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Culture and Detection of Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (BCG)   

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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, Mycobacterium bovis BCG, Culture

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.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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