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MiddleBrook Pharmaceuticals middlebrook 7h9 medium
FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in <t>Middlebrook</t> <t>7H9</t> medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
Middlebrook 7h9 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection"

Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00089

FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
Figure Legend Snippet: FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

Techniques Used: Infection

2) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

3) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

4) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

5) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

6) Product Images from "Copper resistance is essential for virulence of Mycobacterium tuberculosis"

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1009261108

MctB is required for copper resistance of and maintaining a low intracellular copper concentration in M. smegmatis . ( A ) Expression of ms3747 in M. smegmatis . Proteins were extracted with 2% SDS from WT M. smegmatis , the ms3747 mutant ML77, and ML77 complemented with the ms3747 expression vector pML451. Proteins were detected in a Western blot by using the monoclonal antibody 5D1.23. ( B ) Serial dilutions of cultures of M. smegmatis SMR5 (WT), ML77 (Δ mctB ), and ML77 complemented with mctB were spotted on 7H10 agar plates without or with CuSO4 at a concentration of 25 μM. ( C ) M. smegmatis SMR5 (black bars) and the Δ ms3747 mutant ML77 (gray bars) were grown in self-made Middlebrook 7H9 medium with 0, 6.3, or 25 μM CuSO 4 . Samples were taken after growth for 36 h. Copper was determined by measuring the absorption of the Cu(II)–dithizone complex at 553 nm.
Figure Legend Snippet: MctB is required for copper resistance of and maintaining a low intracellular copper concentration in M. smegmatis . ( A ) Expression of ms3747 in M. smegmatis . Proteins were extracted with 2% SDS from WT M. smegmatis , the ms3747 mutant ML77, and ML77 complemented with the ms3747 expression vector pML451. Proteins were detected in a Western blot by using the monoclonal antibody 5D1.23. ( B ) Serial dilutions of cultures of M. smegmatis SMR5 (WT), ML77 (Δ mctB ), and ML77 complemented with mctB were spotted on 7H10 agar plates without or with CuSO4 at a concentration of 25 μM. ( C ) M. smegmatis SMR5 (black bars) and the Δ ms3747 mutant ML77 (gray bars) were grown in self-made Middlebrook 7H9 medium with 0, 6.3, or 25 μM CuSO 4 . Samples were taken after growth for 36 h. Copper was determined by measuring the absorption of the Cu(II)–dithizone complex at 553 nm.

Techniques Used: Concentration Assay, Expressing, Mutagenesis, Plasmid Preparation, Western Blot

7) Product Images from "A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis ▿"

Article Title: A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis ▿

Journal:

doi: 10.1128/JB.00257-07

Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in Middlebrook 7H9 medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation
Figure Legend Snippet: Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in Middlebrook 7H9 medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation

Techniques Used:

8) Product Images from "A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis"

Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Journal: mBio

doi: 10.1128/mBio.01388-17

The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .
Figure Legend Snippet: The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .

Techniques Used: Mutagenesis, Incubation, Expressing

9) Product Images from "Mycobacteriophage SWU1 gp39 can potentiate multiple antibiotics against Mycobacterium via altering the cell wall permeability"

Article Title: Mycobacteriophage SWU1 gp39 can potentiate multiple antibiotics against Mycobacterium via altering the cell wall permeability

Journal: Scientific Reports

doi: 10.1038/srep28701

WT-pAL-gp39 increased bacterial growth defect following exposure to multiple antibiotics. The bacterial strains of WT-pAL, WT-pAL-gp39 ( A ), WT-pAL-E ( C ) and WT-pAL-S ( D ) were grown in M9 medium ( B ) or Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 0.2% glycerinum and 0.25% acetamide, with or without hygromycin (100 μg/ml). The OD 600 were determined at an interval of 3 h. ( E ) Ten-fold serial dilutions of WT-pAL and WT-pAL-gp39 were spotted on Middlebrook 7H10 containing isoniazide (4 μg/ml), erythromycin (16μg/ml), norfloxacin (1μg/ml) and ampicilin (100 μg/ml). Then the result was recorded when incubated at 37 °C for 3 days. The data reported represent the means (n = 3) ± SD (standard deviation).
Figure Legend Snippet: WT-pAL-gp39 increased bacterial growth defect following exposure to multiple antibiotics. The bacterial strains of WT-pAL, WT-pAL-gp39 ( A ), WT-pAL-E ( C ) and WT-pAL-S ( D ) were grown in M9 medium ( B ) or Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 0.2% glycerinum and 0.25% acetamide, with or without hygromycin (100 μg/ml). The OD 600 were determined at an interval of 3 h. ( E ) Ten-fold serial dilutions of WT-pAL and WT-pAL-gp39 were spotted on Middlebrook 7H10 containing isoniazide (4 μg/ml), erythromycin (16μg/ml), norfloxacin (1μg/ml) and ampicilin (100 μg/ml). Then the result was recorded when incubated at 37 °C for 3 days. The data reported represent the means (n = 3) ± SD (standard deviation).

Techniques Used: Incubation, Standard Deviation

10) Product Images from "Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †"

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01402-10

(A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at
Figure Legend Snippet: (A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at

Techniques Used: Cell Culture, Incubation

11) Product Images from "N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis"

Article Title: N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis

Journal: Journal of Bacteriology

doi:

Growth of mutant 49 in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins: MSH and MSH precursor contents are expressed in micromoles per gram of RDW.
Figure Legend Snippet: Growth of mutant 49 in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins: MSH and MSH precursor contents are expressed in micromoles per gram of RDW.

Techniques Used: Mutagenesis

12) Product Images from "Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection"

Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00089

FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
Figure Legend Snippet: FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

Techniques Used: Infection

13) Product Images from "Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy"

Article Title: Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy

Journal: mBio

doi: 10.1128/mBio.00938-18

mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).
Figure Legend Snippet: mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).

Techniques Used: In Vitro, Standard Deviation, Infection

mc 2 7901 and mc 2 7902 generate INH persisters in culture. (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100 and treated with INH (1 mg/liter). Samples were taken at the indicated time points, diluted, and plated for CFU. Mean with standard deviation is plotted ( n = 2). (B) mc 2 7901 and mc 2 7902 cultures treated or not with INH (1 mg/liter) for 2 days were infected with the phage Φ 2 DRM9 and analyzed by flow cytometry. Phage Φ 2 DRM9 contains both the L5 promoter driving GFP (mVenus) expression and the INH persister-specific dnaK promoter fused to the red fluorescent protein gene (RFP, tdTomato). The panels show the high-RFP population back-gated for GFP expression, representing the persister population (low GFP/high RFP).
Figure Legend Snippet: mc 2 7901 and mc 2 7902 generate INH persisters in culture. (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100 and treated with INH (1 mg/liter). Samples were taken at the indicated time points, diluted, and plated for CFU. Mean with standard deviation is plotted ( n = 2). (B) mc 2 7901 and mc 2 7902 cultures treated or not with INH (1 mg/liter) for 2 days were infected with the phage Φ 2 DRM9 and analyzed by flow cytometry. Phage Φ 2 DRM9 contains both the L5 promoter driving GFP (mVenus) expression and the INH persister-specific dnaK promoter fused to the red fluorescent protein gene (RFP, tdTomato). The panels show the high-RFP population back-gated for GFP expression, representing the persister population (low GFP/high RFP).

Techniques Used: Standard Deviation, Infection, Flow Cytometry, Cytometry, Expressing

mc 2 7901- and mc 2 7902-derived MDR strains grow slower than parental strains in vitro and are killed by second-line TB drugs or nutrient starvation. (A) Log-phase cultures were diluted 1/100, and growth was followed by recording optical density at 600 nm. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1. At the indicated time points, macrophages were lysed to determine bacterial titers. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) PLAM-supplemented log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were treated with EOK (ethionamide [25 mg/liter], ofloxacin [5 mg/liter], kanamycin [20 mg/liter]). (D) Log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were washed five times in PBS and resuspended in Middlebrook 7H9-OADC-glycerol-tyloxapol containing PLAM (dilution factor 1/100) or not. In experiments in panels B, C, and D, the strains were initially grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM. Samples were taken at the indicated time points, diluted, and plated for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. Means with standard deviations are plotted ( n = 3).
Figure Legend Snippet: mc 2 7901- and mc 2 7902-derived MDR strains grow slower than parental strains in vitro and are killed by second-line TB drugs or nutrient starvation. (A) Log-phase cultures were diluted 1/100, and growth was followed by recording optical density at 600 nm. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1. At the indicated time points, macrophages were lysed to determine bacterial titers. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) PLAM-supplemented log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were treated with EOK (ethionamide [25 mg/liter], ofloxacin [5 mg/liter], kanamycin [20 mg/liter]). (D) Log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were washed five times in PBS and resuspended in Middlebrook 7H9-OADC-glycerol-tyloxapol containing PLAM (dilution factor 1/100) or not. In experiments in panels B, C, and D, the strains were initially grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM. Samples were taken at the indicated time points, diluted, and plated for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. Means with standard deviations are plotted ( n = 3).

Techniques Used: Derivative Assay, In Vitro, Standard Deviation, Infection

14) Product Images from "N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis"

Article Title: N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis

Journal: Journal of Bacteriology

doi:

Growth of mutant 49 in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins: MSH and MSH precursor contents are expressed in micromoles per gram of RDW.
Figure Legend Snippet: Growth of mutant 49 in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins: MSH and MSH precursor contents are expressed in micromoles per gram of RDW.

Techniques Used: Mutagenesis

15) Product Images from "PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis"

Article Title: PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

Journal: mBio

doi: 10.1128/mBio.01720-16

Characterization of gallium(III)-porphyrin (Ga-PIX) and its toxicity for M. tuberculosis . (A) Fluorescence spectra of PIX, hemin, and synthesized Ga-PIX after excitation at 410 nm. (B) Survival of M. tuberculosis (Mtb) in iron-free Middlebrook 7H9 medium containing 2 µM ferric carboxymycobactin (cMBT) and in the presence of increasing concentrations of gallium chloride (GaCl 3 ), porphyrin IX (PIX), and gallium porphyrin IX (Ga-PIX) was determined by the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates.
Figure Legend Snippet: Characterization of gallium(III)-porphyrin (Ga-PIX) and its toxicity for M. tuberculosis . (A) Fluorescence spectra of PIX, hemin, and synthesized Ga-PIX after excitation at 410 nm. (B) Survival of M. tuberculosis (Mtb) in iron-free Middlebrook 7H9 medium containing 2 µM ferric carboxymycobactin (cMBT) and in the presence of increasing concentrations of gallium chloride (GaCl 3 ), porphyrin IX (PIX), and gallium porphyrin IX (Ga-PIX) was determined by the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates.

Techniques Used: Fluorescence, Synthesized, Alamar Blue Assay

16) Product Images from "An Amidase_3 domain-containing N-acetylmuramyl-L-alanine amidase is required for mycobacterial cell division"

Article Title: An Amidase_3 domain-containing N-acetylmuramyl-L-alanine amidase is required for mycobacterial cell division

Journal: Scientific Reports

doi: 10.1038/s41598-017-01184-7

Time-lapse microscopic to assess the localization of FtsZ-rsEGFP. ( A ) M. smegmatis mc 2 155 and Δ ami1 cells were grown in Middlebrook 7H9 and subjected to time-lapse analysis to monitor FtsZ bundling and constriction, indicated by white arrows. In the Δ ami mutant, FtsZ is able to form rings, but these eventually collapse. ( B ) Time-lapse microscopic to assess the localization of FtsZ-rsEGFP in cells stained with FM4-64. Cells were grown in Middlebrook 7H9 and stained with FM4-64 during time lapse analysis. White color indicates FM4-64 staining. Depicted in panel I is the formation of FtsZ rings in a single cellular compartment, followed by collapse and repolymerization. In panel II, FtsZ rings bundle in two connected daughter cells, but these do not coalese at the septum instead, failure to separate leads to the formation of a lateral bud at the septum (red arrows). In panel III, failure to separate leads to the formation of a lateral bud, not located at the septum, with subsequent formation of a FtsZ ring in the lateral bud (magenta arrows). Scale bar = 5 µm. Time frame is in hours.
Figure Legend Snippet: Time-lapse microscopic to assess the localization of FtsZ-rsEGFP. ( A ) M. smegmatis mc 2 155 and Δ ami1 cells were grown in Middlebrook 7H9 and subjected to time-lapse analysis to monitor FtsZ bundling and constriction, indicated by white arrows. In the Δ ami mutant, FtsZ is able to form rings, but these eventually collapse. ( B ) Time-lapse microscopic to assess the localization of FtsZ-rsEGFP in cells stained with FM4-64. Cells were grown in Middlebrook 7H9 and stained with FM4-64 during time lapse analysis. White color indicates FM4-64 staining. Depicted in panel I is the formation of FtsZ rings in a single cellular compartment, followed by collapse and repolymerization. In panel II, FtsZ rings bundle in two connected daughter cells, but these do not coalese at the septum instead, failure to separate leads to the formation of a lateral bud at the septum (red arrows). In panel III, failure to separate leads to the formation of a lateral bud, not located at the septum, with subsequent formation of a FtsZ ring in the lateral bud (magenta arrows). Scale bar = 5 µm. Time frame is in hours.

Techniques Used: Mutagenesis, Staining

( A ) Time-lapse microscopic analysis of growth in the Δ ami1 mutant strain. Cells were grown and imaged in Middlebrook 7H9 media. Shown is the growth of mc 2 155 (top panel), which displays a growth and cell division pattern that is typical of mycobacteria with polar extension of cells (red arrows), as well as the Δ ami1 strain, which exhibited the formation of lateral buds (yellow arrows) that subsequently formed branches. Scale = 5 µm. Ectopic polar growth and Y-formed cell division was also noted (orange arrows). Time frame is in hours. The histogram depicts the proportion of microcolonies with polar/lateral budding. For the Δ ami1 mutant and wild type, 74 and 59 micro-colonies were assessed.
Figure Legend Snippet: ( A ) Time-lapse microscopic analysis of growth in the Δ ami1 mutant strain. Cells were grown and imaged in Middlebrook 7H9 media. Shown is the growth of mc 2 155 (top panel), which displays a growth and cell division pattern that is typical of mycobacteria with polar extension of cells (red arrows), as well as the Δ ami1 strain, which exhibited the formation of lateral buds (yellow arrows) that subsequently formed branches. Scale = 5 µm. Ectopic polar growth and Y-formed cell division was also noted (orange arrows). Time frame is in hours. The histogram depicts the proportion of microcolonies with polar/lateral budding. For the Δ ami1 mutant and wild type, 74 and 59 micro-colonies were assessed.

Techniques Used: Mutagenesis

Time-lapse microscopic to assess the localization of DivIVA-rsEGFP. M. smegmatis mc 2 155 and Δ ami1 cells were grown in Middlebrook 7H9 and subjected to time-lapse analysis to monitor DivIVA localization, indicated by white arrows in the wild type. In the Δ ami mutant (panel I), DivIVA localizes at midcell, but the failure to separate leads to the formation of ectopic cell poles, which extend into lateral buds (yellow arrow). Alternatively, in the Δ ami mutant (panel II), DivIVA traffics to the lateral axis of the cell and nucleates lateral branching (blue arrows). Scale bar = 5 µm. Time frame is in hours. The histogram depicts the proportion of cells with ectopic DivIVA localization. For the Δ ami1 mutant and wild type, 1404 and 1626 individual cells were assessed. *The assessment of the proportion of cells with branches is subject to underestimation as cellular crowding makes accurate quantification difficult. Reported here is the proportion of cells with clearly visible branching patterns.
Figure Legend Snippet: Time-lapse microscopic to assess the localization of DivIVA-rsEGFP. M. smegmatis mc 2 155 and Δ ami1 cells were grown in Middlebrook 7H9 and subjected to time-lapse analysis to monitor DivIVA localization, indicated by white arrows in the wild type. In the Δ ami mutant (panel I), DivIVA localizes at midcell, but the failure to separate leads to the formation of ectopic cell poles, which extend into lateral buds (yellow arrow). Alternatively, in the Δ ami mutant (panel II), DivIVA traffics to the lateral axis of the cell and nucleates lateral branching (blue arrows). Scale bar = 5 µm. Time frame is in hours. The histogram depicts the proportion of cells with ectopic DivIVA localization. For the Δ ami1 mutant and wild type, 1404 and 1626 individual cells were assessed. *The assessment of the proportion of cells with branches is subject to underestimation as cellular crowding makes accurate quantification difficult. Reported here is the proportion of cells with clearly visible branching patterns.

Techniques Used: Mutagenesis

( A ) Time-lapse microscopic analysis of septal cell wall release in the Δ ami1 mutant strain. Cells were grown and imaged in Middlebrook 7H9 media. ( A ) Shown is the growth of the Δ ami1 strain with release of material from the septum (green arrow) suggestive of dysregulated septal envelope synthesis. ( B ) Depicts growth of the Δ ami1 strain in the presence of FM4-64 (last two frames), the black arrows highlight positive FM4-64 staining of the cell envelope material released from mid-cell. Scale = 5 µm. Time frame is in hours.
Figure Legend Snippet: ( A ) Time-lapse microscopic analysis of septal cell wall release in the Δ ami1 mutant strain. Cells were grown and imaged in Middlebrook 7H9 media. ( A ) Shown is the growth of the Δ ami1 strain with release of material from the septum (green arrow) suggestive of dysregulated septal envelope synthesis. ( B ) Depicts growth of the Δ ami1 strain in the presence of FM4-64 (last two frames), the black arrows highlight positive FM4-64 staining of the cell envelope material released from mid-cell. Scale = 5 µm. Time frame is in hours.

Techniques Used: Mutagenesis, Staining

17) Product Images from "N-acetyl-cysteine exhibits potent anti-mycobacterial activity in addition to its known anti-oxidative functions"

Article Title: N-acetyl-cysteine exhibits potent anti-mycobacterial activity in addition to its known anti-oxidative functions

Journal: BMC Microbiology

doi: 10.1186/s12866-016-0872-7

NAC restrains mycobacterial growth within THP-1 macrophages and exhibits a direct anti-mycobacterial effect on extracellular bacteria in vitro. a Human-THP-1 macrophages were infected with M. tuberculosis , M. avium or M. bovis strains at an MOI of 10 for 3 h. Extracellular bacteria were removed by washing. Cells were then cultivated for 5 days in the presence of NAC at 10 mM. CFU counts were assessed as described in Methods. b and c Mycobacteria strains were grown in Middlebrook 7H9 supplemented with OADC in 96-well plates. Metabolic activity measurements ( b ) and CFU counts ( c ) were performed as described in Methods. Significant differences were observed for the indicated experimental conditions compared to untreated cultures (* p
Figure Legend Snippet: NAC restrains mycobacterial growth within THP-1 macrophages and exhibits a direct anti-mycobacterial effect on extracellular bacteria in vitro. a Human-THP-1 macrophages were infected with M. tuberculosis , M. avium or M. bovis strains at an MOI of 10 for 3 h. Extracellular bacteria were removed by washing. Cells were then cultivated for 5 days in the presence of NAC at 10 mM. CFU counts were assessed as described in Methods. b and c Mycobacteria strains were grown in Middlebrook 7H9 supplemented with OADC in 96-well plates. Metabolic activity measurements ( b ) and CFU counts ( c ) were performed as described in Methods. Significant differences were observed for the indicated experimental conditions compared to untreated cultures (* p

Techniques Used: In Vitro, Infection, Activity Assay

NAC limits mycobacterial proliferation by acting as an anti-mycobacterial compound. Mycobacterium tuberculosis was grown in Middlebrook 7H9 supplemented with OADC as described in Methods. a Kinetic of mycobacteria growth during 7 days in the presence of NAC at indicated concentrations was verified using CFU counts. b Mycobacterial growth was evaluated at different pH as described in Methods. Fold increase of bacterial growth was calculated as the ratio of CFU number counted on days 0 and 5. Significant differences were observed for the indicated experimental conditions (*** p
Figure Legend Snippet: NAC limits mycobacterial proliferation by acting as an anti-mycobacterial compound. Mycobacterium tuberculosis was grown in Middlebrook 7H9 supplemented with OADC as described in Methods. a Kinetic of mycobacteria growth during 7 days in the presence of NAC at indicated concentrations was verified using CFU counts. b Mycobacterial growth was evaluated at different pH as described in Methods. Fold increase of bacterial growth was calculated as the ratio of CFU number counted on days 0 and 5. Significant differences were observed for the indicated experimental conditions (*** p

Techniques Used:

18) Product Images from "A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis"

Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Journal: mBio

doi: 10.1128/mBio.01388-17

The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .
Figure Legend Snippet: The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .

Techniques Used: Mutagenesis, Incubation, Expressing

19) Product Images from "Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †"

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01402-10

(A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at
Figure Legend Snippet: (A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at

Techniques Used: Cell Culture, Incubation

20) Product Images from "Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates"

Article Title: Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates

Journal: Frontiers in Chemistry

doi: 10.3389/fchem.2018.00519

51 V NMR (78.9 MHz) spectra are shown of solution of decavanadate (10 mM V 10 , 100 mM V-atoms). The samples are from the bottom up diluted V 10 stock solution (100 mM V-atom) at pH 3.1; 10 mM V 10 in the presence of 0.48 mM and 0.97 mM citrate at pH 2.8 and 2.2, respectively; 10 mM V 10 in the presence of 24 mM P i at pH 6.9; and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 .
Figure Legend Snippet: 51 V NMR (78.9 MHz) spectra are shown of solution of decavanadate (10 mM V 10 , 100 mM V-atoms). The samples are from the bottom up diluted V 10 stock solution (100 mM V-atom) at pH 3.1; 10 mM V 10 in the presence of 0.48 mM and 0.97 mM citrate at pH 2.8 and 2.2, respectively; 10 mM V 10 in the presence of 24 mM P i at pH 6.9; and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 .

Techniques Used: Nuclear Magnetic Resonance

51 V NMR (78.9 MHz) spectra are shown of solution of colorless oxovanadate (40 mM V 1 , 40 mM V-atoms). The samples are from the bottom up diluted V 1 stock solution (40 mM V-atom) at pH 8.3; 10 mM V 10 in the presence of 24 mM P i at pH 8.1; 10 mM V 10 in the presence of 0.48 mM citrate at pH 6.3, and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 . The spectrum labeled Reference is of the capillary reference alone (top spectrum). The key to the signals: V-oligomers, V 1 monomer; V 2 , dimer; V 4 , tetramer; V 5 , pentamer; VCit, V-citrate complex; PV, vanadate-phosphate complex.
Figure Legend Snippet: 51 V NMR (78.9 MHz) spectra are shown of solution of colorless oxovanadate (40 mM V 1 , 40 mM V-atoms). The samples are from the bottom up diluted V 1 stock solution (40 mM V-atom) at pH 8.3; 10 mM V 10 in the presence of 24 mM P i at pH 8.1; 10 mM V 10 in the presence of 0.48 mM citrate at pH 6.3, and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 . The spectrum labeled Reference is of the capillary reference alone (top spectrum). The key to the signals: V-oligomers, V 1 monomer; V 2 , dimer; V 4 , tetramer; V 5 , pentamer; VCit, V-citrate complex; PV, vanadate-phosphate complex.

Techniques Used: Nuclear Magnetic Resonance, Labeling

21) Product Images from "The prrAB Two-Component System Is Essential for Mycobacterium tuberculosis Viability and Is Induced under Nitrogen-Limiting Conditions"

Article Title: The prrAB Two-Component System Is Essential for Mycobacterium tuberculosis Viability and Is Induced under Nitrogen-Limiting Conditions

Journal: Journal of Bacteriology

doi: 10.1128/JB.06258-11

prrA and prrB transcription and PrrA levels during in vitro growth. (A) Quantitation of prrA and prrB transcripts during M. tuberculosis H37Rv in vitro exponential growth in supplemented Middlebrook 7H9 broth with glycerol. The optical density at 600
Figure Legend Snippet: prrA and prrB transcription and PrrA levels during in vitro growth. (A) Quantitation of prrA and prrB transcripts during M. tuberculosis H37Rv in vitro exponential growth in supplemented Middlebrook 7H9 broth with glycerol. The optical density at 600

Techniques Used: In Vitro, Quantitation Assay

22) Product Images from "PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence"

Article Title: PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence

Journal: Scientific Reports

doi: 10.1038/srep21624

Cell surface of Msmeg-PE11 is highly hydrophobic. ( a ) Pellicle formation of Msmeg-pVV or Msmeg-PE11 was monitored by growing standing cultures of the strains without shaking in Middlebrook 7H9 medium in absence of Tween 80 at 37 °C for various time points. ( b ) Biofilm formation was quantified by crystal violet staining for which Msmeg-pVV or Msmeg-PE11 cells were washed, stained with 1% crystal violet and ethanol extract was spectrophotometrically measured at 570 nm. ( c ) Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 medium with (upper panel) or without (lower panel) 0.05% Tween-80 at 37 °C with shaking for 24 h and 48 h. Cultures were then allowed to settle at room temperature for 30 min. ( d ) In another experiment, Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 broth with congo red (100 μg/ml) and 0.05% Tween 80 for 24 h, 48 h and 72 h at 37 °C. Cells were next washed and re-suspended in acetone. Congo red in the supernatant was spectrophotometrically measured at 490 nm. Data are representative of mean ± SD of three different experiments.
Figure Legend Snippet: Cell surface of Msmeg-PE11 is highly hydrophobic. ( a ) Pellicle formation of Msmeg-pVV or Msmeg-PE11 was monitored by growing standing cultures of the strains without shaking in Middlebrook 7H9 medium in absence of Tween 80 at 37 °C for various time points. ( b ) Biofilm formation was quantified by crystal violet staining for which Msmeg-pVV or Msmeg-PE11 cells were washed, stained with 1% crystal violet and ethanol extract was spectrophotometrically measured at 570 nm. ( c ) Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 medium with (upper panel) or without (lower panel) 0.05% Tween-80 at 37 °C with shaking for 24 h and 48 h. Cultures were then allowed to settle at room temperature for 30 min. ( d ) In another experiment, Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 broth with congo red (100 μg/ml) and 0.05% Tween 80 for 24 h, 48 h and 72 h at 37 °C. Cells were next washed and re-suspended in acetone. Congo red in the supernatant was spectrophotometrically measured at 490 nm. Data are representative of mean ± SD of three different experiments.

Techniques Used: Staining, Cell Culture

23) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

24) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

25) Product Images from "PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis"

Article Title: PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

Journal: mBio

doi: 10.1128/mBio.01720-16

Characterization of gallium(III)-porphyrin (Ga-PIX) and its toxicity for M. tuberculosis . (A) Fluorescence spectra of PIX, hemin, and synthesized Ga-PIX after excitation at 410 nm. (B) Survival of M. tuberculosis (Mtb) in iron-free Middlebrook 7H9 medium containing 2 µM ferric carboxymycobactin (cMBT) and in the presence of increasing concentrations of gallium chloride (GaCl 3 ), porphyrin IX (PIX), and gallium porphyrin IX (Ga-PIX) was determined by the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates.
Figure Legend Snippet: Characterization of gallium(III)-porphyrin (Ga-PIX) and its toxicity for M. tuberculosis . (A) Fluorescence spectra of PIX, hemin, and synthesized Ga-PIX after excitation at 410 nm. (B) Survival of M. tuberculosis (Mtb) in iron-free Middlebrook 7H9 medium containing 2 µM ferric carboxymycobactin (cMBT) and in the presence of increasing concentrations of gallium chloride (GaCl 3 ), porphyrin IX (PIX), and gallium porphyrin IX (Ga-PIX) was determined by the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates.

Techniques Used: Fluorescence, Synthesized, Alamar Blue Assay

26) Product Images from "Glucose 6-Phosphate Accumulation in Mycobacteria"

Article Title: Glucose 6-Phosphate Accumulation in Mycobacteria

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.074310

Intracellular G6P concentration in M. smegmatis decreases under carbon starvation but increases under nitrogen and phosphate starvation. The bacteria grown in Middlebrook 7H9 with 1/10th carbon and nitrogen sources and 1/50th phosphate and sulfate sources at 35 °C were harvested at 72 h. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D. *, significantly different from that of control ( p
Figure Legend Snippet: Intracellular G6P concentration in M. smegmatis decreases under carbon starvation but increases under nitrogen and phosphate starvation. The bacteria grown in Middlebrook 7H9 with 1/10th carbon and nitrogen sources and 1/50th phosphate and sulfate sources at 35 °C were harvested at 72 h. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D. *, significantly different from that of control ( p

Techniques Used: Concentration Assay

Higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase correlates with an accumulation of glucose 6-phosphate in M. smegmatis . The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol at 35 °C, and the cells were collected and harvested at different time intervals to represent cells from different growth phases. Harvesting time for E. coli ( EC ) and B. megaterium ( BM ) were: exponential, 12 h; early stationary, 15 h; stationary, 24 h. Harvesting for M. smegmatis ( MS ) was at 48, 55, and 72 h, respectively. A , concentration of glucose 6-phosphate and related intracellular metabolites at different growth phases in different bacteria. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis at different growth phases in different bacteria. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D.
Figure Legend Snippet: Higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase correlates with an accumulation of glucose 6-phosphate in M. smegmatis . The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol at 35 °C, and the cells were collected and harvested at different time intervals to represent cells from different growth phases. Harvesting time for E. coli ( EC ) and B. megaterium ( BM ) were: exponential, 12 h; early stationary, 15 h; stationary, 24 h. Harvesting for M. smegmatis ( MS ) was at 48, 55, and 72 h, respectively. A , concentration of glucose 6-phosphate and related intracellular metabolites at different growth phases in different bacteria. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis at different growth phases in different bacteria. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D.

Techniques Used: Activity Assay, Mass Spectrometry, Concentration Assay

M. smegmatis , when grown under gluconeogenic conditions, still accumulates G6P, likely via up-regulation of phosphoenolpyruvate carboxykinase activity. The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol, glucose, or acetate at 35 °C and harvested at 72 h. A , concentration of glucose 6-phosphate and related intracellular metabolites in M. smegmatis grown on different carbon substrates. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis in M. smegmatis grown in different carbon substrates. Each bar represents an average from three independent experiments. The error bars indicate S.D. ***, significantly different from that of cells grown in glycerol ( p
Figure Legend Snippet: M. smegmatis , when grown under gluconeogenic conditions, still accumulates G6P, likely via up-regulation of phosphoenolpyruvate carboxykinase activity. The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol, glucose, or acetate at 35 °C and harvested at 72 h. A , concentration of glucose 6-phosphate and related intracellular metabolites in M. smegmatis grown on different carbon substrates. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis in M. smegmatis grown in different carbon substrates. Each bar represents an average from three independent experiments. The error bars indicate S.D. ***, significantly different from that of cells grown in glycerol ( p

Techniques Used: Activity Assay, Concentration Assay

27) Product Images from "Cell-Envelope Remodeling as a Determinant of Phenotypic Antibacterial Tolerance in Mycobacterium tuberculosis"

Article Title: Cell-Envelope Remodeling as a Determinant of Phenotypic Antibacterial Tolerance in Mycobacterium tuberculosis

Journal: ACS Infectious Diseases

doi: 10.1021/acsinfecdis.5b00148

Changes in osmolarity affect Mtb growth. (A) Mtb growth at various NaCl concentrations in Middlebrook 7H9 medium, monitored by OD 600 nm. (B) Effect of various NaCl concentrations on Mtb viability (CFU/mL). (C) Reversible changes in growth observed after exposure to various NaCl concentrations followed by an outgrowth at 10 mM NaCl, monitored by OD 600 nm. (D) Changes in membrane potential over time of Mtb H37Rv cultivated at 10, 125, 250, and 1000 mM NaCl. (E) Association of CoroNa Green (Na + probe) with Mtb-mCherry phagosomes in resting and activated RAW264.7 macrophages. (F) Quantitative analysis of the levels of Na + (arbitrary units) in macrophages treated or not with 5 ng/mL IFN-γ. (∗∗) p ≤ 0.01 (Student’s t test). All data shown are representative of at least two independent experiments. Scale bar = 10 μm.
Figure Legend Snippet: Changes in osmolarity affect Mtb growth. (A) Mtb growth at various NaCl concentrations in Middlebrook 7H9 medium, monitored by OD 600 nm. (B) Effect of various NaCl concentrations on Mtb viability (CFU/mL). (C) Reversible changes in growth observed after exposure to various NaCl concentrations followed by an outgrowth at 10 mM NaCl, monitored by OD 600 nm. (D) Changes in membrane potential over time of Mtb H37Rv cultivated at 10, 125, 250, and 1000 mM NaCl. (E) Association of CoroNa Green (Na + probe) with Mtb-mCherry phagosomes in resting and activated RAW264.7 macrophages. (F) Quantitative analysis of the levels of Na + (arbitrary units) in macrophages treated or not with 5 ng/mL IFN-γ. (∗∗) p ≤ 0.01 (Student’s t test). All data shown are representative of at least two independent experiments. Scale bar = 10 μm.

Techniques Used:

28) Product Images from "Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy"

Article Title: Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy

Journal: mBio

doi: 10.1128/mBio.00938-18

mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).
Figure Legend Snippet: mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).

Techniques Used: In Vitro, Standard Deviation, Infection

mc 2 7901- and mc 2 7902-derived MDR strains grow slower than parental strains in vitro and are killed by second-line TB drugs or nutrient starvation. (A) Log-phase cultures were diluted 1/100, and growth was followed by recording optical density at 600 nm. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1. At the indicated time points, macrophages were lysed to determine bacterial titers. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) PLAM-supplemented log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were treated with EOK (ethionamide [25 mg/liter], ofloxacin [5 mg/liter], kanamycin [20 mg/liter]). (D) Log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were washed five times in PBS and resuspended in Middlebrook 7H9-OADC-glycerol-tyloxapol containing PLAM (dilution factor 1/100) or not. In experiments in panels B, C, and D, the strains were initially grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM. Samples were taken at the indicated time points, diluted, and plated for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. Means with standard deviations are plotted ( n = 3).
Figure Legend Snippet: mc 2 7901- and mc 2 7902-derived MDR strains grow slower than parental strains in vitro and are killed by second-line TB drugs or nutrient starvation. (A) Log-phase cultures were diluted 1/100, and growth was followed by recording optical density at 600 nm. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1. At the indicated time points, macrophages were lysed to determine bacterial titers. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) PLAM-supplemented log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were treated with EOK (ethionamide [25 mg/liter], ofloxacin [5 mg/liter], kanamycin [20 mg/liter]). (D) Log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were washed five times in PBS and resuspended in Middlebrook 7H9-OADC-glycerol-tyloxapol containing PLAM (dilution factor 1/100) or not. In experiments in panels B, C, and D, the strains were initially grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM. Samples were taken at the indicated time points, diluted, and plated for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. Means with standard deviations are plotted ( n = 3).

Techniques Used: Derivative Assay, In Vitro, Standard Deviation, Infection

29) Product Images from "Interfering With DNA Decondensation as a Strategy Against Mycobacteria"

Article Title: Interfering With DNA Decondensation as a Strategy Against Mycobacteria

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02034

M. tuberculosis condenses DNA during starvation. M. tuberculosis mc 2 6030 was cultured in ADC-supplemented Middlebrook 7H9 medium before being starved in PBS. Lipid distribution and DNA localization was imaged using Nile Red (red) and Hoechst (green) respectively at day 0, 6, and 10 (A) and percentage bacteria with condensed DNA was quantified at day 0, 3, 6, and 10. Values represent mean percentage bacteria with condense DNA ± standard error, pooled data of 2 measurements, * P
Figure Legend Snippet: M. tuberculosis condenses DNA during starvation. M. tuberculosis mc 2 6030 was cultured in ADC-supplemented Middlebrook 7H9 medium before being starved in PBS. Lipid distribution and DNA localization was imaged using Nile Red (red) and Hoechst (green) respectively at day 0, 6, and 10 (A) and percentage bacteria with condensed DNA was quantified at day 0, 3, 6, and 10. Values represent mean percentage bacteria with condense DNA ± standard error, pooled data of 2 measurements, * P

Techniques Used: Cell Culture

30) Product Images from "A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress"

Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

Journal:

doi: 10.1128/JB.00393-06

Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the
Figure Legend Snippet: Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the

Techniques Used:

Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.
Figure Legend Snippet: Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.

Techniques Used:

Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.
Figure Legend Snippet: Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.

Techniques Used:

Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2
Figure Legend Snippet: Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2

Techniques Used:

31) Product Images from "Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice"

Article Title: Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006043

Silencing of otsB2 leads to T6P accumulation in M . tuberculosis . (A) Cells of M . tuberculosis wild-type and the c- otsB2 -tet-on mutant were grown in Middlebrook 7H9 liquid medium at different ATc concentrations for 6 days. Hot water extracts obtained from 7.5×10 6 cells were analyzed by thin-layer chromatography, demonstrating the gradual accumulation of a substance co-migrating with an authentic T6P standard. Given that the volume of the cytosol of M . tuberculosis cells is 0.21 μm 3 on average [ 49 ], the cytosolic T6P concentration in stressed cells can be estimated to be in the range from 5–10 mM. (B) 1 H-NMR spectroscopy confirms the presence of T6P in hot water extracts of partially silenced cells of the c- otsB2 -tet-on mutant, whereas no T6P was detectable in extracts of fully induced cells of the c- otsB2 -tet-on mutant or M . tuberculosis wild-type. (C) Silencing of otsB2 in a Δ otsA (u) mutant does not result in a growth defect. Cells were grown in 96-well microtiter plates for 6 days, and growth was quantified using the resazurin microplate assay. Values are means of triplicates ± SEM. (D) Silencing of otsB2 in a Δ otsA (u) mutant does not result in T6P accumulation. Cells were grown in Middlebrook 7H9 liquid medium at different ATc concentrations for 6 days. Hot water extracts obtained from 7.5×10 6 cells were analyzed by thin-layer chromatography.
Figure Legend Snippet: Silencing of otsB2 leads to T6P accumulation in M . tuberculosis . (A) Cells of M . tuberculosis wild-type and the c- otsB2 -tet-on mutant were grown in Middlebrook 7H9 liquid medium at different ATc concentrations for 6 days. Hot water extracts obtained from 7.5×10 6 cells were analyzed by thin-layer chromatography, demonstrating the gradual accumulation of a substance co-migrating with an authentic T6P standard. Given that the volume of the cytosol of M . tuberculosis cells is 0.21 μm 3 on average [ 49 ], the cytosolic T6P concentration in stressed cells can be estimated to be in the range from 5–10 mM. (B) 1 H-NMR spectroscopy confirms the presence of T6P in hot water extracts of partially silenced cells of the c- otsB2 -tet-on mutant, whereas no T6P was detectable in extracts of fully induced cells of the c- otsB2 -tet-on mutant or M . tuberculosis wild-type. (C) Silencing of otsB2 in a Δ otsA (u) mutant does not result in a growth defect. Cells were grown in 96-well microtiter plates for 6 days, and growth was quantified using the resazurin microplate assay. Values are means of triplicates ± SEM. (D) Silencing of otsB2 in a Δ otsA (u) mutant does not result in T6P accumulation. Cells were grown in Middlebrook 7H9 liquid medium at different ATc concentrations for 6 days. Hot water extracts obtained from 7.5×10 6 cells were analyzed by thin-layer chromatography.

Techniques Used: Mutagenesis, Thin Layer Chromatography, Concentration Assay, Nuclear Magnetic Resonance, Spectroscopy

32) Product Images from "Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase"

Article Title: Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01339-16

M. tuberculosis H37Rv in vitro kill kinetics. Cells were incubated with compounds at 20 times their MIC values for different times over 14 days in 10 ml of Middlebrook 7H9 medium containing 10% (vol/vol) ADC and 0.05% (vol/vol) Tween 80. The MIC values used in this experiment were 0.013 μg/ml, 0.6 μg/ml, and 0.06 μg/ml for compound 14, linezolid, and moxifloxacin, respectively. The means and the standard deviations of data from triplicate cultures at each point are shown.
Figure Legend Snippet: M. tuberculosis H37Rv in vitro kill kinetics. Cells were incubated with compounds at 20 times their MIC values for different times over 14 days in 10 ml of Middlebrook 7H9 medium containing 10% (vol/vol) ADC and 0.05% (vol/vol) Tween 80. The MIC values used in this experiment were 0.013 μg/ml, 0.6 μg/ml, and 0.06 μg/ml for compound 14, linezolid, and moxifloxacin, respectively. The means and the standard deviations of data from triplicate cultures at each point are shown.

Techniques Used: In Vitro, Incubation

33) Product Images from "The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth"

Article Title: The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth

Journal: Bioorganic & medicinal chemistry letters

doi: 10.1016/j.bmcl.2011.06.057

Disruption of cysQ attenuates Mtb growth in vitro. Wild-type (◆), Δ cysQ (■), and Δ cysQ :: cysQ (▲) Mtb were grown in Middlebrook 7H9 medium at 37 °C on an orbital shaker. Bacterial growth was monitored over a period of 5 d by measuring optical density (OD 600 ). The addition of either 2 mM cysteine (long dash) or methionine (short dash) to the growth medium did not restore Δ cysQ growth. These results represent the averaged data from triplicate cultures.
Figure Legend Snippet: Disruption of cysQ attenuates Mtb growth in vitro. Wild-type (◆), Δ cysQ (■), and Δ cysQ :: cysQ (▲) Mtb were grown in Middlebrook 7H9 medium at 37 °C on an orbital shaker. Bacterial growth was monitored over a period of 5 d by measuring optical density (OD 600 ). The addition of either 2 mM cysteine (long dash) or methionine (short dash) to the growth medium did not restore Δ cysQ growth. These results represent the averaged data from triplicate cultures.

Techniques Used: In Vitro

34) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

35) Product Images from "Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo "

Article Title: Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

Journal: Infection and Immunity

doi: 10.1128/IAI.72.1.515-526.2004

In vitro growth kinetics of M. tuberculosis Erdman wild type and the five rpf deletion mutant strains in Middlebrook 7H9 medium at 37°C with agitation. The ΔRv1009 strain used in this experiment was mc 2 3126. We also found that strain mc
Figure Legend Snippet: In vitro growth kinetics of M. tuberculosis Erdman wild type and the five rpf deletion mutant strains in Middlebrook 7H9 medium at 37°C with agitation. The ΔRv1009 strain used in this experiment was mc 2 3126. We also found that strain mc

Techniques Used: In Vitro, Mutagenesis

36) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.077297

Subcellular localization and growth phase-dependent expression of MSMEG_4247 and MSMEG_4241 in wild-type cells. A , sucrose density ( dashed line ) and protein concentration ( open circle ) profiles of wild-type cell lysate fractionated by a density sedimentation. B , PIM biosynthetic activities of each fraction measured by GDP-[ 3 H]mannose radiolabeling. PPM , polyprenol-phosphate-mannose. C , Western blotting using anti-MSMEG_4247 ( top ), anti-MSMEG_4241 ( middle ), or anti-PimB′ ( bottom ) antibodies. D , growth phase-dependent changes in LM/LAM levels of wild type cells grown in Middlebrook 7H9 broth. Culture was initiated by 1:100 dilution of a confluent starter culture, and aliquots were collected at the indicated time points, which correspond to logarithmic (18 h), early stationary (41 h), and late stationary (90 h) phases. Purified LM/LAM were separated by SDS-PAGE and visualized by carbohydrate staining. Loading was adjusted for equal cell pellet equivalents. E , growth phase-dependent changes in levels of MSMEG_4247 and MSMEG_4241 detected by Western blotting using anti-MSMEG_4247 or anti MSMEG_4241 antibodies. Loading was adjusted to 15 μg of protein/lane.
Figure Legend Snippet: Subcellular localization and growth phase-dependent expression of MSMEG_4247 and MSMEG_4241 in wild-type cells. A , sucrose density ( dashed line ) and protein concentration ( open circle ) profiles of wild-type cell lysate fractionated by a density sedimentation. B , PIM biosynthetic activities of each fraction measured by GDP-[ 3 H]mannose radiolabeling. PPM , polyprenol-phosphate-mannose. C , Western blotting using anti-MSMEG_4247 ( top ), anti-MSMEG_4241 ( middle ), or anti-PimB′ ( bottom ) antibodies. D , growth phase-dependent changes in LM/LAM levels of wild type cells grown in Middlebrook 7H9 broth. Culture was initiated by 1:100 dilution of a confluent starter culture, and aliquots were collected at the indicated time points, which correspond to logarithmic (18 h), early stationary (41 h), and late stationary (90 h) phases. Purified LM/LAM were separated by SDS-PAGE and visualized by carbohydrate staining. Loading was adjusted for equal cell pellet equivalents. E , growth phase-dependent changes in levels of MSMEG_4247 and MSMEG_4241 detected by Western blotting using anti-MSMEG_4247 or anti MSMEG_4241 antibodies. Loading was adjusted to 15 μg of protein/lane.

Techniques Used: Expressing, Protein Concentration, Sedimentation, Radioactivity, Western Blot, Laser Capture Microdissection, Purification, SDS Page, Staining

37) Product Images from "Glucose 6-Phosphate Accumulation in Mycobacteria"

Article Title: Glucose 6-Phosphate Accumulation in Mycobacteria

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.074310

Intracellular G6P concentration in M. smegmatis decreases under carbon starvation but increases under nitrogen and phosphate starvation. The bacteria grown in Middlebrook 7H9 with 1/10th carbon and nitrogen sources and 1/50th phosphate and sulfate sources at 35 °C were harvested at 72 h. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D. *, significantly different from that of control ( p
Figure Legend Snippet: Intracellular G6P concentration in M. smegmatis decreases under carbon starvation but increases under nitrogen and phosphate starvation. The bacteria grown in Middlebrook 7H9 with 1/10th carbon and nitrogen sources and 1/50th phosphate and sulfate sources at 35 °C were harvested at 72 h. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D. *, significantly different from that of control ( p

Techniques Used: Concentration Assay

Higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase correlates with an accumulation of glucose 6-phosphate in M. smegmatis . The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol at 35 °C, and the cells were collected and harvested at different time intervals to represent cells from different growth phases. Harvesting time for E. coli ( EC ) and B. megaterium ( BM ) were: exponential, 12 h; early stationary, 15 h; stationary, 24 h. Harvesting for M. smegmatis ( MS ) was at 48, 55, and 72 h, respectively. A , concentration of glucose 6-phosphate and related intracellular metabolites at different growth phases in different bacteria. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis at different growth phases in different bacteria. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D.
Figure Legend Snippet: Higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase correlates with an accumulation of glucose 6-phosphate in M. smegmatis . The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol at 35 °C, and the cells were collected and harvested at different time intervals to represent cells from different growth phases. Harvesting time for E. coli ( EC ) and B. megaterium ( BM ) were: exponential, 12 h; early stationary, 15 h; stationary, 24 h. Harvesting for M. smegmatis ( MS ) was at 48, 55, and 72 h, respectively. A , concentration of glucose 6-phosphate and related intracellular metabolites at different growth phases in different bacteria. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis at different growth phases in different bacteria. Each datum represents an average of data obtained from three independent experiments. The error bars indicate S.D.

Techniques Used: Activity Assay, Mass Spectrometry, Concentration Assay

M. smegmatis , when grown under gluconeogenic conditions, still accumulates G6P, likely via up-regulation of phosphoenolpyruvate carboxykinase activity. The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol, glucose, or acetate at 35 °C and harvested at 72 h. A , concentration of glucose 6-phosphate and related intracellular metabolites in M. smegmatis grown on different carbon substrates. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis in M. smegmatis grown in different carbon substrates. Each bar represents an average from three independent experiments. The error bars indicate S.D. ***, significantly different from that of cells grown in glycerol ( p
Figure Legend Snippet: M. smegmatis , when grown under gluconeogenic conditions, still accumulates G6P, likely via up-regulation of phosphoenolpyruvate carboxykinase activity. The bacteria were grown in Middlebrook 7H9 with 0.2% glycerol, glucose, or acetate at 35 °C and harvested at 72 h. A , concentration of glucose 6-phosphate and related intracellular metabolites in M. smegmatis grown on different carbon substrates. B , activities of key enzymes of pentose phosphate pathway and gluconeogenesis in M. smegmatis grown in different carbon substrates. Each bar represents an average from three independent experiments. The error bars indicate S.D. ***, significantly different from that of cells grown in glycerol ( p

Techniques Used: Activity Assay, Concentration Assay

38) Product Images from "A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis"

Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Journal: mBio

doi: 10.1128/mBio.01388-17

The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .
Figure Legend Snippet: The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .

Techniques Used: Mutagenesis, Incubation, Expressing

39) Product Images from "A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress"

Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

Journal:

doi: 10.1128/JB.00393-06

Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the
Figure Legend Snippet: Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the

Techniques Used:

Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.
Figure Legend Snippet: Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.

Techniques Used:

Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.
Figure Legend Snippet: Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.

Techniques Used:

Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2
Figure Legend Snippet: Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2

Techniques Used:

40) Product Images from "Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †"

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01402-10

(A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at
Figure Legend Snippet: (A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at

Techniques Used: Cell Culture, Incubation

41) Product Images from "Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †"

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01402-10

(A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at
Figure Legend Snippet: (A) Resistance of M. smegmatis to CuOOH is dependent in part upon Ohr. Cells were cultured to late stationary phase (17 days) in Middlebrook 7H9 medium. Cells were then suspended in the same medium at an A 600 of 0.1 and incubated with CuOOH for 2 h at

Techniques Used: Cell Culture, Incubation

42) Product Images from "Stress-Induced Reorganization of the Mycobacterial Membrane Domain"

Article Title: Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Journal: mBio

doi: 10.1128/mBio.01823-17

Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P
Figure Legend Snippet: Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P

Techniques Used: Radial Immuno Diffusion, Fluorescence, Imaging, Staining, Marker, Expressing

43) Product Images from "Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense"

Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

Journal: Scientific Reports

doi: 10.1038/srep46716

The effect of PE_PGRS41 on the growth of M. smegmatis . ( A ) PCR amplification of PE_PGRS41 encoding sequence from M. tuberculosis genome approximately 1086 bp. ( B ) M. tuberculosis PE_PGRS41 was expressed in M. smegmatis and detected using Western blotting. Cell lysates of Ms_Vec and Ms_PE_PGRS41 were subjected to Western blot to determine the expression of His-tagged PE_PGRS41 protein in M. smegmatis by anti-His antibody. ( C ) Cell fractionation experiments were performed to determine the sub-cellular localization of PE_PGRS41, GroEL2 protein serves as a cytoplasm marker of M. smegmatis , CW represents cell wall; CP represents cytoplasm. ( D ) The morphology of Ms_Vec and Ms_PE_PGRS41 were detected after induction by acetamide in the presence of hygromycin. ( E ) Ms_Vec and Ms_PE_PGRS41 were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 1% acetamide and 0.2% glycerinum, with hygromycin (100 μg/ml). The OD 600 was determined at an interval of 4 h.
Figure Legend Snippet: The effect of PE_PGRS41 on the growth of M. smegmatis . ( A ) PCR amplification of PE_PGRS41 encoding sequence from M. tuberculosis genome approximately 1086 bp. ( B ) M. tuberculosis PE_PGRS41 was expressed in M. smegmatis and detected using Western blotting. Cell lysates of Ms_Vec and Ms_PE_PGRS41 were subjected to Western blot to determine the expression of His-tagged PE_PGRS41 protein in M. smegmatis by anti-His antibody. ( C ) Cell fractionation experiments were performed to determine the sub-cellular localization of PE_PGRS41, GroEL2 protein serves as a cytoplasm marker of M. smegmatis , CW represents cell wall; CP represents cytoplasm. ( D ) The morphology of Ms_Vec and Ms_PE_PGRS41 were detected after induction by acetamide in the presence of hygromycin. ( E ) Ms_Vec and Ms_PE_PGRS41 were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 1% acetamide and 0.2% glycerinum, with hygromycin (100 μg/ml). The OD 600 was determined at an interval of 4 h.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Western Blot, Mass Spectrometry, Expressing, Cell Fractionation, Marker

44) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

45) Product Images from "Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro"

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro

Journal: Infection and Immunity

doi:

Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).
Figure Legend Snippet: Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).

Techniques Used:

46) Product Images from "Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals"

Article Title: Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Journal: Journal of Veterinary Science

doi: 10.4142/jvs.2015.16.1.31

Detection limit of the SD MPT64 Rapid test for Mycobacterium (M.) bovis . The test was used to analyze a series of diluted suspensions of M. bovis AN5 cultured in Middlebrook 7H9 broth with enrichment. A representative image of three tests is shown. The results were interpreted as positive (+), negative (-), or weak (±). NC, negative control; 1, 2.1 × 10 3 CFU/mL of M. bovis ; 2, 4.2 × 10 3 CFU/mL of M. bovis ; 3, 8.5 × 10 3 CFU/mL of M. bovis ; 4, 1.7 × 10 4 CFU/mL of M. bovis ; 5, 3.4 × 10 4 CFU/mL of M. bovis ; 6, 6.8 × 10 4 CFU/mL of M. bovis ; 7, 1.3 × 10 5 CFU/mL of M. bovis ; 8, 2.7 × 10 5 CFU/mL of M. bovis ; 9, 5.4 × 10 5 CFU/mL of M. bovis ; 10, 1.0 × 10 6 CFU/mL of M. bovis ; 11, 2.0 × 10 6 CFU/mL of M. bovis .
Figure Legend Snippet: Detection limit of the SD MPT64 Rapid test for Mycobacterium (M.) bovis . The test was used to analyze a series of diluted suspensions of M. bovis AN5 cultured in Middlebrook 7H9 broth with enrichment. A representative image of three tests is shown. The results were interpreted as positive (+), negative (-), or weak (±). NC, negative control; 1, 2.1 × 10 3 CFU/mL of M. bovis ; 2, 4.2 × 10 3 CFU/mL of M. bovis ; 3, 8.5 × 10 3 CFU/mL of M. bovis ; 4, 1.7 × 10 4 CFU/mL of M. bovis ; 5, 3.4 × 10 4 CFU/mL of M. bovis ; 6, 6.8 × 10 4 CFU/mL of M. bovis ; 7, 1.3 × 10 5 CFU/mL of M. bovis ; 8, 2.7 × 10 5 CFU/mL of M. bovis ; 9, 5.4 × 10 5 CFU/mL of M. bovis ; 10, 1.0 × 10 6 CFU/mL of M. bovis ; 11, 2.0 × 10 6 CFU/mL of M. bovis .

Techniques Used: Cell Culture, Negative Control

47) Product Images from "PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis"

Article Title: PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

Journal: mBio

doi: 10.1128/mBio.01720-16

Characterization of gallium(III)-porphyrin (Ga-PIX) and its toxicity for M. tuberculosis . (A) Fluorescence spectra of PIX, hemin, and synthesized Ga-PIX after excitation at 410 nm. (B) Survival of M. tuberculosis (Mtb) in iron-free Middlebrook 7H9 medium containing 2 µM ferric carboxymycobactin (cMBT) and in the presence of increasing concentrations of gallium chloride (GaCl 3 ), porphyrin IX (PIX), and gallium porphyrin IX (Ga-PIX) was determined by the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates.
Figure Legend Snippet: Characterization of gallium(III)-porphyrin (Ga-PIX) and its toxicity for M. tuberculosis . (A) Fluorescence spectra of PIX, hemin, and synthesized Ga-PIX after excitation at 410 nm. (B) Survival of M. tuberculosis (Mtb) in iron-free Middlebrook 7H9 medium containing 2 µM ferric carboxymycobactin (cMBT) and in the presence of increasing concentrations of gallium chloride (GaCl 3 ), porphyrin IX (PIX), and gallium porphyrin IX (Ga-PIX) was determined by the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates.

Techniques Used: Fluorescence, Synthesized, Alamar Blue Assay

48) Product Images from "A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis"

Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Journal: mBio

doi: 10.1128/mBio.01388-17

The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .
Figure Legend Snippet: The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .

Techniques Used: Mutagenesis, Incubation, Expressing

49) Product Images from "A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival"

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021941

Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).
Figure Legend Snippet: Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).

Techniques Used: Plasmid Preparation, Expressing, Over Expression, Construct, Incubation, Western Blot, Purification, Positive Control

50) Product Images from "Stress-Induced Reorganization of the Mycobacterial Membrane Domain"

Article Title: Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Journal: mBio

doi: 10.1128/mBio.01823-17

Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P
Figure Legend Snippet: Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P

Techniques Used: Radial Immuno Diffusion, Fluorescence, Imaging, Staining, Marker, Expressing

51) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

52) Product Images from "Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro"

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro

Journal: Infection and Immunity

doi:

Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).
Figure Legend Snippet: Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).

Techniques Used:

53) Product Images from "Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates"

Article Title: Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates

Journal: Frontiers in Chemistry

doi: 10.3389/fchem.2018.00519

51 V NMR (78.9 MHz) spectra are shown of solution of colorless oxovanadate (40 mM V 1 , 40 mM V-atoms). The samples are from the bottom up diluted V 1 stock solution (40 mM V-atom) at pH 8.3; 10 mM V 10 in the presence of 24 mM P i at pH 8.1; 10 mM V 10 in the presence of 0.48 mM citrate at pH 6.3, and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 . The spectrum labeled Reference is of the capillary reference alone (top spectrum). The key to the signals: V-oligomers, V 1 monomer; V 2 , dimer; V 4 , tetramer; V 5 , pentamer; VCit, V-citrate complex; PV, vanadate-phosphate complex.
Figure Legend Snippet: 51 V NMR (78.9 MHz) spectra are shown of solution of colorless oxovanadate (40 mM V 1 , 40 mM V-atoms). The samples are from the bottom up diluted V 1 stock solution (40 mM V-atom) at pH 8.3; 10 mM V 10 in the presence of 24 mM P i at pH 8.1; 10 mM V 10 in the presence of 0.48 mM citrate at pH 6.3, and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 . The spectrum labeled Reference is of the capillary reference alone (top spectrum). The key to the signals: V-oligomers, V 1 monomer; V 2 , dimer; V 4 , tetramer; V 5 , pentamer; VCit, V-citrate complex; PV, vanadate-phosphate complex.

Techniques Used: Nuclear Magnetic Resonance, Labeling

51 V NMR (78.9 MHz) spectra are shown of solution of decavanadate (10 mM V 10 , 100 mM V-atoms). The samples are from the bottom up diluted V 10 stock solution (100 mM V-atom) at pH 3.1; 10 mM V 10 in the presence of 0.48 mM and 0.97 mM citrate at pH 2.8 and 2.2, respectively; 10 mM V 10 in the presence of 24 mM P i at pH 6.9; and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 .
Figure Legend Snippet: 51 V NMR (78.9 MHz) spectra are shown of solution of decavanadate (10 mM V 10 , 100 mM V-atoms). The samples are from the bottom up diluted V 10 stock solution (100 mM V-atom) at pH 3.1; 10 mM V 10 in the presence of 0.48 mM and 0.97 mM citrate at pH 2.8 and 2.2, respectively; 10 mM V 10 in the presence of 24 mM P i at pH 6.9; and finally 10 mM V 10 in the presence of Middlebrook 7H9 broth medium supplemented with 10% ADC enrichment (5% BSA, 2% dextrose, 5% catalase), glycerol (0.2%, v/v) and Tween 80 (0.05%, v/v) recorded both in the absence and the presence of a capillary reference of 100 mM Na 3 VO 4 .

Techniques Used: Nuclear Magnetic Resonance

54) Product Images from "Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis"

Article Title: Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

Journal: BMC Microbiology

doi: 10.1186/1471-2180-10-128

Regulation of heterologous promoter by IGPr . dps promoter activity under induced conditions in different constructs in terms of β -gal activity units expressed as nmol ONPG converted to o-nitrophenol per min per milligram of protein. The transformants were grown in Middlebrook 7H9 medium supplemented with 0.02% glucose (Induced). Each experiment was carried out in triplicates and S.D is indicated as error bars.
Figure Legend Snippet: Regulation of heterologous promoter by IGPr . dps promoter activity under induced conditions in different constructs in terms of β -gal activity units expressed as nmol ONPG converted to o-nitrophenol per min per milligram of protein. The transformants were grown in Middlebrook 7H9 medium supplemented with 0.02% glucose (Induced). Each experiment was carried out in triplicates and S.D is indicated as error bars.

Techniques Used: Activity Assay, Construct

55) Product Images from "Stress-Induced Reorganization of the Mycobacterial Membrane Domain"

Article Title: Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Journal: mBio

doi: 10.1128/mBio.01823-17

Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P
Figure Legend Snippet: Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P

Techniques Used: Radial Immuno Diffusion, Fluorescence, Imaging, Staining, Marker, Expressing

56) Product Images from "Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages"

Article Title: Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

Journal: Memórias do Instituto Oswaldo Cruz

doi: 10.1590/0074-02760170105

growth curve (A) and macrophage infection (B) with wild-type H37Rv (WT), knockout for cdd gene (KO), and complemented (CP) Mycobacterium tuberculosis strains. (A) Growth curves were started at an OD 600 of 0.01 in Middlebrook 7H9 medium, in triplicate. (B) The macrophage infection results are expressed as mean numbers of the logarithms of colony-forming unit (CFU) per well of each strain of three independent measurements.
Figure Legend Snippet: growth curve (A) and macrophage infection (B) with wild-type H37Rv (WT), knockout for cdd gene (KO), and complemented (CP) Mycobacterium tuberculosis strains. (A) Growth curves were started at an OD 600 of 0.01 in Middlebrook 7H9 medium, in triplicate. (B) The macrophage infection results are expressed as mean numbers of the logarithms of colony-forming unit (CFU) per well of each strain of three independent measurements.

Techniques Used: Infection, Knock-Out

57) Product Images from "PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis"

Article Title: PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006399

Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard Middlebrook 7H9 medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.
Figure Legend Snippet: Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard Middlebrook 7H9 medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.

Techniques Used: Mass Spectrometry, Standard Deviation

garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p
Figure Legend Snippet: garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p

Techniques Used: In Vitro, Mouse Assay, Plasmid Preparation, Variant Assay, Standard Deviation, Infection

58) Product Images from "Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection"

Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00089

FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
Figure Legend Snippet: FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

Techniques Used: Infection

59) Product Images from "Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro"

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro

Journal: Infection and Immunity

doi:

Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).
Figure Legend Snippet: Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).

Techniques Used:

60) Product Images from "Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis"

Article Title: Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.71.6.3077-3084.2005

Growth phenotypes of conditional auxotrophs. (A to C) Three independent recombinant strains were grown in liquid media containing hygromycin. (D) Control strain growth. The control strain carried both hygromycin and kanamycin resistance genes integrated into the chromosome via homologous recombination. Symbols: ♦, Middlebrook 7H9 medium; ▪, medium containing tetracycline (200 ng/ml); ▴, medium containing tryptophan (50 μg/ml); ×, medium containing tetracycline and tryptophan.
Figure Legend Snippet: Growth phenotypes of conditional auxotrophs. (A to C) Three independent recombinant strains were grown in liquid media containing hygromycin. (D) Control strain growth. The control strain carried both hygromycin and kanamycin resistance genes integrated into the chromosome via homologous recombination. Symbols: ♦, Middlebrook 7H9 medium; ▪, medium containing tetracycline (200 ng/ml); ▴, medium containing tryptophan (50 μg/ml); ×, medium containing tetracycline and tryptophan.

Techniques Used: Recombinant, Homologous Recombination

61) Product Images from "Interfering With DNA Decondensation as a Strategy Against Mycobacteria"

Article Title: Interfering With DNA Decondensation as a Strategy Against Mycobacteria

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02034

M. tuberculosis condenses DNA during starvation. M. tuberculosis mc 2 6030 was cultured in ADC-supplemented Middlebrook 7H9 medium before being starved in PBS. Lipid distribution and DNA localization was imaged using Nile Red (red) and Hoechst (green) respectively at day 0, 6, and 10 (A) and percentage bacteria with condensed DNA was quantified at day 0, 3, 6, and 10. Values represent mean percentage bacteria with condense DNA ± standard error, pooled data of 2 measurements, * P
Figure Legend Snippet: M. tuberculosis condenses DNA during starvation. M. tuberculosis mc 2 6030 was cultured in ADC-supplemented Middlebrook 7H9 medium before being starved in PBS. Lipid distribution and DNA localization was imaged using Nile Red (red) and Hoechst (green) respectively at day 0, 6, and 10 (A) and percentage bacteria with condensed DNA was quantified at day 0, 3, 6, and 10. Values represent mean percentage bacteria with condense DNA ± standard error, pooled data of 2 measurements, * P

Techniques Used: Cell Culture

62) Product Images from "A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival"

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021941

Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).
Figure Legend Snippet: Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).

Techniques Used: Plasmid Preparation, Expressing, Over Expression, Construct, Incubation, Western Blot, Purification, Positive Control

63) Product Images from "A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival"

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021941

Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).
Figure Legend Snippet: Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).

Techniques Used: Plasmid Preparation, Expressing, Over Expression, Construct, Incubation, Western Blot, Purification, Positive Control

64) Product Images from "A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria"

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

Journal: Nature Communications

doi: 10.1038/ncomms12527

Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).
Figure Legend Snippet: Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).

Techniques Used: Concentration Assay, Transferring

65) Product Images from "A Peptide Permease Mutant of Mycobacterium bovis BCG Resistant to the Toxic Peptides Glutathione and S-Nitrosoglutathione"

Article Title: A Peptide Permease Mutant of Mycobacterium bovis BCG Resistant to the Toxic Peptides Glutathione and S-Nitrosoglutathione

Journal: Infection and Immunity

doi:

Inhibition of growth of wild-type and ( opp Δ-19) BCG by GSH. Exponentially growing cells (OD 600 , 0.4 to 0.6) were diluted to an OD 600 of 0.1 and incubated in 24-well plates in Middlebrook 7H9 in the presence or absence of GSH at the concentrations indicated. After 3 days, the OD 600 of the cultures was measured. Black bars, wild-type BCG; hatched bars, BCG( opp Δ-19). The experiment is representative of three similar experiments.
Figure Legend Snippet: Inhibition of growth of wild-type and ( opp Δ-19) BCG by GSH. Exponentially growing cells (OD 600 , 0.4 to 0.6) were diluted to an OD 600 of 0.1 and incubated in 24-well plates in Middlebrook 7H9 in the presence or absence of GSH at the concentrations indicated. After 3 days, the OD 600 of the cultures was measured. Black bars, wild-type BCG; hatched bars, BCG( opp Δ-19). The experiment is representative of three similar experiments.

Techniques Used: Inhibition, Incubation

66) Product Images from "Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis"

Article Title: Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis

Journal: Infection and Immunity

doi: 10.1128/IAI.01196-13

Fatty acids for LB and ILI formation are provided through TAG (from VLDL) breakdown in host lysosomes. (A and B) M. avium cultured in vitro in Middlebrook 7H9 medium for 4 days was exposed to VLDL for 24 h. Bacteria were processed for EM and examined
Figure Legend Snippet: Fatty acids for LB and ILI formation are provided through TAG (from VLDL) breakdown in host lysosomes. (A and B) M. avium cultured in vitro in Middlebrook 7H9 medium for 4 days was exposed to VLDL for 24 h. Bacteria were processed for EM and examined

Techniques Used: Cell Culture, In Vitro

67) Product Images from "A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis"

Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Journal: mBio

doi: 10.1128/mBio.01388-17

The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .
Figure Legend Snippet: The MHK1 mutant shows defects in cell surface integrity. (A) The MHK1 mutant strain shows hypersensitivity to polymyxin B. The cultures were grown in Middlebrook 7H9 medium containing the indicated concentrations of polymyxin B and CaCl 2 . Cell growth was monitored after 72 h of incubation at 37°C by measuring absorbance at 600 nm. (B) MHK1 shows increased cell aggregation. All strains, Mycobacterium smegmatis mc 2 155 (WT+pSMT3), the mutant (MHK1+pSMT3), and mutant complemented with expression of M. smegmatis ctpE (MHK1+pRHK2), were grown in Middlebrook 7H9 medium up to mid-log phase and diluted in fresh medium with 1.0 mM EGTA or without EGTA. The cells were grown for up to 48 h at 37°C with shaking and left standing for 1.0 h. (C) MHK1 shows enhanced reticulation in biofilm formation. All the strains were grown in Sauton’s medium supplemented with 2.0% glucose in microtiter plates for 5 days at 37°C without any additive or with 1.0 mM EGTA or CaCl 2 .

Techniques Used: Mutagenesis, Incubation, Expressing

68) Product Images from "Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages"

Article Title: Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

Journal: Memórias do Instituto Oswaldo Cruz

doi: 10.1590/0074-02760170105

growth curve (A) and macrophage infection (B) with wild-type H37Rv (WT), knockout for cdd gene (KO), and complemented (CP) Mycobacterium tuberculosis strains. (A) Growth curves were started at an OD 600 of 0.01 in Middlebrook 7H9 medium, in triplicate. (B) The macrophage infection results are expressed as mean numbers of the logarithms of colony-forming unit (CFU) per well of each strain of three independent measurements.
Figure Legend Snippet: growth curve (A) and macrophage infection (B) with wild-type H37Rv (WT), knockout for cdd gene (KO), and complemented (CP) Mycobacterium tuberculosis strains. (A) Growth curves were started at an OD 600 of 0.01 in Middlebrook 7H9 medium, in triplicate. (B) The macrophage infection results are expressed as mean numbers of the logarithms of colony-forming unit (CFU) per well of each strain of three independent measurements.

Techniques Used: Infection, Knock-Out

69) Product Images from "Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo "

Article Title: Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

Journal: Infection and Immunity

doi: 10.1128/IAI.72.1.515-526.2004

In vitro growth kinetics of M. tuberculosis Erdman wild type and the five rpf deletion mutant strains in Middlebrook 7H9 medium at 37°C with agitation. The ΔRv1009 strain used in this experiment was mc 2 3126. We also found that strain mc
Figure Legend Snippet: In vitro growth kinetics of M. tuberculosis Erdman wild type and the five rpf deletion mutant strains in Middlebrook 7H9 medium at 37°C with agitation. The ΔRv1009 strain used in this experiment was mc 2 3126. We also found that strain mc

Techniques Used: In Vitro, Mutagenesis

70) Product Images from "Immunogenicity of Proteome-Determined Mycobacterium avium subsp. paratuberculosis-Specific Proteins in Sheep with Paratuberculosis ▿"

Article Title: Immunogenicity of Proteome-Determined Mycobacterium avium subsp. paratuberculosis-Specific Proteins in Sheep with Paratuberculosis ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00099-08

(I) Proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium cultured in Middlebrook 7H9 broth. Silver-stained 2-D electrophoretograms of M. avium subsp. avium (A) and M. avium subsp. paratuberculosis (B) are shown. The organisms used to generate proteomes were grown in Middlebrook 7H9 broth and harvested during exponential phase. (II) Magnification of areas of the proteomic profiles showing proteins that are specific or predominant in M. avium subsp. paratuberculosi s. Composites of analogous regions of 2-D electrophoretograms of either M. avium subsp. avium (A) or M. avium subsp. paratuberculosis (B) are presented. Regions of the gels shown in part I are magnified to demonstrate the differences observed. This figure was compiled by using Phoretix 2D Elite (part I) and CorelDraw 9 software (parts I and II).
Figure Legend Snippet: (I) Proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium cultured in Middlebrook 7H9 broth. Silver-stained 2-D electrophoretograms of M. avium subsp. avium (A) and M. avium subsp. paratuberculosis (B) are shown. The organisms used to generate proteomes were grown in Middlebrook 7H9 broth and harvested during exponential phase. (II) Magnification of areas of the proteomic profiles showing proteins that are specific or predominant in M. avium subsp. paratuberculosi s. Composites of analogous regions of 2-D electrophoretograms of either M. avium subsp. avium (A) or M. avium subsp. paratuberculosis (B) are presented. Regions of the gels shown in part I are magnified to demonstrate the differences observed. This figure was compiled by using Phoretix 2D Elite (part I) and CorelDraw 9 software (parts I and II).

Techniques Used: Cell Culture, Staining, Software

71) Product Images from "Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence"

Article Title: Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1721545115

Deletion of NADH dehydrogenase genes impacts NADH dehydrogenase expression levels and NADH/NAD + ratio. ( A ) Expression levels of ndh, ndhA , and nuoH in the NADH dehydrogenase mutants relative to their parental strain CDC1551. ( B ) NADH/NAD + ratio in NADH dehydrogenase mutants relative to their parental strain CDC1551. In these experiments, the strains were grown in Middlebrook 7H9, supplemented with OADC, glycerol, and tyloxapol to OD 600 nm ≈ 1. The complemented strains Δ ndh pMV361:: ndh and Δ ndhA pMV361:: ndhA are shown as Δ ndh c and Δ ndhA c, respectively. Δ nuo stands for Δ nuoAN . Average of three independent experiments is shown with SD.
Figure Legend Snippet: Deletion of NADH dehydrogenase genes impacts NADH dehydrogenase expression levels and NADH/NAD + ratio. ( A ) Expression levels of ndh, ndhA , and nuoH in the NADH dehydrogenase mutants relative to their parental strain CDC1551. ( B ) NADH/NAD + ratio in NADH dehydrogenase mutants relative to their parental strain CDC1551. In these experiments, the strains were grown in Middlebrook 7H9, supplemented with OADC, glycerol, and tyloxapol to OD 600 nm ≈ 1. The complemented strains Δ ndh pMV361:: ndh and Δ ndhA pMV361:: ndhA are shown as Δ ndh c and Δ ndhA c, respectively. Δ nuo stands for Δ nuoAN . Average of three independent experiments is shown with SD.

Techniques Used: Expressing

72) Product Images from "Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro"

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro

Journal: Infection and Immunity

doi:

Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).
Figure Legend Snippet: Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).

Techniques Used:

73) Product Images from "A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress"

Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

Journal:

doi: 10.1128/JB.00393-06

Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the
Figure Legend Snippet: Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the

Techniques Used:

Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.
Figure Legend Snippet: Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.

Techniques Used:

Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.
Figure Legend Snippet: Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.

Techniques Used:

Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2
Figure Legend Snippet: Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2

Techniques Used:

74) Product Images from "Stress-Induced Reorganization of the Mycobacterial Membrane Domain"

Article Title: Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Journal: mBio

doi: 10.1128/mBio.01823-17

Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P
Figure Legend Snippet: Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6 . The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6 . The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P

Techniques Used: Radial Immuno Diffusion, Fluorescence, Imaging, Staining, Marker, Expressing

75) Product Images from "Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress"

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00463

Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.
Figure Legend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

Techniques Used: Live Cell Imaging, Generated

Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .
Figure Legend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

Techniques Used: Live Cell Imaging, Generated, Staining, Incubation

Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.
Figure Legend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

Techniques Used: Live Cell Imaging

Related Articles

Centrifugation:

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †
Article Snippet: M. smegmatis mutants and mc2 155 were cultured to an A 600 value of 1.4 to 2 in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween 80. .. The extract was cleared by centrifugation at 13,000 × g for 5 min, and the supernatant was dried in a SpeedVac.

Amplification:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: The unmarked mctB mutant Mtb ) and sequencing of a PCR fragment amplified from chromosomal DNA. .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Construct:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: To examine the function of mctB in Mtb , we constructed a mutant in a similar manner as done for M. smegmatis ( ). .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Incubation:

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †
Article Snippet: The reaction mixture was incubated for 10 min at 60°C and acidified with 10 mM methanesulfonic acid (Fluka). .. M. smegmatis mutants and mc2 155 were cultured to an A 600 value of 1.4 to 2 in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween 80.

Article Title: A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis
Article Snippet: .. The antibiotic sensitivity assays were performed by growing M. smegmatis strains in Middlebrook 7H9 medium supplemented with ADC for 24 h and then inoculating the cultures as 1:200 dilutions in fresh medium containing a range of antibiotic concentrations, followed by incubation at 37°C and 200 rpm for 72 h. Growth was monitored at 600 nm. ..

Infection:

Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection
Article Snippet: .. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. ..

Expressing:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ). .. Expression of mctBMtb ) at the attB site of mycobacteriophage L5 ( ).

Western Blot:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ). .. Expression of mctBMtb ) at the attB site of mycobacteriophage L5 ( ).

Derivative Assay:

Article Title: N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis
Article Snippet: M. smegmatis mc2 155 was obtained from W. R. Jacobs, Jr. (Albert Einstein College of Medicine, Bronx, N.Y.) and cultured at 37°C in Middlebrook 7H9 medium supplemented with 0.5% Tween 80 (Fisher) and 0.4% glucose. .. M. smegmatis mutant 49 is an MSH-deficient mutant derived from mc2 155 by chemical mutagenesis and was cultured in Middlebrook 7H9 medium at 37°C as previously described ( ).

Electron Microscopy:

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: Interestingly, morphological characterisation of M. smegmatis SCF and NCF cells using scanning and transmission electron microscopy showed the presence of high density of MVs on the surface of SCF cells as compared to the negligible density of the MVs on the surface of NCF cells (Figures ). .. The cells from SCF1 (Figures ), upon reinoculation into fresh Middlebrook 7H9 medium and upon directly plating on Middlebrook 7H10 agar, gave rise to populations (Figures , respectively), which were similar in composition to the MLP population (Figures ), containing about 10% SCs and 90% NCs (n = 300).

Gas Chromatography:

Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection
Article Snippet: .. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. ..

Chromatography:

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †
Article Snippet: Lipoamide-(mB)2 and lipoate-(mB)2 were analyzed in method 1 chromatography ( ) with buffer A (0.25% acetic acid) adjusted to pH 4.0 with NaOH and acetonitrile substituted for methanol as buffer B; lipoate-(mB)2 and lipoamide-(mB)2 eluted at 32 and 38.5 min, respectively. .. M. smegmatis mutants and mc2 155 were cultured to an A 600 value of 1.4 to 2 in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween 80.

Cell Culture:

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †
Article Snippet: .. M. smegmatis mutants and mc2 155 were cultured to an A 600 value of 1.4 to 2 in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween 80. ..

Article Title: N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis
Article Snippet: .. M. smegmatis mc2 155 was obtained from W. R. Jacobs, Jr. (Albert Einstein College of Medicine, Bronx, N.Y.) and cultured at 37°C in Middlebrook 7H9 medium supplemented with 0.5% Tween 80 (Fisher) and 0.4% glucose. .. M. smegmatis mutant 49 is an MSH-deficient mutant derived from mc2 155 by chemical mutagenesis and was cultured in Middlebrook 7H9 medium at 37°C as previously described ( ).

Generated:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: To obtain an MctB-specific antibody, hybridomas were generated from B cells harvested from mice immunized with recombinant Mtb MctB protein purified from E. coli . .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

other:

Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection
Article Snippet: Statistical analysis of the FAs composition of both Map isolates grown in Middlebrook 7H9 medium (Figure ) enabled the distinction between both Map strains based on the presence of the TBSA (18:0 10Me) exclusively in the C-type isolate of Map .

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: Exposure of the mycobacterial cells to antibiotic, oxidative, and nitrite stress The Msm cells from the different Percoll fractions (64, 66, and 78%) were suspended in 400 μl of 1x PBS or 0.5% Tween 80 or Middlebrook 7H9 medium (did not make a difference in the results) and exposed to different stress agents.

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: In order to obtain 105 cells/ml of the preparative scale Percoll gradient fractionated SCF1 and SCF2 samples, the cells from the 64 and 66%, respectively, were initially pooled in 400 μl of 1x PBS or 0.5% Tween 80 or Middlebrook 7H9 medium.

Transmission Assay:

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: Interestingly, morphological characterisation of M. smegmatis SCF and NCF cells using scanning and transmission electron microscopy showed the presence of high density of MVs on the surface of SCF cells as compared to the negligible density of the MVs on the surface of NCF cells (Figures ). .. The cells from SCF1 (Figures ), upon reinoculation into fresh Middlebrook 7H9 medium and upon directly plating on Middlebrook 7H10 agar, gave rise to populations (Figures , respectively), which were similar in composition to the MLP population (Figures ), containing about 10% SCs and 90% NCs (n = 300).

Sequencing:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: The unmarked mctB mutant Mtb ) and sequencing of a PCR fragment amplified from chromosomal DNA. .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Recombinant:

Article Title: Mycobacteriophage SWU1 gp39 can potentiate multiple antibiotics against Mycobacterium via altering the cell wall permeability
Article Snippet: .. Bacteria strain, plasmid, and growth conditions M. smegmatis mc2 155 and recombinant bacteria grown in Middlebrook 7H9 medium supplemented with 0.05% Tween80 and 0.2% glycerinum or Middlebrook 7H10 plates supplemented with 0.5% glycerinum. ..

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: To obtain an MctB-specific antibody, hybridomas were generated from B cells harvested from mice immunized with recombinant Mtb MctB protein purified from E. coli . .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Mutagenesis:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: The unmarked mctB mutant Mtb ) and sequencing of a PCR fragment amplified from chromosomal DNA. .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Article Title: N-Acetyl-1-d-myo-Inosityl-2-Amino-2-Deoxy-?-d-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis
Article Snippet: M. smegmatis mc2 155 was obtained from W. R. Jacobs, Jr. (Albert Einstein College of Medicine, Bronx, N.Y.) and cultured at 37°C in Middlebrook 7H9 medium supplemented with 0.5% Tween 80 (Fisher) and 0.4% glucose. .. M. smegmatis mutant 49 is an MSH-deficient mutant derived from mc2 155 by chemical mutagenesis and was cultured in Middlebrook 7H9 medium at 37°C as previously described ( ).

Microscopy:

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: .. Determination of the regeneration potential, heat sensitivity, and viability of SCs and NCs The cells from SCF1 and SCF2 mixture and from NCF were independently inoculated into fresh Middlebrook 7H9 medium as well as plated on Middlebrook 7H10 agar medium, and examined the cells from MLP culture and from colonies from the plates, under microscope (DIC). .. Heat susceptibility was verified at 75°C for 15 min, followed by plating on Middlebrook 7H10 agar, with an equivalent number of heat-unexposed control cells, as mentioned (Traag et al., ) and cfu was determined.

Mouse Assay:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: To obtain an MctB-specific antibody, hybridomas were generated from B cells harvested from mice immunized with recombinant Mtb MctB protein purified from E. coli . .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Polymerase Chain Reaction:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: The unmarked mctB mutant Mtb ) and sequencing of a PCR fragment amplified from chromosomal DNA. .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Purification:

Article Title: Copper resistance is essential for virulence of Mycobacterium tuberculosis
Article Snippet: To obtain an MctB-specific antibody, hybridomas were generated from B cells harvested from mice immunized with recombinant Mtb MctB protein purified from E. coli . .. A Western blot using the 5D1.23 antibody showed that mctB is expressed in Mtb H37Rv grown in Middlebrook 7H9 medium, but absent in ML256 ( ).

Plasmid Preparation:

Article Title: Mycobacteriophage SWU1 gp39 can potentiate multiple antibiotics against Mycobacterium via altering the cell wall permeability
Article Snippet: .. Bacteria strain, plasmid, and growth conditions M. smegmatis mc2 155 and recombinant bacteria grown in Middlebrook 7H9 medium supplemented with 0.05% Tween80 and 0.2% glycerinum or Middlebrook 7H10 plates supplemented with 0.5% glycerinum. ..

Functional Assay:

Article Title: A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis ▿
Article Snippet: These results demonstrated that functional uptake systems exist for these sugars and that these sugars are metabolized by M. smegmatis . .. The generation times ranged from 132 to 174 min, which is similar to growth rates of M. smegmatis in Middlebrook 7H9 medium that contains glycerol as the main carbon source ( ).

Dehydrogenase Assay:

Article Title: Organic Hydroperoxide Resistance Protein and Ergothioneine Compensate for Loss of Mycothiol in Mycobacterium smegmatis Mutants ▿ Mutants ▿ †
Article Snippet: M. smegmatis mutants and mc2 155 were cultured to an A 600 value of 1.4 to 2 in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween 80. .. The pellet was dissolved in 200 μl of lipoamide dehydrogenase assay buffer (100 mM NaCl, 1 mM EDTA, 50 mM NaPO4 , pH 7.0), and the insoluble material was removed by centrifugation at 13,000 × g for 5 min. A 100-μl aliquot of extract was reduced with 25 μg of lipoamide dehydrogenase in the presence of 1 mM NADH at room temperature.

Live Cell Imaging:

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: Live cell imaging of Msm MLP cells showed growth (elongation) followed by SCD and ACD of SCs and their daughter cells to generate microcolony (Video ; Figure ). .. The cells from SCF1 (Figures ), upon reinoculation into fresh Middlebrook 7H9 medium and upon directly plating on Middlebrook 7H10 agar, gave rise to populations (Figures , respectively), which were similar in composition to the MLP population (Figures ), containing about 10% SCs and 90% NCs (n = 300).

BAC Assay:

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: Determination of the regeneration potential, heat sensitivity, and viability of SCs and NCs The cells from SCF1 and SCF2 mixture and from NCF were independently inoculated into fresh Middlebrook 7H9 medium as well as plated on Middlebrook 7H10 agar medium, and examined the cells from MLP culture and from colonies from the plates, under microscope (DIC). .. Vital staining was carried out using commercially available viable/nonviable cell staining kit (LIVE/DEAD® Bac Light™, Molecular Probes, Invitrogen, USA), containing a combination of SYTO9 and propidium iodide (Stocks, ; Lahiri et al., ).

Staining:

Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress
Article Snippet: Determination of the regeneration potential, heat sensitivity, and viability of SCs and NCs The cells from SCF1 and SCF2 mixture and from NCF were independently inoculated into fresh Middlebrook 7H9 medium as well as plated on Middlebrook 7H10 agar medium, and examined the cells from MLP culture and from colonies from the plates, under microscope (DIC). .. Vital staining was carried out using commercially available viable/nonviable cell staining kit (LIVE/DEAD® Bac Light™, Molecular Probes, Invitrogen, USA), containing a combination of SYTO9 and propidium iodide (Stocks, ; Lahiri et al., ).

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    MiddleBrook Pharmaceuticals middlebrook 7h9
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    MiddleBrook Pharmaceuticals middlebrook 7h9 medium
    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in <t>Middlebrook</t> <t>7H9</t> medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
    Middlebrook 7h9 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/middlebrook 7h9 medium/product/MiddleBrook Pharmaceuticals
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    middlebrook 7h9 medium - by Bioz Stars, 2020-04
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    Image Search Results


    (A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

    Journal: Journal of Bacteriology

    Article Title: The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

    doi: 10.1128/JB.01346-13

    Figure Lengend Snippet: (A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

    Article Snippet: M. tuberculosis H37Rv and its derivative, TB38, were grown at 37°C in Middlebrook 7H9 (liquid medium) or 7H10 (solid medium) (Difco) supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), 0.2% (vol/vol) glycerol (Sigma-Aldrich), and 10% ADN (2% glucose, 5% bovine serum albumin, 0.85% NaCl).

    Techniques: Mutagenesis

    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

    doi: 10.3389/fcimb.2017.00089

    Figure Lengend Snippet: FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

    Article Snippet: The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium.

    Techniques: Infection

    Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

    Journal: Frontiers in Microbiology

    Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

    doi: 10.3389/fmicb.2017.00463

    Figure Lengend Snippet: Live cell imaging of Msm MLP cell undergoing ACD in the presence of rifampicin, its lineage and data on the time taken by the rifampicin-stressed sister daughter cells (post-birth) generated from ACD . (A) Live cell imaging panels of Msm cells exposed to 12.5 μg/ml rifampicin for a period of 50 min. Rifampicin exposure was given for 50 min in agarose pad, t = 3:10-4:00 (red square), replaced with Middlebrook 7H9 medium after t = 4:00 and monitored for 10 h. Cell, marked with blue color, underwent asymmetric division ge nerating a short daughter cell and a normal/long-sized daughter cell. The arrows show the division sites in the respective frames. Each cell is represented in different color and as the cell divided, the daughter cells have been given a different color in the cartoon representation. The panel (A) corresponds to the images in the Video S2 . (B) The lineage of the growth and division of the rifampicin-stressed Msm cell undergoing ACD (shown in the panel A ). (C) Time taken for the onset of the next division of rifampicin-treated sister daughter cells (normal/long-sized cell and short cell) generated from ACD, data shown in (D) . The sister cells have been marked in the same color, in (C) . The * sign in (D) indicates the ACD-generated short cell that never grew in the observed time period.

    Article Snippet: Determination of the regeneration potential, heat sensitivity, and viability of SCs and NCs The cells from SCF1 and SCF2 mixture and from NCF were independently inoculated into fresh Middlebrook 7H9 medium as well as plated on Middlebrook 7H10 agar medium, and examined the cells from MLP culture and from colonies from the plates, under microscope (DIC).

    Techniques: Live Cell Imaging, Generated

    Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

    Journal: Frontiers in Microbiology

    Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

    doi: 10.3389/fmicb.2017.00463

    Figure Lengend Snippet: Panels from Video S1 on the live cell imaging of Msm short cells generating micro colony and population regeneration potential of the cells in the SCF and NCF . (A) An Msm cell elongates and divides symmetrically to generate two short daughter cells. One of the short daughter cells (green), grew and divided asymmetrically to generate two unequal-sized daughter cells (indicated in cyan and pink). The other short daughter cell (red), grew and divided symmetrically to generate two comparably-sized short daughter cells (indicated in white and yellow). The daughter cells from the earlier asymmetric division (cyan) further divided symmetrically to generate two daughter cells. The short daughter cell (yellow) further grew and divided symmetrically to generate two daughter cells. Arrows show the site of constriction. Cells of length ≤ 2.60 ± 0.25 μm were considered short cells (as per Figure 1B ). (B) Fraction enriched for Msm short cells. (C) Cells from mid-log phase population. (D) Population generated from the short-cells-enriched fraction after reinoculation into Middlebrook 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook 7H10 agar. The compositions of the cells in (D,E) are similar to that in (C) . Arrows indicate short cells. Length of Msm short cells are shown in (B) . (F–I) Acid-fast stained cells in the SCF and NCF and the respective population generated from them after reinoculation into Middlebrook 7H9 media and incubated till the cultures reached 0.6 OD 600nm .

    Article Snippet: Determination of the regeneration potential, heat sensitivity, and viability of SCs and NCs The cells from SCF1 and SCF2 mixture and from NCF were independently inoculated into fresh Middlebrook 7H9 medium as well as plated on Middlebrook 7H10 agar medium, and examined the cells from MLP culture and from colonies from the plates, under microscope (DIC).

    Techniques: Live Cell Imaging, Generated, Staining, Incubation

    Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

    Journal: Frontiers in Microbiology

    Article Title: Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress

    doi: 10.3389/fmicb.2017.00463

    Figure Lengend Snippet: Live cell imaging of Msm MLP cells undergoing ACD and SCD in the presence of H 2 O 2 and their respective lineage. (A) Live cell imaging panels of Msm cells exposed to 0.8 mM H 2 O 2 for a period of 1 h, immediately after the first division and was replaced with Middlebrook 7H9 medium after 1 h of exposure. (ACD; the cell on the right side in the panels, SCD; the lower of the two cells on the left side in the panels). H 2 O 2 was introduced at 50th min (indicated by the red square at the lower bottom of the panels) and was replaced with the medium at 1 h 50th min (indicated by the green square at the lower bottom of the panels). The treated cells were observed for about 9 h. The panel (A) corresponds to the images in the Video S3 . (B) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing ACD (shown in the right side of the panel A ). (C) The lineage of the growth and division of the H 2 O 2 -stressed Msm cell undergoing SCD (shown in the left side of the panel A ). The zero time point does not correlate with the birth of the starting mother cell. The cell lengths given are from the DIC images, but not drawn to scale. The time of generation of daughter cells from asymmetric cell division (ACD) and symmetric cell division (SCD) has been indicated for each division. The time point of exposure to the stress and its withdrawal have been indicated with red arrows.

    Article Snippet: Determination of the regeneration potential, heat sensitivity, and viability of SCs and NCs The cells from SCF1 and SCF2 mixture and from NCF were independently inoculated into fresh Middlebrook 7H9 medium as well as plated on Middlebrook 7H10 agar medium, and examined the cells from MLP culture and from colonies from the plates, under microscope (DIC).

    Techniques: Live Cell Imaging