and second upper is 5% unless the slide indicates a higher
amount of tissue contamination. Qualified purified cells may
be stored at À80
C or lyophilized.
66. 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.
67. 2.5% Vesphene contact time to inactivate M. tuberculosis is
20 min. 70% ethanol is used here to remove residual Vesphene.
68. BioFlo bioreactors include Digital Mettler Toledo
® ISM sensor
technology which allows remote monitoring of pH, redox,
dissolved oxygen (polarographic and/or optical), and carbon
dioxide measurements. This system, or a related one, should
always be used for remote monitoring and alarm functions
when used for pathogen fermentation.
69. This step will likely be done outside the BSC. In that instance,
make sure to take extreme precaution, as unnoticed leaks or
spills from syringe sampling could lead to laboratory personnel
exposure.
70. This timeline is optimal for protein, lipid, and metabolite analysis. Other analyses, such as RNA interrogation, may require
optimization of timelines.
71. Pellet is not washed with sterile water prior to γ-irradiation.
72. Chloroform:methanol (2:1 v/v) needs to be added inside of a
Class 2 BII BSC before removal from BSL-3 facility.
References
1. Hazbon MH, Rigouts L, Schito M,
Ezewudo M, Kudo T, Itoh T, Ohkuma M,
Kiss K, Wu L, Ma J, Hamada M, Strong M,
Salfinger M, Daley CL, Nick JA, Lee JS,
Rastogi N, Couvin D, Hurtado-Ortiz R,
Bizet C, Suresh A, Rodwell T, Albertini A,
Lacourciere
KA,
Deheer-Graham
A,
Alexander S, Russell JE, Bradford R, Riojas
MA (2018) Mycobacterial biomaterials and
resources for researchers. Pathog Dis 76(4):
fty042
2. Sharma R, Singh P, McCoy RC, Lenz SM,
Donovan K, Ochoa MT, Estrada-Garcia I,
Silva-Miranda M, Jurado-Santa Cruz F, Balagon MF, Stryjewska B, Scollard DM, Pena MT,
Lahiri R, Williams DL, Truman RW, Adams LB
(2019) Isolation of Mycobacterium lepromatosis
and development of molecular diagnostic
assays to distinguish M. leprae and
M. lepromatosis. Clin Infect Dis 71(8):
e262–e269
3. Tortoli E (2006) The new mycobacteria: an
update. FEMS Immunol Med Microbiol
48:159–178
4. Gupta RS, Lo B, Son J (2018) Phylogenomics
and comparative genomic studies robustly support division of the genus Mycobacterium into
an emended genus Mycobacterium and four
novel genera. Front Microbiol 9:67
5. Tortoli E, Brown-Elliott BA, Chalmers JD,
Cirillo DM, Daley CL, Emler S, Floto RA,
Garcia MJ, Hoefsloot W, Koh WJ, Lange C,
Loebinger M, Maurer FP, Morimoto K,
Niemann S, Richter E, Turenne CY,
Vasireddy R, Vasireddy S, Wagner D, Wallace
RJ Jr, Wengenack N, van Ingen J (2019) Same
meat, different gravy: ignore the new names of
mycobacteria. Eur Respir J 54:1900795
56
Elizabeth Wallace et al.
amount of tissue contamination. Qualified purified cells may
be stored at À80
C or lyophilized.
66. 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.
67. 2.5% Vesphene contact time to inactivate M. tuberculosis is
20 min. 70% ethanol is used here to remove residual Vesphene.
68. BioFlo bioreactors include Digital Mettler Toledo
® ISM sensor
technology which allows remote monitoring of pH, redox,
dissolved oxygen (polarographic and/or optical), and carbon
dioxide measurements. This system, or a related one, should
always be used for remote monitoring and alarm functions
when used for pathogen fermentation.
69. This step will likely be done outside the BSC. In that instance,
make sure to take extreme precaution, as unnoticed leaks or
spills from syringe sampling could lead to laboratory personnel
exposure.
70. This timeline is optimal for protein, lipid, and metabolite analysis. Other analyses, such as RNA interrogation, may require
optimization of timelines.
71. Pellet is not washed with sterile water prior to γ-irradiation.
72. Chloroform:methanol (2:1 v/v) needs to be added inside of a
Class 2 BII BSC before removal from BSL-3 facility.
References
1. Hazbon MH, Rigouts L, Schito M,
Ezewudo M, Kudo T, Itoh T, Ohkuma M,
Kiss K, Wu L, Ma J, Hamada M, Strong M,
Salfinger M, Daley CL, Nick JA, Lee JS,
Rastogi N, Couvin D, Hurtado-Ortiz R,
Bizet C, Suresh A, Rodwell T, Albertini A,
Lacourciere
KA,
Deheer-Graham
A,
Alexander S, Russell JE, Bradford R, Riojas
MA (2018) Mycobacterial biomaterials and
resources for researchers. Pathog Dis 76(4):
fty042
2. Sharma R, Singh P, McCoy RC, Lenz SM,
Donovan K, Ochoa MT, Estrada-Garcia I,
Silva-Miranda M, Jurado-Santa Cruz F, Balagon MF, Stryjewska B, Scollard DM, Pena MT,
Lahiri R, Williams DL, Truman RW, Adams LB
(2019) Isolation of Mycobacterium lepromatosis
and development of molecular diagnostic
assays to distinguish M. leprae and
M. lepromatosis. Clin Infect Dis 71(8):
e262–e269
3. Tortoli E (2006) The new mycobacteria: an
update. FEMS Immunol Med Microbiol
48:159–178
4. Gupta RS, Lo B, Son J (2018) Phylogenomics
and comparative genomic studies robustly support division of the genus Mycobacterium into
an emended genus Mycobacterium and four
novel genera. Front Microbiol 9:67
5. Tortoli E, Brown-Elliott BA, Chalmers JD,
Cirillo DM, Daley CL, Emler S, Floto RA,
Garcia MJ, Hoefsloot W, Koh WJ, Lange C,
Loebinger M, Maurer FP, Morimoto K,
Niemann S, Richter E, Turenne CY,
Vasireddy R, Vasireddy S, Wagner D, Wallace
RJ Jr, Wengenack N, van Ingen J (2019) Same
meat, different gravy: ignore the new names of
mycobacteria. Eur Respir J 54:1900795
56
Elizabeth Wallace et al.