13. A perimeter ring of sterile dH 2 O is used in microtiter assays to
insulate the plate from condensation and prevent disparity of
cultures in the outer wells.
14. Ensure that plates remain at approximately 37
C so that
condensation does not build up on the seal, as this will affect
the OD reading. After reading, experimental plates should be
placed immediately back into the anaerobic cabinet.
15. Due to the demanding nature of resuscitation, the lag phase is
quite extended. In rich media, the lag phase will last approximately 300 h and in minimal cholesterol media, the lag phase
will last 200 h. After this extended lag phase, the cultures will
enter log phase.
16. Parafilm should be used to maintain NRP in the cultures whilst
outside the hypoxic conditions of the anaerobic cabinet.
Ensure sufficient parafilm is used to seal centrifuge tube when
centrifuging cultures, as the stress of centrifugation at 4
C can
cause rips in the parafilm and could compromise the hypoxic
nature of the culture.
References
1. Wayne LG, Sohaskey CD (2001) Nonreplicating persistence of Mycobacterium Tuberculosis.
Annu Rev Microbiol 55:139–163
2. Gibson SER, Harrison J, Cox JAG (2018)
Modelling a silent epidemic: a review of the
in vitro models of latent tuberculosis. Pathogens 7(4):88. https://doi.org/10.3390/
pathogens7040088
3. Martin CJ, Carey AF, Fortune SM (2016) A
bug’s life in the granuloma. Semin Immunopathol 38:213–220
4. Parish T, Stoker NG, Wayne LG (2003) In
vitro model of hypoxically induced nonreplicating persistence of Mycobacterium tuberculosis. Methods Mol Med 54:247–269. https://
doi.org/10.1385/1-59259-147-7:247
5. Betts JC, Lukey PT, Robb LC, McAdam RA,
Duncan K (2002) Evaluation of a nutrient starvation model of Mycobacterium tuberculosis
persistence by gene and protein expression
profiling. Mol Microbiol 43:717–731
6. Wipperman MF, Sampson NS, Thomas ST
(2014) Pathogen roid rage: cholesterol utilization by Mycobacterium tuberculosis. Crit Rev
Biochem Mol Biol 49:269–293
7. Voskuil MI, Schnappinger D, Visconti KC et al
(2003) Inhibition of respiration by nitric oxide
induces a Mycobacterium tuberculosis dormancy
program. J Exp Med 198:705–713
8. Griffin JE, Pandey AK, Gilmore SA et al (2012)
Cholesterol catabolism by Mycobacterium
tuberculosis requires transcriptional and metabolic adaptations. Chem Biol 19:218–227
9. Schnappinger D, Ehrt S, Voskuil MI et al
(2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages:
insights into the Phagosomal environment. J
Exp Med 198(5):693–704. https://doi.org/
10.1084/jem.20030846
10. Houben RMGJ, Dodd PJ (2016) The global
burden of latent tuberculosis infection: a
re-estimation using mathematical modelling.
PLoS Med 13:e1002152
11. WHO (2019) Global TB Rep 2019
12. Wayne LG, Hayes LG (1996) An in vitro
model for sequential study of Shiftdown of
Mycobacterium tuberculosis through two stages
of nonreplicating persistence. Infect Immun 64
(6):2062–2069
13. Cho SH, Goodlett D, Franzblau S (2006)
ICAT-based comparative proteomic analysis of
non-replicating persistent Mycobacterium
tuberculosis. Tuberculosis 86:445–460
14. Dickinson JM, Aber VR, Mitchison DA (1977)
Bactericidal activity of streptomycin, isoniazid,
rifampin, ethambutol, and pyrazinamide alone
and in combination against Mycobacterium
tuberculosis. Am Rev Respir Dis 116:627–635
15. Cho SH, Warit S, Wan B et al (2007) Lowoxygen-recovery assay for high-throughput
screening of compounds against nonreplicating
In vitro Drug Screening of Hypoxic NRP Mycobacteria
259
insulate the plate from condensation and prevent disparity of
cultures in the outer wells.
14. Ensure that plates remain at approximately 37
C so that
condensation does not build up on the seal, as this will affect
the OD reading. After reading, experimental plates should be
placed immediately back into the anaerobic cabinet.
15. Due to the demanding nature of resuscitation, the lag phase is
quite extended. In rich media, the lag phase will last approximately 300 h and in minimal cholesterol media, the lag phase
will last 200 h. After this extended lag phase, the cultures will
enter log phase.
16. Parafilm should be used to maintain NRP in the cultures whilst
outside the hypoxic conditions of the anaerobic cabinet.
Ensure sufficient parafilm is used to seal centrifuge tube when
centrifuging cultures, as the stress of centrifugation at 4
C can
cause rips in the parafilm and could compromise the hypoxic
nature of the culture.
References
1. Wayne LG, Sohaskey CD (2001) Nonreplicating persistence of Mycobacterium Tuberculosis.
Annu Rev Microbiol 55:139–163
2. Gibson SER, Harrison J, Cox JAG (2018)
Modelling a silent epidemic: a review of the
in vitro models of latent tuberculosis. Pathogens 7(4):88. https://doi.org/10.3390/
pathogens7040088
3. Martin CJ, Carey AF, Fortune SM (2016) A
bug’s life in the granuloma. Semin Immunopathol 38:213–220
4. Parish T, Stoker NG, Wayne LG (2003) In
vitro model of hypoxically induced nonreplicating persistence of Mycobacterium tuberculosis. Methods Mol Med 54:247–269. https://
doi.org/10.1385/1-59259-147-7:247
5. Betts JC, Lukey PT, Robb LC, McAdam RA,
Duncan K (2002) Evaluation of a nutrient starvation model of Mycobacterium tuberculosis
persistence by gene and protein expression
profiling. Mol Microbiol 43:717–731
6. Wipperman MF, Sampson NS, Thomas ST
(2014) Pathogen roid rage: cholesterol utilization by Mycobacterium tuberculosis. Crit Rev
Biochem Mol Biol 49:269–293
7. Voskuil MI, Schnappinger D, Visconti KC et al
(2003) Inhibition of respiration by nitric oxide
induces a Mycobacterium tuberculosis dormancy
program. J Exp Med 198:705–713
8. Griffin JE, Pandey AK, Gilmore SA et al (2012)
Cholesterol catabolism by Mycobacterium
tuberculosis requires transcriptional and metabolic adaptations. Chem Biol 19:218–227
9. Schnappinger D, Ehrt S, Voskuil MI et al
(2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages:
insights into the Phagosomal environment. J
Exp Med 198(5):693–704. https://doi.org/
10.1084/jem.20030846
10. Houben RMGJ, Dodd PJ (2016) The global
burden of latent tuberculosis infection: a
re-estimation using mathematical modelling.
PLoS Med 13:e1002152
11. WHO (2019) Global TB Rep 2019
12. Wayne LG, Hayes LG (1996) An in vitro
model for sequential study of Shiftdown of
Mycobacterium tuberculosis through two stages
of nonreplicating persistence. Infect Immun 64
(6):2062–2069
13. Cho SH, Goodlett D, Franzblau S (2006)
ICAT-based comparative proteomic analysis of
non-replicating persistent Mycobacterium
tuberculosis. Tuberculosis 86:445–460
14. Dickinson JM, Aber VR, Mitchison DA (1977)
Bactericidal activity of streptomycin, isoniazid,
rifampin, ethambutol, and pyrazinamide alone
and in combination against Mycobacterium
tuberculosis. Am Rev Respir Dis 116:627–635
15. Cho SH, Warit S, Wan B et al (2007) Lowoxygen-recovery assay for high-throughput
screening of compounds against nonreplicating
In vitro Drug Screening of Hypoxic NRP Mycobacteria
259