28. Most isolates of M. tuberculosis require 4 weeks to form a lawn,
and 2 weeks per liquid large-scale pass using the ratios
provided. M. tuberculosis var. bovis requires roughly 6 weeks
to form a lawn and 3 weeks per liquid large-scale pass. M. bovis
also requires media with sodium pyruvate added (this should
replace the glycerol). Specific time points for harvest should be
confirmed empirically.
29. Small-scale growth can also be started from plates.
30. If a warm room maintained at 37
C is unavailable, use an
incubator, environmental shaker tables, and/or environmental
roller bottle apparatus as needed.
31. If roller bottles are not used, repeat steps 2–13 with eight
more flasks. Steps 14–18 are only required if roller bottles
are needed.
32. Each bottle can hold 4 L (so four bottles will be needed for
harvest). Carefully watch the bottle begin to fill. If more than
an inch of foam forms, the bottle was not rinsed correctly. Start
over with a new bottle and discard old one and the 0.2 μm
filtration unit. Also beware of crack formation due to the
pressure.
33. Do not allow the filtration unit to dry out, replace with a new
filtration unit if it does dry out.
34. Occasionally, the sides of roller bottle cultures will compress
inward as the internal temperature changes from 37 to 20
C.
When this occurs, hold compressed area of roller bottle with
one hand while slowly opening the cap with the other. This will
control the re-expansion of the roller bottle and reduce the
aerosolizing of bacteria.
35. To avoid contamination, add 1.25 g sodium azide (NaN 3 ) per
each 1 L of CFP if it will be in storage at 4
C for more than
a week.
36. If there are more than 100 g of cells, use two conical vials.
37. Label the CFP and cell pellet with the appropriate information
(strain, lot number, date, medium, and technician name) and
parafilm the lid. Write down the weight of the cell pellet on the
label and on a large-scale growth data sheet.
38. Label the outside of the package and freeze in a À80
C freezer
until the sample can be transported for irradiation. The cell
pellet is ready for γ-irradiation, heat kill, or chemical inactivation (chloroform/methanol, for example). Confirmation of
inactivation using an appropriate method (such as the Alamar
Blue dye reduction assay or incubation of 1 to 10% of the total
cell paste material for 4–6 weeks on L-J media slants) is necessary prior to any downstream manipulation in a non BSL-3
facility. Dangerous goods transport certification, for the transport infectious material, may be needed based on international,
federal, state, and local transportation laws.
Culturing Mycobacteria
53
and 2 weeks per liquid large-scale pass using the ratios
provided. M. tuberculosis var. bovis requires roughly 6 weeks
to form a lawn and 3 weeks per liquid large-scale pass. M. bovis
also requires media with sodium pyruvate added (this should
replace the glycerol). Specific time points for harvest should be
confirmed empirically.
29. Small-scale growth can also be started from plates.
30. If a warm room maintained at 37
C is unavailable, use an
incubator, environmental shaker tables, and/or environmental
roller bottle apparatus as needed.
31. If roller bottles are not used, repeat steps 2–13 with eight
more flasks. Steps 14–18 are only required if roller bottles
are needed.
32. Each bottle can hold 4 L (so four bottles will be needed for
harvest). Carefully watch the bottle begin to fill. If more than
an inch of foam forms, the bottle was not rinsed correctly. Start
over with a new bottle and discard old one and the 0.2 μm
filtration unit. Also beware of crack formation due to the
pressure.
33. Do not allow the filtration unit to dry out, replace with a new
filtration unit if it does dry out.
34. Occasionally, the sides of roller bottle cultures will compress
inward as the internal temperature changes from 37 to 20
C.
When this occurs, hold compressed area of roller bottle with
one hand while slowly opening the cap with the other. This will
control the re-expansion of the roller bottle and reduce the
aerosolizing of bacteria.
35. To avoid contamination, add 1.25 g sodium azide (NaN 3 ) per
each 1 L of CFP if it will be in storage at 4
C for more than
a week.
36. If there are more than 100 g of cells, use two conical vials.
37. Label the CFP and cell pellet with the appropriate information
(strain, lot number, date, medium, and technician name) and
parafilm the lid. Write down the weight of the cell pellet on the
label and on a large-scale growth data sheet.
38. Label the outside of the package and freeze in a À80
C freezer
until the sample can be transported for irradiation. The cell
pellet is ready for γ-irradiation, heat kill, or chemical inactivation (chloroform/methanol, for example). Confirmation of
inactivation using an appropriate method (such as the Alamar
Blue dye reduction assay or incubation of 1 to 10% of the total
cell paste material for 4–6 weeks on L-J media slants) is necessary prior to any downstream manipulation in a non BSL-3
facility. Dangerous goods transport certification, for the transport infectious material, may be needed based on international,
federal, state, and local transportation laws.
Culturing Mycobacteria
53