13. Liver and spleen samples are stored at À80
C to prevent
degradation. Livers vary in yields from 2 to 37 mg/tissues
with an average of 0.3 mg of whole cells per gram of tissue.
Spleens give higher yields ranging from 18 to 164 mg per
tissue with an average of 1.9 mg of whole cells per gram of
tissue.
14. All glassware, tubes, stir bars, scissors, and homogenizer chamber used for purification of the bacteria should be thoroughly
washed and autoclaved at 120
C for 50 min prior to use to
ensure sterility. Cover beakers with foil, use sterilization
pouches for tubes, stir bars, and scissors before autoclaving to
maintain sterility. Use of glass is not recommended in most
high containment facilities, but if the downstream products are
to be used for MALDI-TOF applications, glass containers are
recommended. In these cases, special exemptions and procedures should be in place.
15. Other types of media can be used but care should be taken to
optimize the protocol for best conditions (i.e., time of harvest).
16. Many bioreactor vessels will trap mycobacteria cells in crevices
and filters or result in adherent or sheared cells that are difficult
or unable to be harvested reproducibly. This protocol supports
the use of BioBLU single-pitched blade vessels with a
macrosparger only.
17. Autoclave DO probe at 15 min liquid cycle. This only needs to
be done once if the probe is new or has not been stored and
unused for a long period of time. The DO probe must be
polarized and charged prior to use. Plug the probe into the
control unit via the DO probe cable and turn on the control
unit. Allow the probe to charge for at least 8 h, overnight is
preferred. Calibrate probe: Unplug the DO line and set zero
when the number stabilizes. Insert DO probe and temperature
probe into the vessel. Attach exhaust heater and incubator
jacket around the vessel. Attach gas line to the sparge filter
and place the rotor motor on top of the vessel. Sparge air at
1 VVM. Run the agitator at 65. Set span at 100 for the DO
calibration when the number stabilizes.
18. Autoclave pH probe at 15 min liquid cycle. Remove from bag
and remove cap. Calibrate probe: Attach pH probe to the pH
cable and plug into the control unit. Rinse with water, measure
pH 7 standard, set ero as pH 7 when the number stabilizes,
rinse with water, measure pH 4 standard, set Span as pH 4
when the number stabilizes. Take a 30 mL sample of media
from the sample port of the vessel using a syringe. Expel the
media into a 50 mL Falcon tube, measure pH with probe.
Using a separate pH meter, measure the pH of the media.
Culturing Mycobacteria
51
C to prevent
degradation. Livers vary in yields from 2 to 37 mg/tissues
with an average of 0.3 mg of whole cells per gram of tissue.
Spleens give higher yields ranging from 18 to 164 mg per
tissue with an average of 1.9 mg of whole cells per gram of
tissue.
14. All glassware, tubes, stir bars, scissors, and homogenizer chamber used for purification of the bacteria should be thoroughly
washed and autoclaved at 120
C for 50 min prior to use to
ensure sterility. Cover beakers with foil, use sterilization
pouches for tubes, stir bars, and scissors before autoclaving to
maintain sterility. Use of glass is not recommended in most
high containment facilities, but if the downstream products are
to be used for MALDI-TOF applications, glass containers are
recommended. In these cases, special exemptions and procedures should be in place.
15. Other types of media can be used but care should be taken to
optimize the protocol for best conditions (i.e., time of harvest).
16. Many bioreactor vessels will trap mycobacteria cells in crevices
and filters or result in adherent or sheared cells that are difficult
or unable to be harvested reproducibly. This protocol supports
the use of BioBLU single-pitched blade vessels with a
macrosparger only.
17. Autoclave DO probe at 15 min liquid cycle. This only needs to
be done once if the probe is new or has not been stored and
unused for a long period of time. The DO probe must be
polarized and charged prior to use. Plug the probe into the
control unit via the DO probe cable and turn on the control
unit. Allow the probe to charge for at least 8 h, overnight is
preferred. Calibrate probe: Unplug the DO line and set zero
when the number stabilizes. Insert DO probe and temperature
probe into the vessel. Attach exhaust heater and incubator
jacket around the vessel. Attach gas line to the sparge filter
and place the rotor motor on top of the vessel. Sparge air at
1 VVM. Run the agitator at 65. Set span at 100 for the DO
calibration when the number stabilizes.
18. Autoclave pH probe at 15 min liquid cycle. Remove from bag
and remove cap. Calibrate probe: Attach pH probe to the pH
cable and plug into the control unit. Rinse with water, measure
pH 7 standard, set ero as pH 7 when the number stabilizes,
rinse with water, measure pH 4 standard, set Span as pH 4
when the number stabilizes. Take a 30 mL sample of media
from the sample port of the vessel using a syringe. Expel the
media into a 50 mL Falcon tube, measure pH with probe.
Using a separate pH meter, measure the pH of the media.
Culturing Mycobacteria
51