has been mostly decolorized, this can appear that entry into
NRP stage I has been achieved. However, upon treatment with
a fast-acting antimicrobial such as rifampicin (which is not
active against NRP mycobacteria), the culture will lyse. This
indicates that the culture had not yet entered the NRP state, as
the culture was still susceptible to rifampicin. After, 48–72 h in
the hypoxia-induced rich media NRP assay, the culture is no
longer susceptible to rifampicin, confirming entry into NRP. In
the hypoxic minimal cholesterol media NRP assay, the culture
is no longer susceptible to rifampicin after 32–48 h.
9. Parafilm will prevent aeration of the culture tube outside the
anaerobic cabinet but only for a short amount of time. Make
sure that the culture is sealed fully with paraffin wax or placed
back into the anaerobic cabinet before 30 min has passed or
re-aeration of the culture is likely.
10. Mycobacteria grown in minimal cholesterol media tolerate the
pressures of entering NRP more so than mycobacteria in rich
media. Cultures in minimal cholesterol medium enter NRP
stage I faster, after 32 h in the anaerobic cabinet. As this is
faster than in rich medium, if running the two assays simultaneously, care must be taken as to when to challenge with
antimicrobials. Dosing in rich medium at 32 h is too early as
bacteria are mostly in a logarithmic state. A potential middle
ground is dosing at 48 h, which is sufficient for bacteria to
reach NRP stage I in rich medium and before they enter NRP
stage II in minimal cholesterol medium.
11. The hypoxic minimal cholesterol medium NRP assay combines
hypoxia and nutrient deprivation—both key elements of the
granuloma—to produce a phenotype which combines elements from both the hypoxia-induced rich medium NRP
assay and the nutrient starvation model. This phenotype represents a more physiologically accurate model of the clinical
phenotype. In this model, methylene blue is decolorized, as
in the Wayne model. Nutrient starvation models even when
sealed do not decolorize methylene blue. In addition, the
Wayne model shows susceptibility to MET, whereas nutrient
starvation models do not. The hypoxic minimal cholesterol
medium NRP assay likewise does not show any susceptibility
to MET.
12. After entry into NRP stage I, mycobacterial cells will swell in
size due to lipid changes at the cell wall. This will alter the
optical density reading at 600 nm, resulting in a change from
0.3 to 0.5 [5, 17, 18]. This is despite the mycobacteria not
replicating, as can be seen using CFU/mL counts.
258
Savannah E. R. Gibson et al.
NRP stage I has been achieved. However, upon treatment with
a fast-acting antimicrobial such as rifampicin (which is not
active against NRP mycobacteria), the culture will lyse. This
indicates that the culture had not yet entered the NRP state, as
the culture was still susceptible to rifampicin. After, 48–72 h in
the hypoxia-induced rich media NRP assay, the culture is no
longer susceptible to rifampicin, confirming entry into NRP. In
the hypoxic minimal cholesterol media NRP assay, the culture
is no longer susceptible to rifampicin after 32–48 h.
9. Parafilm will prevent aeration of the culture tube outside the
anaerobic cabinet but only for a short amount of time. Make
sure that the culture is sealed fully with paraffin wax or placed
back into the anaerobic cabinet before 30 min has passed or
re-aeration of the culture is likely.
10. Mycobacteria grown in minimal cholesterol media tolerate the
pressures of entering NRP more so than mycobacteria in rich
media. Cultures in minimal cholesterol medium enter NRP
stage I faster, after 32 h in the anaerobic cabinet. As this is
faster than in rich medium, if running the two assays simultaneously, care must be taken as to when to challenge with
antimicrobials. Dosing in rich medium at 32 h is too early as
bacteria are mostly in a logarithmic state. A potential middle
ground is dosing at 48 h, which is sufficient for bacteria to
reach NRP stage I in rich medium and before they enter NRP
stage II in minimal cholesterol medium.
11. The hypoxic minimal cholesterol medium NRP assay combines
hypoxia and nutrient deprivation—both key elements of the
granuloma—to produce a phenotype which combines elements from both the hypoxia-induced rich medium NRP
assay and the nutrient starvation model. This phenotype represents a more physiologically accurate model of the clinical
phenotype. In this model, methylene blue is decolorized, as
in the Wayne model. Nutrient starvation models even when
sealed do not decolorize methylene blue. In addition, the
Wayne model shows susceptibility to MET, whereas nutrient
starvation models do not. The hypoxic minimal cholesterol
medium NRP assay likewise does not show any susceptibility
to MET.
12. After entry into NRP stage I, mycobacterial cells will swell in
size due to lipid changes at the cell wall. This will alter the
optical density reading at 600 nm, resulting in a change from
0.3 to 0.5 [5, 17, 18]. This is despite the mycobacteria not
replicating, as can be seen using CFU/mL counts.
258
Savannah E. R. Gibson et al.