar plates

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].

References

  1. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., 3rd, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S. and Barrell, B. G. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393(6685): 537-544.
  2. Frieden, T. R., Sterling, T. R., Munsiff, S. S., Watt, C. J. and Dye, C. (2003). Tuberculosis. Lancet 362(9387): 887-899.

简介

结核分枝杆菌(MTB)是负责结核病(一种人类肺部传染病)的细菌病原体。 牛分枝杆菌(Mycobacterium bovis)(BCG)是牛中结核病的致病因子,并且通常用作人类的疫苗染色剂。 在该方案中描述了用于培养MTB和BCG的具体配方和方法。

关键字:结核分枝杆菌, 卡介苗, 文化

材料和试剂

  1. Middlebrook 7H9肉汤粉末(Sigma-Aldrich,目录号:M0178)
  2. Middlebrook 7H11琼脂基粉末(Sigma-Aldrich,目录号:M0428)
  3. Middlebrook OADC生长补充剂(Sigma-Aldrich,目录号:M0678)
  4. BD TB快速染色试剂盒(BD Biosciences,目录号:212522)
  5. 一般化学品(Sigma-Aldrich)
  6. ADNaCl(参见配方)
  7. 7H9液体培养基(见配方)
  8. 7H11琼脂平板(见配方)

设备

  1. T75组织培养瓶(Sigma-Aldrich,目录号:Z707546)
  2. 标准玻璃显微镜载玻片
  3. 光学显微镜
  4. 本生灯或固定火源
  5. 标准无菌培养皿

程序

  1. 文化
    1. 准备7H9液体培养基
    2. 第1天:在T75烧瓶中,将1ml(4.5亿)MTB或BCG(储存在15%甘油和85%7H9液体培养基中)的1个冷冻瓶重悬于20ml 7H9液体培养基中。
    3. 在37℃,无CO 2湿润的培养箱中水平培养5-14天。
    4. 每5天测量OD <600>,直到OD <600> <2.0为止。
    5. 可通过将细菌悬浮液以不同稀释度接种在补充有OADC的Middlebrook 7H11琼脂平板上,然后在两周后计数菌落,计数可行的MTB/BCG集落。
    6. 在室温下通过自旋下降收集细菌,3,000×g/min,7分钟
  2. 检测:Ziehl-Neelsen酸快速染色检测程序
    1. 吸取10微升MTB/BCG文化在玻璃显微镜载玻片和热在本生火焰的顶部,直到它完全干燥,以固定细菌。
    2. 用卡布芬品红染料(BD试剂盒试剂A)填充载玻片。
    3. 轻轻加热载玻片,直到蒸汽(5分钟)。
    4. 倒掉卡布芬品红。
    5. 用自来水彻底洗涤载玻片(5分钟)。
    6. 用酸 - 醇脱色(5分钟)。
    7. 用自来水彻底洗涤载玻片(5分钟)。
    8. 用亚甲基蓝(BD试剂盒试剂B)复染(1分钟)。
    9. 用自来水冲洗。
    10. 多余的水,并干燥在本生火焰。
    11. 幻灯片现在准备在标准光学显微镜下观察。

食谱

  1. ADNaCl
    15 g BSA
    6 g葡萄糖
    2.55g NaCl
    溶解在300ml ddH 2 O中 注意:BSA需要一些时间才能进入解决方案。 过滤灭菌补充品,并在4℃下保存在铝箔上 。 不要存储超过一个月。
  2. 7H9液体培养基
    将4.7g 7H9粉末溶解在900ml dH 2 O中 加入2毫升甘油并混匀
    高压灭菌,让完全冷却。
    在室温下在黑暗中储存培养基
    注意:请勿使用已储存超过一个月的介质。
    使用前,加入2.5毫升20%吐温80(过滤灭菌)和10%ADNaCl补充。 在这一点上,介质可以过滤灭菌或直接使用。 添加了补充剂和吐温的培养基应在4℃保存,并在几周内使用。
  3. 7H11琼脂平板上 将21g 7H11粉末溶解在900ml dH 2 O中 加入5毫升甘油并旋转以获得平滑的悬浮液
    注意:如有必要,煮沸以完全溶解粉末。
    121℃高压灭菌15分钟
    当冷却至50-55℃时,向培养基中加入100ml Middlebrook OADC富集和2.5ml 20%Tween 80(过滤灭菌)。
    混合均匀,倒入琼脂平板

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。该方案是在美国加利福尼亚州斯坦福大学遗传学系的Cohen实验室开发的[Chen等人(未公开)]。

参考文献

  1. Cole,ST,Brosch,R.,Parkhill,J.,Garnier,T.,Churcher,C.,Harris,D.,Gordon,SV,Eiglmeier,K.,Gas,S.,Barry,CE,3rd,Tekaia ,F.,Badcock,K.,Basham,D.,Brown,D.,Chillingworth,T.,Connor,R.,Davies,R.,Devlin,K.,Feltwell,T.,Gentles, ,N.,Holroyd,S.,Hornsby,T.,Jagels,K.,Krogh,A.,McLean,J.,Moule,S.,Murphy,L.,Oliver,K.,Osborne,J.,Quail ,MA,Rajandream,MA,Rogers,J.,Rutter,S.,Seeger,K.,Skelton,J.,Squares,R.,Squares,S.,Sulston,JE,Taylor,K.,Whitehead,和Barrell,BG(1998)。 解密来自完整基因组序列的结核分枝杆菌的生物学。 a> 393(6685):537-544。
  2. Frieden,T.R.,Sterling,T.R.,Munsiff,S.S.,Watt,C.J.and Dye,C。(2003)。 Tuberculosis 。 Lancet 362(9387):887-899。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, R. (2012). Culture and Detection of Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (BCG). Bio-protocol 2(1): e49. DOI: 10.21769/BioProtoc.49.
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12/1/2014 12:54:18 AM Reply
Question on protein concentration in the media:
ADNaCl contains 50 mg/mL BSA, dilute to 10% in the media to final 5 mg/mL BSA protein. Please confirm.

So MIC or any drug test in the media depends on the compound protein binding or free drug concentration?
2/22/2013 8:11:16 AM Reply
RAN CHEN
Stanford

The answer for the final concentration of BSA is yes. If the drug can be neutralized by BSA, of course you need to consider about the protein binding.

2/28/2013 5:30:16 PM


1/10/2012 7:36:30 PM Reply
RAN CHEN
Stanford

BCG???????????????????: “culture horizontally in incubator humidified at 37°C without CO2 for 5 - 14 days.”,???????,?????????????????,?????CO2????,??????????

2/28/2013 5:29:38 PM


关 向前
广东医学院
11/29/2011 9:47:11 AM Reply
Ran Chen
Stanford University

OD600 2.0 约 = 2X10e5/ul
需要严格计数时,才涂平板、数菌落。
悬液梯度稀释后,分别涂平板,再数菌落,是为了取得最佳的精确度。

12/23/2011 9:34:31 AM