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MiddleBrook Pharmaceuticals 7h10 agar plates
Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook <t>7H10</t> agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.
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1) Product Images from "A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis"

Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01846-18

Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.
Figure Legend Snippet: Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

Techniques Used: Activity Assay, Incubation

Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.
Figure Legend Snippet: Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

Techniques Used: Activity Assay, Recombinant, Alamar Blue Assay, Infection, Negative Control

Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.
Figure Legend Snippet: Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

Techniques Used: Recombinant, Infection, Incubation

2) Product Images from "Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds"

Article Title: Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

Journal: BMC Infectious Diseases

doi: 10.1186/1471-2334-11-195

Bacterial viability after exposure to malachite green . Bacterial strains grown to mid-log phase were diluted to 10 7 cells/ml and 0.1 ml was plated on 7H10 medium with (green bars) or without (yellow bars) 1 mg/liter of malachite green. Data points show means +/- standard deviations of duplicate. Significantly different from values of H37Rv ( p
Figure Legend Snippet: Bacterial viability after exposure to malachite green . Bacterial strains grown to mid-log phase were diluted to 10 7 cells/ml and 0.1 ml was plated on 7H10 medium with (green bars) or without (yellow bars) 1 mg/liter of malachite green. Data points show means +/- standard deviations of duplicate. Significantly different from values of H37Rv ( p

Techniques Used:

3) Product Images from "A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis"

Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01846-18

Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.
Figure Legend Snippet: Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

Techniques Used: Activity Assay, Incubation

Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.
Figure Legend Snippet: Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

Techniques Used: Activity Assay, Recombinant, Alamar Blue Assay, Infection, Negative Control

Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.
Figure Legend Snippet: Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

Techniques Used: Recombinant, Infection, Incubation

4) Product Images from "Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds"

Article Title: Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

Journal: BMC Infectious Diseases

doi: 10.1186/1471-2334-11-195

Bacterial viability after exposure to malachite green . Bacterial strains grown to mid-log phase were diluted to 10 7 cells/ml and 0.1 ml was plated on 7H10 medium with (green bars) or without (yellow bars) 1 mg/liter of malachite green. Data points show means +/- standard deviations of duplicate. Significantly different from values of H37Rv ( p
Figure Legend Snippet: Bacterial viability after exposure to malachite green . Bacterial strains grown to mid-log phase were diluted to 10 7 cells/ml and 0.1 ml was plated on 7H10 medium with (green bars) or without (yellow bars) 1 mg/liter of malachite green. Data points show means +/- standard deviations of duplicate. Significantly different from values of H37Rv ( p

Techniques Used:

5) Product Images from "Mycobacterium llatzerense, a waterborne Mycobacterium, that resists phagocytosis by Acanthamoeba castellanii"

Article Title: Mycobacterium llatzerense, a waterborne Mycobacterium, that resists phagocytosis by Acanthamoeba castellanii

Journal: Scientific Reports

doi: 10.1038/srep46270

M. llatzerense resists A. castellanii predation. For the droplet test, bacterial suspension (10 μL) were spotted on Middlebrook 7H10, 10% OADC, with or without an A. castellanii lawn. Mll: Mycobacterium llatzerense isolates EDP_1 to EDP_5; Ms: Mycobacterium septicum; Ec: Escherichia coli.
Figure Legend Snippet: M. llatzerense resists A. castellanii predation. For the droplet test, bacterial suspension (10 μL) were spotted on Middlebrook 7H10, 10% OADC, with or without an A. castellanii lawn. Mll: Mycobacterium llatzerense isolates EDP_1 to EDP_5; Ms: Mycobacterium septicum; Ec: Escherichia coli.

Techniques Used: Mass Spectrometry

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 and maintenance of a low intracellular copper concentration in M. tuberculosis . ( A ) Schematic representation of the chromosomal rv1698 region of M. tuberculosis H37Rv (WT). A fragment of 39 bp was replaced by the 45-bp loxP site in the 5′ part of the gene introducing stop codons in all three reading frames to construct the rv1698 deletion mutant ML256 by homologous recombination. The genes are drawn to scale. ( B ) Expression of mctB in Mtb . Proteins were extracted with 2% SDS from WT Mtb and the mctB mutant ML256. ML257 is an ML256 derivative carrying the integrative mctB expression vector pML955. Proteins were detected in a Western blot by using the MctB-specific monoclonal antibody 5D1.23. ( C ) Copper susceptibility. Serial dilutions of log-phase cultures of Mtb H37Rv (WT), ML256 (Δ mctB ), and ML257 ( +mctB ) were spotted on 7H11/OADC agar without or with CuSO 4 at a concentration of 150 μM. ( D ) Growth defect of Mtb Δ mctB mutant. Mtb WT, the Δ mctB mutant ML256, and the complemented mutant ML257 were grown in 7H10 medium supplemented with peptone (1 g/L) with or without 100 μM CuSO 4 . The optical density at 600 nm was measured at the indicated time points. ( E ) Mtb (black bars), the Δ mctB mutant ML256 (light gray bars), and the complemented mutant ML257 (dark gray bars) were grown in minimal medium with 0.8, 6.3, or 25 μM CuSO 4 . 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 and maintenance of a low intracellular copper concentration in M. tuberculosis . ( A ) Schematic representation of the chromosomal rv1698 region of M. tuberculosis H37Rv (WT). A fragment of 39 bp was replaced by the 45-bp loxP site in the 5′ part of the gene introducing stop codons in all three reading frames to construct the rv1698 deletion mutant ML256 by homologous recombination. The genes are drawn to scale. ( B ) Expression of mctB in Mtb . Proteins were extracted with 2% SDS from WT Mtb and the mctB mutant ML256. ML257 is an ML256 derivative carrying the integrative mctB expression vector pML955. Proteins were detected in a Western blot by using the MctB-specific monoclonal antibody 5D1.23. ( C ) Copper susceptibility. Serial dilutions of log-phase cultures of Mtb H37Rv (WT), ML256 (Δ mctB ), and ML257 ( +mctB ) were spotted on 7H11/OADC agar without or with CuSO 4 at a concentration of 150 μM. ( D ) Growth defect of Mtb Δ mctB mutant. Mtb WT, the Δ mctB mutant ML256, and the complemented mutant ML257 were grown in 7H10 medium supplemented with peptone (1 g/L) with or without 100 μM CuSO 4 . The optical density at 600 nm was measured at the indicated time points. ( E ) Mtb (black bars), the Δ mctB mutant ML256 (light gray bars), and the complemented mutant ML257 (dark gray bars) were grown in minimal medium with 0.8, 6.3, or 25 μM CuSO 4 . Copper was determined by measuring the absorption of the Cu(II)-dithizone complex at 553 nm.

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

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 "Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG"

Article Title: Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152051

NK cells markedly enhance the ability of macrophages to eradicate BCG. RAW 264.7 murine macrophage cells were infected with BCG (MOI = 3) at 37°C for 2 h, washed with PBS three times, and then plated at 1 × 10 6 cells/mL in a 12 well plate. Purified NK cells (3 × 10 5 ), which had been stimulated with BCG (MOI = 1) at 37°C for 4 h, were added to the BCG-infected RAW cell culture. As controls, unstimulated naïve NK cells were added to the BCG-infected RAW cell culture, and the BCG-infected RAW cells alone were additionally prepared. Forty eight hours later, these cells were harvested and lysed with 1 mL of 0.067% SDS solution. Serial dilutions were plated on Middlebrook 7H10 agar plates, and 3 weeks later, the number of bacterial colonies grown on the agar plates were counted (A). As in (A), IFNγ (B) and TNF-α (C) in the culture supernatants were measured by ELISA. Purified naïve NK cells were cultured in medium supplemented with either the culture supernatant of the BCG-infected RAW cells or uninfected control RAW cells at a ratio of 1:1 at 37°C for 24 h, and IFNγ in the culture supernatants was measured using ELISA (D). The data are presented as mean ± standard deviation, and p values
Figure Legend Snippet: NK cells markedly enhance the ability of macrophages to eradicate BCG. RAW 264.7 murine macrophage cells were infected with BCG (MOI = 3) at 37°C for 2 h, washed with PBS three times, and then plated at 1 × 10 6 cells/mL in a 12 well plate. Purified NK cells (3 × 10 5 ), which had been stimulated with BCG (MOI = 1) at 37°C for 4 h, were added to the BCG-infected RAW cell culture. As controls, unstimulated naïve NK cells were added to the BCG-infected RAW cell culture, and the BCG-infected RAW cells alone were additionally prepared. Forty eight hours later, these cells were harvested and lysed with 1 mL of 0.067% SDS solution. Serial dilutions were plated on Middlebrook 7H10 agar plates, and 3 weeks later, the number of bacterial colonies grown on the agar plates were counted (A). As in (A), IFNγ (B) and TNF-α (C) in the culture supernatants were measured by ELISA. Purified naïve NK cells were cultured in medium supplemented with either the culture supernatant of the BCG-infected RAW cells or uninfected control RAW cells at a ratio of 1:1 at 37°C for 24 h, and IFNγ in the culture supernatants was measured using ELISA (D). The data are presented as mean ± standard deviation, and p values

Techniques Used: Infection, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

8) Product Images from "The First Report of Mycobacterium celatum Isolation from Domestic Pig (Sus scrofa domestica) and Roe Deer (Capreolus capreolus) and an Overview of Human Infections in Slovenia"

Article Title: The First Report of Mycobacterium celatum Isolation from Domestic Pig (Sus scrofa domestica) and Roe Deer (Capreolus capreolus) and an Overview of Human Infections in Slovenia

Journal: Veterinary Medicine International

doi: 10.4061/2011/432954

Colony morphology of pig M. celatum isolate grown on Middlebrook 7H10 agar plates and viewed under the microscope (×100).
Figure Legend Snippet: Colony morphology of pig M. celatum isolate grown on Middlebrook 7H10 agar plates and viewed under the microscope (×100).

Techniques Used: Microscopy

9) Product Images from "Increased Phagocytosis of Mycobacterium marinum Mutants Defective in Lipooligosaccharide Production"

Article Title: Increased Phagocytosis of Mycobacterium marinum Mutants Defective in Lipooligosaccharide Production

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.525550

Screening for LOS biosynthetic mutants in M. marinum . A , selection of rough morphotypes. Screening the Tn library on Middlebrook 7H10 led to six rough variants carrying Tn insertions in losA ( MMAR _ 2313 ) (two independent mutants), MMAR _ 2315 , MMAR _ 2319
Figure Legend Snippet: Screening for LOS biosynthetic mutants in M. marinum . A , selection of rough morphotypes. Screening the Tn library on Middlebrook 7H10 led to six rough variants carrying Tn insertions in losA ( MMAR _ 2313 ) (two independent mutants), MMAR _ 2315 , MMAR _ 2319

Techniques Used: Selection

10) Product Images from "Mycobacterium avium subsp hominissuis biofilm is composed of distinct phenotypes and influenced by the presence of antimicrobials"

Article Title: Mycobacterium avium subsp hominissuis biofilm is composed of distinct phenotypes and influenced by the presence of antimicrobials

Journal: Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases

doi: 10.1111/j.1469-0691.2010.03307.x

Selection of resistant and susceptible bacteria. Planktonic and sessile subpopulations were plated onto 7H10 agar. The 7H10 agar plates contained 20 mg/L of clarithromycin. The figure shows how many of the original colonies were resistant to antibiotics.
Figure Legend Snippet: Selection of resistant and susceptible bacteria. Planktonic and sessile subpopulations were plated onto 7H10 agar. The 7H10 agar plates contained 20 mg/L of clarithromycin. The figure shows how many of the original colonies were resistant to antibiotics.

Techniques Used: Selection

11) Product Images from "Persimmon-derived tannin has bacteriostatic and anti-inflammatory activity in a murine model of Mycobacterium avium complex (MAC) disease"

Article Title: Persimmon-derived tannin has bacteriostatic and anti-inflammatory activity in a murine model of Mycobacterium avium complex (MAC) disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0183489

The effect of soluble tannin in a MAC-infected pulmonary granuloma model. Diets were started 1 week before MAC infection. All mice were analyzed at 8 weeks after MAC infection. (a) Microscopic morphology of granulomas in the H E-stained lungs of MAC-infected BALB/c mice fed control and 2% soluble tannin diets. Dotted lines indicate the border of granulomas. Original magnification, ×40 and ×200. (b) Analysis of the size of lung granulomas between control and 2% soluble tannin diet. (c) MAC CFU counts in one lobe of lungs using Middlebrook 7H10 agar plates as described in the Materials and Methods. The values are presented as means ± SEM (uninfected mice, n = 5; MAC-infected mice, n = 8). * P
Figure Legend Snippet: The effect of soluble tannin in a MAC-infected pulmonary granuloma model. Diets were started 1 week before MAC infection. All mice were analyzed at 8 weeks after MAC infection. (a) Microscopic morphology of granulomas in the H E-stained lungs of MAC-infected BALB/c mice fed control and 2% soluble tannin diets. Dotted lines indicate the border of granulomas. Original magnification, ×40 and ×200. (b) Analysis of the size of lung granulomas between control and 2% soluble tannin diet. (c) MAC CFU counts in one lobe of lungs using Middlebrook 7H10 agar plates as described in the Materials and Methods. The values are presented as means ± SEM (uninfected mice, n = 5; MAC-infected mice, n = 8). * P

Techniques Used: Infection, Mouse Assay, Staining

12) Product Images from "Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori"

Article Title: Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori

Journal: Journal of Traditional and Complementary Medicine

doi: 10.1016/j.jtcme.2021.07.009

Effect of QSK on cell surface integrity (A) ZN staining images of control and cells treated with QSK. Scale bar depicts 20 μm. (B) Colony morphology of control and cells treated with QSK observed on 7H10-based medium at 10x magnification. Scale bar depicts 20 μm. (C) Cell sedimentation. Left panel shows O.D 600 of control cells and treated with QSK depicted on y axis with respect to time (hours) on x-axis. (D) Nitrocefin hydrolysis of control and cells treated with QSK. Mean of O.D 485 ± SD of three independent sets are depicted on y axis.
Figure Legend Snippet: Effect of QSK on cell surface integrity (A) ZN staining images of control and cells treated with QSK. Scale bar depicts 20 μm. (B) Colony morphology of control and cells treated with QSK observed on 7H10-based medium at 10x magnification. Scale bar depicts 20 μm. (C) Cell sedimentation. Left panel shows O.D 600 of control cells and treated with QSK depicted on y axis with respect to time (hours) on x-axis. (D) Nitrocefin hydrolysis of control and cells treated with QSK. Mean of O.D 485 ± SD of three independent sets are depicted on y axis.

Techniques Used: Staining, Sedimentation

13) Product Images from "The Environment of “ Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells"

Article Title: The Environment of “ Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells

Journal: Infection and Immunity

doi: 10.1128/IAI.02699-14

In vivo and in vitro characterization of microaggregates. (A) C57BL/6 mice were infected intranasally with equal concentrations of microaggregate and planktonic MAC104 for 1 day. Mice were sacrificed, and lungs were homogenized and plated on 7H10 plates
Figure Legend Snippet: In vivo and in vitro characterization of microaggregates. (A) C57BL/6 mice were infected intranasally with equal concentrations of microaggregate and planktonic MAC104 for 1 day. Mice were sacrificed, and lungs were homogenized and plated on 7H10 plates

Techniques Used: In Vivo, In Vitro, Mouse Assay, Infection

14) Product Images from "Type I interferon decreases macrophage energy metabolism during mycobacterial infection"

Article Title: Type I interferon decreases macrophage energy metabolism during mycobacterial infection

Journal: Cell reports

doi: 10.1016/j.celrep.2021.109195

Type I IFN restrains macrophage metabolism during live Mtb infection (A) OCR of WT BMDMs (left) or IFNAR KO BMDMs (right) either mock infected or infected with live H37v at an MOI of 1 or 10 for 24 h. A single representative plate is shown. (B) Quantification of mitochondrial parameters in BMDMs infected with live H37Rv (MOI of 10) derived from (A). Each point represents a single well and the bar is the mean from seven plates across two independent experiments. (C) ECAR from the same conditions as in (A). A single representative plate is shown. (D) Quantification of glycolytic parameters in BMDMs infected with live H37Rv (MOI of 10) derived from (C). Each point represents a single well and the bar is the mean from seven plates across two independent experiments. (E) Log 2 FC in expression (RNA-seq) of the 13 protein coding genes encoded on mtDNA in WT or IFNAR KO BMDMs infected with live H37Rv (MOI of 10) for 24 h compared to mock-infected cells of each genotype. (F) mROS in WT or IFNAR KO BMDMs infected with an MOI of 10 live H37Rv or HK H37Rv for either 24 or 48 h. BMDMs were either untreated or treated with 500 U/mL IFNβ. mROS measured with MSR MFI (flow cytometry) normalized to untreated, mock-infected controls at each time point. A representative experiment of two independent experiments is shown. Statistical tests shown comparing genotypes within the same infection and treatment conditions unless otherwise specified. (G) OCR during a mitochondrial stress test of alveolar macrophages (AMs) isolated by BAL from WT or IFNAR KO mice either uninfected or infected for 15 days with a high-dose aerosol challenge of H37Rv. One of six plates from two independent experiments is shown. (H) OCR during a mitochondrial stress test of AMs sorted by fluorescence-activated cell sorting (FACS) from WT or IFNAR KO mice 15 days after aerosol infection. Each line is a technical replicate (across three plates) from one of three independent experiments. . (K) Bacterial burden in the lung and spleen of WT and IFNAR KO mice 14–15 days after high-dose aerosol infection. CFU were enumerated by serial dilution on 7H10 plates. Each dot is an individual mouse from three independent experiments and the bars represent the mean.
Figure Legend Snippet: Type I IFN restrains macrophage metabolism during live Mtb infection (A) OCR of WT BMDMs (left) or IFNAR KO BMDMs (right) either mock infected or infected with live H37v at an MOI of 1 or 10 for 24 h. A single representative plate is shown. (B) Quantification of mitochondrial parameters in BMDMs infected with live H37Rv (MOI of 10) derived from (A). Each point represents a single well and the bar is the mean from seven plates across two independent experiments. (C) ECAR from the same conditions as in (A). A single representative plate is shown. (D) Quantification of glycolytic parameters in BMDMs infected with live H37Rv (MOI of 10) derived from (C). Each point represents a single well and the bar is the mean from seven plates across two independent experiments. (E) Log 2 FC in expression (RNA-seq) of the 13 protein coding genes encoded on mtDNA in WT or IFNAR KO BMDMs infected with live H37Rv (MOI of 10) for 24 h compared to mock-infected cells of each genotype. (F) mROS in WT or IFNAR KO BMDMs infected with an MOI of 10 live H37Rv or HK H37Rv for either 24 or 48 h. BMDMs were either untreated or treated with 500 U/mL IFNβ. mROS measured with MSR MFI (flow cytometry) normalized to untreated, mock-infected controls at each time point. A representative experiment of two independent experiments is shown. Statistical tests shown comparing genotypes within the same infection and treatment conditions unless otherwise specified. (G) OCR during a mitochondrial stress test of alveolar macrophages (AMs) isolated by BAL from WT or IFNAR KO mice either uninfected or infected for 15 days with a high-dose aerosol challenge of H37Rv. One of six plates from two independent experiments is shown. (H) OCR during a mitochondrial stress test of AMs sorted by fluorescence-activated cell sorting (FACS) from WT or IFNAR KO mice 15 days after aerosol infection. Each line is a technical replicate (across three plates) from one of three independent experiments. . (K) Bacterial burden in the lung and spleen of WT and IFNAR KO mice 14–15 days after high-dose aerosol infection. CFU were enumerated by serial dilution on 7H10 plates. Each dot is an individual mouse from three independent experiments and the bars represent the mean.

Techniques Used: Infection, Derivative Assay, Expressing, RNA Sequencing Assay, Flow Cytometry, Affinity Magnetic Separation, Isolation, Mouse Assay, Fluorescence, FACS, Serial Dilution

15) Product Images from "Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region, Ethiopia"

Article Title: Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region, Ethiopia

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.03566-14

Growth of Mycobacterium tuberculosis lineage 7 versus other lineages on agar plates. M. tuberculosis isolates belonging to lineages 3, 4, and 7 were streaked onto Middlebrook 7H10 plates and incubated at 37°C for 5 weeks, and colony morphology
Figure Legend Snippet: Growth of Mycobacterium tuberculosis lineage 7 versus other lineages on agar plates. M. tuberculosis isolates belonging to lineages 3, 4, and 7 were streaked onto Middlebrook 7H10 plates and incubated at 37°C for 5 weeks, and colony morphology

Techniques Used: Incubation

16) Product Images from "The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH"

Article Title: The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00273-19

Survival of nonreplicating (NR) cultures of M. tuberculosis after exposure to drugs (alone and in combination) as estimated by CFU counts. Twelve-day-old (H12) cultures adjusted to pH 7.3 were incubated with drugs. Rifampin (R), 8 μg/ml; sutezolid (SZ), 1 μg/ml; nitazoxanide (NZ), 10 μg/ml; PA-824 (pretomanid; PA), 2 μg/ml; isoniazid (I), 2 μg/ml; pyrazinamide (Z), 100 μg/ml; ethambutol (E), 4 μg/ml. Dashed lines indicate the limit of detection (5 CFU/ml). Means and standard deviations from two experiments are shown. In each experiment, two Wayne tubes per condition per time point were used, from which at least two 7H10 agar plates were inoculated. (A) Single drugs, 2-drug combinations, and R-I-Z-E; (B) 3- and 4-drug combinations.
Figure Legend Snippet: Survival of nonreplicating (NR) cultures of M. tuberculosis after exposure to drugs (alone and in combination) as estimated by CFU counts. Twelve-day-old (H12) cultures adjusted to pH 7.3 were incubated with drugs. Rifampin (R), 8 μg/ml; sutezolid (SZ), 1 μg/ml; nitazoxanide (NZ), 10 μg/ml; PA-824 (pretomanid; PA), 2 μg/ml; isoniazid (I), 2 μg/ml; pyrazinamide (Z), 100 μg/ml; ethambutol (E), 4 μg/ml. Dashed lines indicate the limit of detection (5 CFU/ml). Means and standard deviations from two experiments are shown. In each experiment, two Wayne tubes per condition per time point were used, from which at least two 7H10 agar plates were inoculated. (A) Single drugs, 2-drug combinations, and R-I-Z-E; (B) 3- and 4-drug combinations.

Techniques Used: Incubation

17) Product Images from "Experimental Evolution Reveals Redox State Modulates Mycobacterial Pathogenicity"

Article Title: Experimental Evolution Reveals Redox State Modulates Mycobacterial Pathogenicity

Journal: bioRxiv

doi: 10.1101/2021.12.23.474061

Survival in hypoxic environments and resuscitation of M. smegmatis mc 2 155 and mc 2 51 (A) The oxygen tension indicator Methylene blue of mc 2 51 culture (left) changes to colorless on day 6th, on the contrast, the indicator of mc 2 155 culture (right) maintains blue in the Wayne dormancy model. The cultures were initially inoculated in anaerobic tubes at OD 600 of 0.01 with 0.5 head-space ratio. The cultures were stirred at 120 rpm. Methylene blue (1.5 mg/L) was used as an oxygen tension indicator. Methylene blue changes in color from blue to colorless under reducing conditions. Data present results of three biological replicates. Growth rates of strains mc 2 155 (navy blue) and mc 2 51 (brown) were measured by measuring the OD 600 (B) and by determination of CFUs (C) after plating on 7H10. Data are presented as the mean ± standard deviation of three independent replicates. ** p
Figure Legend Snippet: Survival in hypoxic environments and resuscitation of M. smegmatis mc 2 155 and mc 2 51 (A) The oxygen tension indicator Methylene blue of mc 2 51 culture (left) changes to colorless on day 6th, on the contrast, the indicator of mc 2 155 culture (right) maintains blue in the Wayne dormancy model. The cultures were initially inoculated in anaerobic tubes at OD 600 of 0.01 with 0.5 head-space ratio. The cultures were stirred at 120 rpm. Methylene blue (1.5 mg/L) was used as an oxygen tension indicator. Methylene blue changes in color from blue to colorless under reducing conditions. Data present results of three biological replicates. Growth rates of strains mc 2 155 (navy blue) and mc 2 51 (brown) were measured by measuring the OD 600 (B) and by determination of CFUs (C) after plating on 7H10. Data are presented as the mean ± standard deviation of three independent replicates. ** p

Techniques Used: Standard Deviation

18) 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

Msmeg-PE11 are more resistant to SDS, lysozyme, hydrogen peroxide, low pH and antibiotics treatment when compared with Msmeg-pVV . The Msmeg-pVV or Msmeg-PE11 were either treated with medium alone or subjected to ( a ) 0.1% SDS, ( b ) lysozyme, ( c ) 5 mM hydrogen peroxide, ( d ) low pH (5.5 for 24 h), ( e ) 20 μg/ml ethambutol, ( f ) 20 μg/ml isoniazid, ( g ) rifampicin (20 μg/ml), ( h ) vancomycin (5 μg/ml) and ( i ) ampicillin (750 μg/ml) treatment. Bacterial CFUs were counted on 7H10 agar plates. Data are representative of mean ± SD of three different experiments.
Figure Legend Snippet: Msmeg-PE11 are more resistant to SDS, lysozyme, hydrogen peroxide, low pH and antibiotics treatment when compared with Msmeg-pVV . The Msmeg-pVV or Msmeg-PE11 were either treated with medium alone or subjected to ( a ) 0.1% SDS, ( b ) lysozyme, ( c ) 5 mM hydrogen peroxide, ( d ) low pH (5.5 for 24 h), ( e ) 20 μg/ml ethambutol, ( f ) 20 μg/ml isoniazid, ( g ) rifampicin (20 μg/ml), ( h ) vancomycin (5 μg/ml) and ( i ) ampicillin (750 μg/ml) treatment. Bacterial CFUs were counted on 7H10 agar plates. Data are representative of mean ± SD of three different experiments.

Techniques Used:

PE11 alters colony morphology, surface architecture and width of M. smegmatis . ( a ) Msmeg-pVV or Msmeg-PE11 bacteria were plated on 7H10 agar plates supplemented with OADC and incubated for 5–6 days and the mycobacterial colonies were photographed. ( b ) For transmission electron microscopy (TEM) analysis, Msmeg-pVV or Msmeg-PE11 strains were cultured on 7H10 agar for 5–6 days and surface architecture of these bacteria were analyzed. Scale bar, 100 nm. ( c ) In another experiment, Msmeg-pVV or Msmeg-PE11 bacteria were harvested for scanning electron microscopy (SEM) analysis to measure the diameters of Msmeg-pVV or Msmeg-PE11 and mean width of 100 bacilli each of Msmeg-pVV and Msmeg-PE11 is graphically represented in nm.
Figure Legend Snippet: PE11 alters colony morphology, surface architecture and width of M. smegmatis . ( a ) Msmeg-pVV or Msmeg-PE11 bacteria were plated on 7H10 agar plates supplemented with OADC and incubated for 5–6 days and the mycobacterial colonies were photographed. ( b ) For transmission electron microscopy (TEM) analysis, Msmeg-pVV or Msmeg-PE11 strains were cultured on 7H10 agar for 5–6 days and surface architecture of these bacteria were analyzed. Scale bar, 100 nm. ( c ) In another experiment, Msmeg-pVV or Msmeg-PE11 bacteria were harvested for scanning electron microscopy (SEM) analysis to measure the diameters of Msmeg-pVV or Msmeg-PE11 and mean width of 100 bacilli each of Msmeg-pVV and Msmeg-PE11 is graphically represented in nm.

Techniques Used: Incubation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Cell Culture

19) Product Images from "A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis"

Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01846-18

Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.
Figure Legend Snippet: Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

Techniques Used: Activity Assay, Incubation

Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.
Figure Legend Snippet: Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

Techniques Used: Activity Assay, Recombinant, Alamar Blue Assay, Infection, Negative Control

Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.
Figure Legend Snippet: Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

Techniques Used: Recombinant, Infection, Incubation

20) Product Images from "Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region, Ethiopia"

Article Title: Mycobacterium tuberculosis Lineage 7 Strains Are Associated with Prolonged Patient Delay in Seeking Treatment for Pulmonary Tuberculosis in Amhara Region, Ethiopia

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.03566-14

Growth of Mycobacterium tuberculosis lineage 7 versus other lineages on agar plates. M. tuberculosis isolates belonging to lineages 3, 4, and 7 were streaked onto Middlebrook 7H10 plates and incubated at 37°C for 5 weeks, and colony morphology
Figure Legend Snippet: Growth of Mycobacterium tuberculosis lineage 7 versus other lineages on agar plates. M. tuberculosis isolates belonging to lineages 3, 4, and 7 were streaked onto Middlebrook 7H10 plates and incubated at 37°C for 5 weeks, and colony morphology

Techniques Used: Incubation

21) Product Images from "Comparative Genomic and Transcriptomic Analyses of Mycobacterium kansasii Subtypes Provide New Insights Into Their Pathogenicity and Taxonomy"

Article Title: Comparative Genomic and Transcriptomic Analyses of Mycobacterium kansasii Subtypes Provide New Insights Into Their Pathogenicity and Taxonomy

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2020.00122

Complementation of the espACD operon in M. kansasii subtype II with that from M. kansasii subtype I is crucial for its virulence. The functional complementation experiment revealed that the espACD operon plays a significant role in M. kansasii subtype II pathogenicity, at least in THP-1 cells. Intracellular CFU count showing the bacterial load in THP-1 macrophages infected with KAUST-I (•), KAUST-II (•), KAUST-II-pSMT3 (▴) and KAUST-II-pSMT3- espACD (▾) at a MOI of 5. The infected macrophages were lysed at 0, 24, 48, and 72 h time-points post-infection and three dilutions of the released mycobacterial cells were plated on 7H10 agar plates. CFU were counted and recorded after 15 days of plating. Experiments were performed with three replicates, and Student's t -test for significance was calculated with the level of significance shown (***highly significant difference, p
Figure Legend Snippet: Complementation of the espACD operon in M. kansasii subtype II with that from M. kansasii subtype I is crucial for its virulence. The functional complementation experiment revealed that the espACD operon plays a significant role in M. kansasii subtype II pathogenicity, at least in THP-1 cells. Intracellular CFU count showing the bacterial load in THP-1 macrophages infected with KAUST-I (•), KAUST-II (•), KAUST-II-pSMT3 (▴) and KAUST-II-pSMT3- espACD (▾) at a MOI of 5. The infected macrophages were lysed at 0, 24, 48, and 72 h time-points post-infection and three dilutions of the released mycobacterial cells were plated on 7H10 agar plates. CFU were counted and recorded after 15 days of plating. Experiments were performed with three replicates, and Student's t -test for significance was calculated with the level of significance shown (***highly significant difference, p

Techniques Used: Functional Assay, Infection

22) Product Images from "Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis"

Article Title: Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005768

Compensatory flux of ADP-glucose through GlgA and OtsA links the GlgC-GlgA and TreS-Pep2 routes for α-glucan production. (A) α-Glucan visualization in M . smegmatis mutant strains. Cells were cultivated on Middlebrook 7H10 agar plates for 3 days and exposed to iodine vapor for staining of α-glucans. Branched α-glucans give a pale red-brown color. In the absence of branching enzyme GlgB, long linear glucans are produced resulting in a dark blue color of cells with the intensity of staining correlating with the amount of total cellular α-glucans. (B) Analysis of nucleotide sugar diphosphates in cell extracts of M . smegmatis mutant strains. Cells were cultivated for 24 h in Middlebrook 7H9 liquid medium. Due to trehalose auxotrophy of the M . smegmatis Δ glgA (u) Δ otsA mutant, 50 μM trehalose was added to all cultures. Cell suspensions were normalized to OD 600 nm , washed with PBS, concentrated 50-fold and disrupted by bead beating. Cell-free extracts were heat inactivated for 15 min at 100°C, and 1 H NMR spectroscopy was used to detect the anomeric protons of ADP-glucose.
Figure Legend Snippet: Compensatory flux of ADP-glucose through GlgA and OtsA links the GlgC-GlgA and TreS-Pep2 routes for α-glucan production. (A) α-Glucan visualization in M . smegmatis mutant strains. Cells were cultivated on Middlebrook 7H10 agar plates for 3 days and exposed to iodine vapor for staining of α-glucans. Branched α-glucans give a pale red-brown color. In the absence of branching enzyme GlgB, long linear glucans are produced resulting in a dark blue color of cells with the intensity of staining correlating with the amount of total cellular α-glucans. (B) Analysis of nucleotide sugar diphosphates in cell extracts of M . smegmatis mutant strains. Cells were cultivated for 24 h in Middlebrook 7H9 liquid medium. Due to trehalose auxotrophy of the M . smegmatis Δ glgA (u) Δ otsA mutant, 50 μM trehalose was added to all cultures. Cell suspensions were normalized to OD 600 nm , washed with PBS, concentrated 50-fold and disrupted by bead beating. Cell-free extracts were heat inactivated for 15 min at 100°C, and 1 H NMR spectroscopy was used to detect the anomeric protons of ADP-glucose.

Techniques Used: Mutagenesis, Staining, Produced, Nuclear Magnetic Resonance, Spectroscopy

Evidence for an alternative route to M1P in M . smegmatis independent of TreS-Pep2 and trehalose. (A) Heterologous expression of the M . tuberculosis glgC gene (Rv1213) restores M1P accumulation in the M . smegmatis Δ glgE Δ pep2 double mutant harboring a spontaneous IS 1096 element insertion in the endogenous glgC locus (i.e. M . smegmatis Δ glgE Δ pep2 glgC :IS 1096 ). Equivalent quantities of crude extracts of M . smegmatis strains were analyzed using 1 H NMR spectroscopy. The assignment of the peaks was based on our previous studies [ 16 , 18 ]. (B) Hot water extracts from 1 ml culture aliquots of M . smegmatis strains (normalized to OD 600 nm = 0.5) were analyzed by TLC, demonstrating M1P accumulation in the M . smegmatis Δ glgE Δ pep2 glgC :IS 1096 strain expressing the M . tuberculosis glgC gene (Rv1213). M1P and trehalose (5 μg each) were used as standards. (C) M . smegmatis Δ pep2 (u) Δ glgE and Δ treS (u) Δ glgE double mutants accumulate M1P. TLC analysis was performed as described in (B). (D) The M . smegmatis Δ pep2 (u) Δ glgE mutant is trehalose resistant despite accumulating M1P, indicating trehalose-independent M1P formation. Strains were grown on Middlebrook 7H10 agar plates with or without 1 mM trehalose and incubated at 37°C for 3 days. (E) Conditional silencing of the glgE gene in M . smegmatis mutant strains reveals the requirement of GlgC and GlgA for the alternative route to M1P synthesis. Cells of the indicated conditional c- glgE -tet-off mutant strains were cultivated for 24 h with or without 1 μg ml -1 ATc as indicated, and hot water extracts from 1 ml culture aliquots (normalized to OD 600 nm = 0.5) were analyzed by TLC.
Figure Legend Snippet: Evidence for an alternative route to M1P in M . smegmatis independent of TreS-Pep2 and trehalose. (A) Heterologous expression of the M . tuberculosis glgC gene (Rv1213) restores M1P accumulation in the M . smegmatis Δ glgE Δ pep2 double mutant harboring a spontaneous IS 1096 element insertion in the endogenous glgC locus (i.e. M . smegmatis Δ glgE Δ pep2 glgC :IS 1096 ). Equivalent quantities of crude extracts of M . smegmatis strains were analyzed using 1 H NMR spectroscopy. The assignment of the peaks was based on our previous studies [ 16 , 18 ]. (B) Hot water extracts from 1 ml culture aliquots of M . smegmatis strains (normalized to OD 600 nm = 0.5) were analyzed by TLC, demonstrating M1P accumulation in the M . smegmatis Δ glgE Δ pep2 glgC :IS 1096 strain expressing the M . tuberculosis glgC gene (Rv1213). M1P and trehalose (5 μg each) were used as standards. (C) M . smegmatis Δ pep2 (u) Δ glgE and Δ treS (u) Δ glgE double mutants accumulate M1P. TLC analysis was performed as described in (B). (D) The M . smegmatis Δ pep2 (u) Δ glgE mutant is trehalose resistant despite accumulating M1P, indicating trehalose-independent M1P formation. Strains were grown on Middlebrook 7H10 agar plates with or without 1 mM trehalose and incubated at 37°C for 3 days. (E) Conditional silencing of the glgE gene in M . smegmatis mutant strains reveals the requirement of GlgC and GlgA for the alternative route to M1P synthesis. Cells of the indicated conditional c- glgE -tet-off mutant strains were cultivated for 24 h with or without 1 μg ml -1 ATc as indicated, and hot water extracts from 1 ml culture aliquots (normalized to OD 600 nm = 0.5) were analyzed by TLC.

Techniques Used: Expressing, Mutagenesis, Nuclear Magnetic Resonance, Spectroscopy, Thin Layer Chromatography, Incubation

23) Product Images from "Natural killer activation for bladder cancer elimination can be achieved in vitro by heat-killed BCG"

Article Title: Natural killer activation for bladder cancer elimination can be achieved in vitro by heat-killed BCG

Journal: bioRxiv

doi: 10.1101/2020.06.04.129361

The number of BCG cells estimated by flow cytometry. Serial dilutions, as indicated, of BCG grown in Middlebrook 7H10 medium were prepared and analysed by flow cytometry at constant speed during 30 seconds. The number of events within the FSC vs SSC region corresponding to bacteria (A) were used to build a calibration curve (B).
Figure Legend Snippet: The number of BCG cells estimated by flow cytometry. Serial dilutions, as indicated, of BCG grown in Middlebrook 7H10 medium were prepared and analysed by flow cytometry at constant speed during 30 seconds. The number of events within the FSC vs SSC region corresponding to bacteria (A) were used to build a calibration curve (B).

Techniques Used: Flow Cytometry

24) Product Images from "Mycobacterium tuberculosis Infection Induces HDAC1-Mediated Suppression of IL-12B Gene Expression in Macrophages"

Article Title: Mycobacterium tuberculosis Infection Induces HDAC1-Mediated Suppression of IL-12B Gene Expression in Macrophages

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2015.00090

Survival of MTB decreases when HDAC1 is knocked down in macrophages . THP-1 monocytes (1 × 10 6 cells) seeded in a 6-well plate were treated with scrambled siRNA and siHDAC1, and infected with MTB. Macrophages not treated with siRNA but infected with MTB were also kept as control. The number of bacteria recovered from the macrophages 4 h after infection was considered as the number of bacteria that gained entry into the macrophages (and the number at 0 h for studying the viability inside macrophages); and subsequent isolation of bacilli from the macrophages was carried out at 24, 48, and 72 h. The number of viable bacilli in each of the wells was assayed by plating on a 7H10 agar plates and incubating the plates at 37°C, and counting the colony forming unit (cfu). Data shown here are the mean ± SD of three independent experiments.
Figure Legend Snippet: Survival of MTB decreases when HDAC1 is knocked down in macrophages . THP-1 monocytes (1 × 10 6 cells) seeded in a 6-well plate were treated with scrambled siRNA and siHDAC1, and infected with MTB. Macrophages not treated with siRNA but infected with MTB were also kept as control. The number of bacteria recovered from the macrophages 4 h after infection was considered as the number of bacteria that gained entry into the macrophages (and the number at 0 h for studying the viability inside macrophages); and subsequent isolation of bacilli from the macrophages was carried out at 24, 48, and 72 h. The number of viable bacilli in each of the wells was assayed by plating on a 7H10 agar plates and incubating the plates at 37°C, and counting the colony forming unit (cfu). Data shown here are the mean ± SD of three independent experiments.

Techniques Used: Infection, Isolation

25) Product Images from "Experimental Evolution Reveals Redox State Modulates Mycobacterial Pathogenicity"

Article Title: Experimental Evolution Reveals Redox State Modulates Mycobacterial Pathogenicity

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2022.758304

Survival in hypoxic environments and resuscitation of M. smegmatis mc 2 155 and mc 2 51. (A) The oxygen tension indicator methylene blue of mc 2 51 culture (left) changes to colorless on day 6, while the indicator of mc 2 155 culture (right) stays blue in the Wayne dormancy model. The cultures were initially inoculated in anaerobic tubes at an OD 600 of 0.01 with a headspace ratio of 0.5. The cultures were stirred at 120 rpm. Methylene blue (1.5 mg/L) was used as an oxygen tension indicator. Methylene blue changes from blue to colorless under reducing conditions. Data present results of three biological replicates. Growth rates of strains mc 2 155 (navy blue) and mc 2 51 (brown) were measured by measuring the OD 600 (B) and by determination of CFUs (C) after plating on 7H10. Data are presented as the mean ± standard deviation of three independent replicates. ** p
Figure Legend Snippet: Survival in hypoxic environments and resuscitation of M. smegmatis mc 2 155 and mc 2 51. (A) The oxygen tension indicator methylene blue of mc 2 51 culture (left) changes to colorless on day 6, while the indicator of mc 2 155 culture (right) stays blue in the Wayne dormancy model. The cultures were initially inoculated in anaerobic tubes at an OD 600 of 0.01 with a headspace ratio of 0.5. The cultures were stirred at 120 rpm. Methylene blue (1.5 mg/L) was used as an oxygen tension indicator. Methylene blue changes from blue to colorless under reducing conditions. Data present results of three biological replicates. Growth rates of strains mc 2 155 (navy blue) and mc 2 51 (brown) were measured by measuring the OD 600 (B) and by determination of CFUs (C) after plating on 7H10. Data are presented as the mean ± standard deviation of three independent replicates. ** p

Techniques Used: Standard Deviation

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    MiddleBrook Pharmaceuticals 7h10 agar plates
    Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook <t>7H10</t> agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.
    7h10 Agar Plates, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals middlebrook 7h10 agar
    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 <t>Middlebrook</t> 7H9 liquid medium. (E) Population generated from plating of the cells in the short-cells-enriched fraction on Middlebrook <t>7H10</t> 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 .
    Middlebrook 7h10 Agar, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    middlebrook pharmaceuticals 7h10 agar plate
    The effect of the mutation in the N104 strain on its antimicrobial susceptibility and copper resistance. (a) Susceptibility to antimicrobials was assessed by growing the cells for 15 days on <t>7H10</t> agar containing the following antimicrobial concentrations: 0 µg ml −1 (control plate), 0.06 µg ml −1 (rifampicin and clarithromycin), 0.25 µg ml −1 (levofloxacin) and 1 µg ml −1 (ethambutol). Inocula were prepared using 10-fold dilutions of cultures adjusted to 0.2 at OD 530 and spotted (2 µl/spot). (b) The Congo red-binding ability of the P104, N104, NP and NN MAH 104 strains. Congo red binding was quantified by measuring A 488 of Congo red divided by the OD 650 of the bacterial cell suspensions. Data are represented as the mean± sd obtained from n =4. (c) Copper resistance was determined after growing the cells for 10 days on 7H10 agar containing 0, 25, 50, or 100 µM CuSO 4 . Inocula were prepared using 10-fold dilutions of cultures adjusted to 0.2 at O D 530 and spotted (2 µl/spot). Tukey’s HSD test was used for statistical analysis. *** P
    7h10 Agar Plate, supplied by middlebrook pharmaceuticals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    7h10 agar plate - by Bioz Stars, 2022-05
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    Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

    doi: 10.1128/AAC.01846-18

    Figure Lengend Snippet: Bactericidal activity of HAMLET (HL) against M. tuberculosis . (A) M. tuberculosis H37Rv was grown in HdB minimal medium to an OD 600 of 1.3, and then 5 ml of culture was seeded into 12-well microplates. M. tuberculosis was treated with 300 μg/ml HAMLET on days 0, 1, and 2 as indicated by the arrows. The bactericidal activity of HAMLET was determined by measuring the optical density at 600 nm. Error bars represent standard errors of mean values of biological triplicates. (B) Images of the wells were taken on the second day of HAMLET treatment. (C) Tenfold serial dilutions of cultures of untreated and HAMLET-treated M. tuberculosis strains were plated on Middlebrook 7H10 agar after the first treatment with HAMLET (day 0). Agar plates were imaged after incubation at 37°C for 15 days.

    Article Snippet: Each day, the OD600 was measured, and 10 μl of each sample of serial 10-fold dilutions was dropped on Middlebrook 7H10 agar plates.

    Techniques: Activity Assay, Incubation

    Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

    doi: 10.1128/AAC.01846-18

    Figure Lengend Snippet: Activity of recombinant HAMLET against M. tuberculosis in macrophages. The viability of differentiated THP-1 macrophages in the presence of increasing concentrations of recombinant α-lactalbumin (A), oleic acid (B), and recombinant HAMLET (C) was determined using the microplate alamarBlue assay. Error bars represent standard errors of mean values of biological triplicates. The MIC 90 is represented by dotted lines. (D) Differentiated THP-1 macrophages were infected with M. tuberculosis H37Rv at an MOI of 10 for 4 h. Then, recombinant HAMLET was added at the indicated amounts, and the viability of M. tuberculosis was determined for two days using a drop assay on Middlebrook 7H10 agar plates. The uninfected macrophages are shown as a negative control.

    Article Snippet: Each day, the OD600 was measured, and 10 μl of each sample of serial 10-fold dilutions was dropped on Middlebrook 7H10 agar plates.

    Techniques: Activity Assay, Recombinant, Alamar Blue Assay, Infection, Negative Control

    Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis

    doi: 10.1128/AAC.01846-18

    Figure Lengend Snippet: Recombinant HAMLET increases the efficacy of bedaquiline, delamanid, and clarithromycin against M. tuberculosis in infected macrophages. Differentiated THP-1 cells were infected with M. tuberculosis H37Rv at an MOI of 10, washed, and treated with serial dilutions of bedaquiline (A), delamanid (B), and clarithromycin (C) in the presence and absence of 200 μg/ml recombinant HAMLET for two days. Ten-microliter drops of lysed macrophages were then plated on Middlebrook 7H10 agar plates which were incubated for 13 days at 37°C.

    Article Snippet: Each day, the OD600 was measured, and 10 μl of each sample of serial 10-fold dilutions was dropped on Middlebrook 7H10 agar plates.

    Techniques: Recombinant, Infection, Incubation

    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: 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).

    Techniques: Live Cell Imaging, Generated, Staining, Incubation

    K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

    Journal: Antibiotics

    Article Title: Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

    doi: 10.3390/antibiotics11040509

    Figure Lengend Snippet: K + effect on the growth of mc 2 155 and its ∆ mchK derivative under different conditions. ( a ) Effect of K + addition (10–200 mM KCl) on the growth of mc 2 155 and ∆ mchK at neutral pH. The ability of high K + concentrations to restore the growth of the ∆ mchK mutant was measured in cultures at mid-exponential phase (12 h). ( b ) Effect of extracellular pH (5–8) on the growth of mc 2 155 and the ∆ mchK mutant in Middlebrook 7H9 liquid medium after 12 h of incubation (initial OD 600 0.05). ( c ) Effect of K + addition (10–100 mM KCl) on the growth of mc 2 155 and ∆ mchK at pH 5.5. The OD 600 was measured after 12 h of incubation at 37 °C. Error bars indicate 95% CI (confidence intervals); (*) p

    Article Snippet: The plasmid with the in-frame deletion of mchK , termed p2NIL-ΔmchK , was introduced into M. smegmatis mc2 155 and plated on Middlebrook 7H10 agar supplemented with kanamycin (25 µg/mL) and hygromycin (50 µg/mL).

    Techniques: Mutagenesis, Incubation

    The effect of the mutation in the N104 strain on its antimicrobial susceptibility and copper resistance. (a) Susceptibility to antimicrobials was assessed by growing the cells for 15 days on 7H10 agar containing the following antimicrobial concentrations: 0 µg ml −1 (control plate), 0.06 µg ml −1 (rifampicin and clarithromycin), 0.25 µg ml −1 (levofloxacin) and 1 µg ml −1 (ethambutol). Inocula were prepared using 10-fold dilutions of cultures adjusted to 0.2 at OD 530 and spotted (2 µl/spot). (b) The Congo red-binding ability of the P104, N104, NP and NN MAH 104 strains. Congo red binding was quantified by measuring A 488 of Congo red divided by the OD 650 of the bacterial cell suspensions. Data are represented as the mean± sd obtained from n =4. (c) Copper resistance was determined after growing the cells for 10 days on 7H10 agar containing 0, 25, 50, or 100 µM CuSO 4 . Inocula were prepared using 10-fold dilutions of cultures adjusted to 0.2 at O D 530 and spotted (2 µl/spot). Tukey’s HSD test was used for statistical analysis. *** P

    Journal: Microbiology

    Article Title: Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis

    doi: 10.1099/mic.0.001007

    Figure Lengend Snippet: The effect of the mutation in the N104 strain on its antimicrobial susceptibility and copper resistance. (a) Susceptibility to antimicrobials was assessed by growing the cells for 15 days on 7H10 agar containing the following antimicrobial concentrations: 0 µg ml −1 (control plate), 0.06 µg ml −1 (rifampicin and clarithromycin), 0.25 µg ml −1 (levofloxacin) and 1 µg ml −1 (ethambutol). Inocula were prepared using 10-fold dilutions of cultures adjusted to 0.2 at OD 530 and spotted (2 µl/spot). (b) The Congo red-binding ability of the P104, N104, NP and NN MAH 104 strains. Congo red binding was quantified by measuring A 488 of Congo red divided by the OD 650 of the bacterial cell suspensions. Data are represented as the mean± sd obtained from n =4. (c) Copper resistance was determined after growing the cells for 10 days on 7H10 agar containing 0, 25, 50, or 100 µM CuSO 4 . Inocula were prepared using 10-fold dilutions of cultures adjusted to 0.2 at O D 530 and spotted (2 µl/spot). Tukey’s HSD test was used for statistical analysis. *** P

    Article Snippet: Colony morphologyColony morphology was observed using a light microscope (CKX41, OLYMPUS, Tokyo, Japan) after 5, 8 and 14 days of culture on a 7H10 agar plate.

    Techniques: Mutagenesis, Binding Assay

    Assessment of the phenotype of N104-specific SNP using the revertant parent strain. (a) Schematic representing the introduction of the spanning region of MAV_RS14660 and MAV_RS14655 from the P104 genome into the N104 genome. The target codon for recombination is represented using red letters. Regions filled with pink represents res sites. (b) Representative images of colonies of MAH 104 strains on 7H10 agar after 8 days of growth. Left: revertant parent strain (NP). Right: strain with the N104 sequence into the N104 genome as control (NN). Scale bars represent 0.5 mm. (c) Diameter of colonies on agar were measured after n days of culturing, as indicated in the figure. Data are represented as the mean± sd of 20 samples in each group. (d, e) Motility of the MAH 104 strains on 0.3 % (w/v) agar plate. (f) Colonies on 0.3 % agar after 5 days of growth. Scale bars represent 1 cm. (g) Diameter of colonies were measured after 5 days. Data are represented as the mean± sd obtained from n =4. (f, g) Growth of the MAH 104 strains in 7H9 medium under conditions of shaking at 37 °C was assessed at OD 530 (f), and c.f.u. (g) were enumerated after n days of culturing, as indicated in the figure. Data are represented as the mean± sd obtained from (f) n =5 and (g) n =4. Tukey’s HSD test was used for statistical analysis. *** P

    Journal: Microbiology

    Article Title: Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis

    doi: 10.1099/mic.0.001007

    Figure Lengend Snippet: Assessment of the phenotype of N104-specific SNP using the revertant parent strain. (a) Schematic representing the introduction of the spanning region of MAV_RS14660 and MAV_RS14655 from the P104 genome into the N104 genome. The target codon for recombination is represented using red letters. Regions filled with pink represents res sites. (b) Representative images of colonies of MAH 104 strains on 7H10 agar after 8 days of growth. Left: revertant parent strain (NP). Right: strain with the N104 sequence into the N104 genome as control (NN). Scale bars represent 0.5 mm. (c) Diameter of colonies on agar were measured after n days of culturing, as indicated in the figure. Data are represented as the mean± sd of 20 samples in each group. (d, e) Motility of the MAH 104 strains on 0.3 % (w/v) agar plate. (f) Colonies on 0.3 % agar after 5 days of growth. Scale bars represent 1 cm. (g) Diameter of colonies were measured after 5 days. Data are represented as the mean± sd obtained from n =4. (f, g) Growth of the MAH 104 strains in 7H9 medium under conditions of shaking at 37 °C was assessed at OD 530 (f), and c.f.u. (g) were enumerated after n days of culturing, as indicated in the figure. Data are represented as the mean± sd obtained from (f) n =5 and (g) n =4. Tukey’s HSD test was used for statistical analysis. *** P

    Article Snippet: Colony morphologyColony morphology was observed using a light microscope (CKX41, OLYMPUS, Tokyo, Japan) after 5, 8 and 14 days of culture on a 7H10 agar plate.

    Techniques: Sequencing

    Bacterial morphology. (a, b) Bacterial cells on solid 7H10 medium after more than 20 days of culturing were observed using scanning electron microscopy. (a) Electron micrographs of P104 and N104 are shown. Scale bar represents 1 µm. (b) Length of bacterial cells measured from using the scanning electron micrographs. Bar indicates median ( n =100). (c, d) Bacterial cells harvested from 7H9 liquid medium at 0.5 OD 530 and observed using cryo-transmission electron microscopy (Cryo-TEM). (c) Representative micrographs of strains P104 and N104. Scale bar represents 0.5 µm. (d) Length of bacterial cells was measured using the Cryo-TEM (P104, closed circle; N104, open circle). Bar indicates median (P104, n =49; N104, n =73). he Mann–Whitney U test (b, d) was used for statistical analysis. *** P

    Journal: Microbiology

    Article Title: Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis

    doi: 10.1099/mic.0.001007

    Figure Lengend Snippet: Bacterial morphology. (a, b) Bacterial cells on solid 7H10 medium after more than 20 days of culturing were observed using scanning electron microscopy. (a) Electron micrographs of P104 and N104 are shown. Scale bar represents 1 µm. (b) Length of bacterial cells measured from using the scanning electron micrographs. Bar indicates median ( n =100). (c, d) Bacterial cells harvested from 7H9 liquid medium at 0.5 OD 530 and observed using cryo-transmission electron microscopy (Cryo-TEM). (c) Representative micrographs of strains P104 and N104. Scale bar represents 0.5 µm. (d) Length of bacterial cells was measured using the Cryo-TEM (P104, closed circle; N104, open circle). Bar indicates median (P104, n =49; N104, n =73). he Mann–Whitney U test (b, d) was used for statistical analysis. *** P

    Article Snippet: Colony morphologyColony morphology was observed using a light microscope (CKX41, OLYMPUS, Tokyo, Japan) after 5, 8 and 14 days of culture on a 7H10 agar plate.

    Techniques: Electron Microscopy, Transmission Assay, Transmission Electron Microscopy, MANN-WHITNEY

    Phenotype of parent (P104) and variant (N104) MAH 104 strains. (a) Diameter of colonies of MAH 104 strains on agar plates were measured after n days of culturing, as indicated in the figure. Data are represented as he mean±standard deviation ( sd ) of 10 samples in each group. (b) Representative images of colonies of the two strains on 7H10 agar after 8 days of growth. Scale bars represent 0.5 mm. (c, d) Motility of the two strains on 0.3 % agar. (c) Colonies on 0.3 % agar after 5 days of growth. Scale bars represent 10 mm. (d) Diameter of colonies as measured after 5 days of culture. Data are represented as the mean± sd obtained from n =4. (e, f) Growth of MAH 104 strains in 7H9 broth under conditions of shaking at 37 °C, analysed at OD 530 (e) and based on colony-forming units (c.f.u.) (f) after n days of culturing as indicated in the figure. Data are represented as the mean± sd obtained from (c) n =3 and (d) n =4. Student’s t -test was used for statistical analysis. *** P

    Journal: Microbiology

    Article Title: Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis

    doi: 10.1099/mic.0.001007

    Figure Lengend Snippet: Phenotype of parent (P104) and variant (N104) MAH 104 strains. (a) Diameter of colonies of MAH 104 strains on agar plates were measured after n days of culturing, as indicated in the figure. Data are represented as he mean±standard deviation ( sd ) of 10 samples in each group. (b) Representative images of colonies of the two strains on 7H10 agar after 8 days of growth. Scale bars represent 0.5 mm. (c, d) Motility of the two strains on 0.3 % agar. (c) Colonies on 0.3 % agar after 5 days of growth. Scale bars represent 10 mm. (d) Diameter of colonies as measured after 5 days of culture. Data are represented as the mean± sd obtained from n =4. (e, f) Growth of MAH 104 strains in 7H9 broth under conditions of shaking at 37 °C, analysed at OD 530 (e) and based on colony-forming units (c.f.u.) (f) after n days of culturing as indicated in the figure. Data are represented as the mean± sd obtained from (c) n =3 and (d) n =4. Student’s t -test was used for statistical analysis. *** P

    Article Snippet: Colony morphologyColony morphology was observed using a light microscope (CKX41, OLYMPUS, Tokyo, Japan) after 5, 8 and 14 days of culture on a 7H10 agar plate.

    Techniques: Variant Assay, Standard Deviation