often wise to set up duplicate cultures in case one becomes
contaminated. Cultures can be checked for purity using acidfast staining.
11. We routinely generate starter cultures of M. tuberculosis using
10 mL of medium in a 50-mL conical tube and incubating for
10–15 days as standing/stationary. Other methods of culture
using small stirred cultures in glass tubes, rolling/rotating
tubes or tissue culture flasks are equally acceptable. Selection
of the culture method will depend on the equipment and
resources available. Cultures can also be started from frozen
stocks.
12. We use roller bottles to generate larger cultures. Under these
conditions, the doubling time is about 24 h. Using a 1/100
dilution, we obtain late stationary cultures in about 7 days.
13. In general, mycobacterial cultures should be removed from the
incubator when in the logarithmic phase of growth. However,
for M. avium the transformation frequency is maximal at early
log phase of growth [10].
14. For slow-growing species, the addition of glycine (to a final
concentration of 1.5%) improves transformation efficiencies
[7, 10, 20–22]. Glycine is incorporated into peptidoglycan
synthesis in place of alanine [23]. Since glycine is a poor substrate for transpeptidation, it results in decreased cell wall
cross-linking. Glycine should be added 1–2 days prior to
harvesting.
15. For slow-growing species, cells can be kept at room temperature, but electroporation at 37
C will increase the transformation efficiency [7, 10]. For the highest transformation
efficiency, we recommend pre-warming the glycerol washes to
37
C.
16. The final volume of cells depends on the DNA to be transformed. For transformation of replicating or integrating plasmids, we resuspend in a final volume of 5 mL. For
transformation of suicide vectors, or co-transformations,
where a higher transformation efficiency is required, we resuspend in 1 mL.
17. The electroporation medium also has an effect on the time
constant. Use of glycerol provides a high-resistance medium,
allowing longer time constants to be achieved.
18. The efficiency of electroporation depends on the choice of
DNA for transformation; some vectors have been unable to
transform particular mycobacterial species and the efficiency
depends on the choice of the selectable marker. For the
M. tuberculosis complex, the efficiency of transformation is
not affected by the amount of DNA added in the range of
280
Tanya Parish
contaminated. Cultures can be checked for purity using acidfast staining.
11. We routinely generate starter cultures of M. tuberculosis using
10 mL of medium in a 50-mL conical tube and incubating for
10–15 days as standing/stationary. Other methods of culture
using small stirred cultures in glass tubes, rolling/rotating
tubes or tissue culture flasks are equally acceptable. Selection
of the culture method will depend on the equipment and
resources available. Cultures can also be started from frozen
stocks.
12. We use roller bottles to generate larger cultures. Under these
conditions, the doubling time is about 24 h. Using a 1/100
dilution, we obtain late stationary cultures in about 7 days.
13. In general, mycobacterial cultures should be removed from the
incubator when in the logarithmic phase of growth. However,
for M. avium the transformation frequency is maximal at early
log phase of growth [10].
14. For slow-growing species, the addition of glycine (to a final
concentration of 1.5%) improves transformation efficiencies
[7, 10, 20–22]. Glycine is incorporated into peptidoglycan
synthesis in place of alanine [23]. Since glycine is a poor substrate for transpeptidation, it results in decreased cell wall
cross-linking. Glycine should be added 1–2 days prior to
harvesting.
15. For slow-growing species, cells can be kept at room temperature, but electroporation at 37
C will increase the transformation efficiency [7, 10]. For the highest transformation
efficiency, we recommend pre-warming the glycerol washes to
37
C.
16. The final volume of cells depends on the DNA to be transformed. For transformation of replicating or integrating plasmids, we resuspend in a final volume of 5 mL. For
transformation of suicide vectors, or co-transformations,
where a higher transformation efficiency is required, we resuspend in 1 mL.
17. The electroporation medium also has an effect on the time
constant. Use of glycerol provides a high-resistance medium,
allowing longer time constants to be achieved.
18. The efficiency of electroporation depends on the choice of
DNA for transformation; some vectors have been unable to
transform particular mycobacterial species and the efficiency
depends on the choice of the selectable marker. For the
M. tuberculosis complex, the efficiency of transformation is
not affected by the amount of DNA added in the range of
280
Tanya Parish