oadc supplemented middlebrook 7h10-kan selective agar plates  (MiddleBrook Pharmaceuticals)

Journal: Communications Biology

Article Title: Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice

doi: 10.1038/s42003-024-06006-x

Figure Lengend Snippet: Serial dilutions (10 2 –10 4 pfu) of different strains of bacteriophages were mixed with 1 × 10 5 CFUs M. smegmatis ( A ) or H37Rv ( B ) in 7H9 medium and incubated at 37 °C with shaking for 1 h. The infection cultures were then mixed with 0.8% top agar and spread on 12-well 7H10 agar plates supplemented with 10% OADC enrichment. C – F : H37Rv (1 × 10 5 CFUs) were infected with various bacteriophages at an MOI of 1 for 1 h, then inoculated into 20 mL 7H9 media supplemented with 10% ADC and incubated at 37 °C for 9 days. The cultures were sampled at days 3, 6, and 9 post infection by plating 10 μL of 10-fold serial dilutions of on the 7H10 agar plates and cultured at 37 °C for CFU determination. C : OD 600 of sampled liquid cultures was measured at different time points as indicated. D : Statistics of the CFUs of each sampled liquid culture sampled at various time points. E : Titering assay of phage- Mtb liquid culture sampled at different time points. The data are representative results of two independent experiments. F : Phage infection of H37Rv that expresses a GFP reporter gene. An MOI of 1 was used to infect H37Rv-GFP. GFP expression was pictured under fluorescence microscopy after nine days of phage infection.

Article Snippet: M. smegmatis was streaked on 7H10 agar plate supplemented with 10% Middlebrook Oleic Albumin Dextrose Catalase (OADC), 1 mM CaCl 2 , 10 μg/mL of carbenicillin, and 100 μg/mL of cycloheximide and cultured at 37 °C for about 3–4 days for single colony growth.

Techniques: Incubation, Infection, Cell Culture, Expressing, Fluorescence, Microscopy

Journal: Communications Biology

Article Title: Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice

doi: 10.1038/s42003-024-06006-x

Figure Lengend Snippet: A Experimental procedures of the assay. B , C : Half million macrophages were infected with H37Rv at an MOI of 1. After 4 h of Mtb infection, three different phages (5 × 10 6 , MOI of 10) were applied to the Mtb -infected primary human macrophages, and cultured at 37 °C. The cells were sampled at days 0, 5 and 10 to determine the bacillary load for the evaluation the Mtb -killing ability of the phages by plating the ultrasound-broken cells on 7H10 plates (supplemented with 10% OADC) and the CFUs were counted on Day 8. B shows the statistics of the CFUs of all phages, and ( C ) shows the representative pictures (duplicated) of the 7H10 plates taken at day 5 and day 10. D , E : Testing the Mtb -killing ability of phage DS6A in Mtb -infected macrophages derived from four different healthy donors, and phage D29 was used as a negative control. The infected macrophages were sampled at different time points, sonicated, and plated on 7H10 agar plates using different dilutions to titer the bacillary load. D shows the statistics of five donors, and ( E ) shows the representative pictures taken from 10x diluted plates for days 4 and 7 cultures. F : Confocal macroscopy images show the Mtb -killing efficacy of phage DS6A in H37Rv-GFP infected macrophages. Cellmask plasma membrane stain (red) was used to localize the cell membrane, GFP (green) was used for tracking the Mtb bacilli, and DAPI (blue) was used to stain the nuclei of the macrophages. The red arrows show the Mtb in the macrophages. Unpaired student T-tests were used to analyze the differences between groups in Figure D . n = 5 independent donors. All statistical data are represented as mean ± SEM. Statistical significance was defined as * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: M. smegmatis was streaked on 7H10 agar plate supplemented with 10% Middlebrook Oleic Albumin Dextrose Catalase (OADC), 1 mM CaCl 2 , 10 μg/mL of carbenicillin, and 100 μg/mL of cycloheximide and cultured at 37 °C for about 3–4 days for single colony growth.

Techniques: Infection, Cell Culture, Derivative Assay, Negative Control, Sonication, Clinical Proteomics, Membrane, Staining

Journal: Communications Biology

Article Title: Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice

doi: 10.1038/s42003-024-06006-x

Figure Lengend Snippet: A Reconstitution of human immune cells in splenocytes (upper panels) and PBMC (lower panels) from humanized NSG-SGM3 mice. NSG-SGM3 mice were irradiated and intravenously injected with 2 × 10 5 hCD34+ HSC. After 10–12 weeks, reconstitution of human T cells, B cells, myeloid cells, and natural killer (NK) cells within human CD45-gated population was analyzed by flow cytometry. B Statistics of different immune cells before and after infection. C Five mice were sacrificed to check Mtb infection in lungs and spleens by determining the organ CFUs by plating serially diluted organ homogenates on 7H10 agar plates as described above.

Article Snippet: M. smegmatis was streaked on 7H10 agar plate supplemented with 10% Middlebrook Oleic Albumin Dextrose Catalase (OADC), 1 mM CaCl 2 , 10 μg/mL of carbenicillin, and 100 μg/mL of cycloheximide and cultured at 37 °C for about 3–4 days for single colony growth.

Techniques: Irradiation, Injection, Flow Cytometry, Infection

Journal: Communications Biology

Article Title: Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice

doi: 10.1038/s42003-024-06006-x

Figure Lengend Snippet: A Schematic presentation of the animal experiment procedures. H37Rv aerosol infected humanized mice were treated three days after infection with 1 × 10 11 pfu of phage DS6A per mouse or equal volume of sterile PBS every other day for eight times. B The mouse body weights were monitored over time, and the body weight change over the initial body weight of the mice are shown as percentages ( n = 9 for each group). C The growth of H37Rv in the mice lung and spleen homogenates together with initial infection dose (left panel in purple) are shown ( n = 3 for initial infection dose determination; n = 9 for Mtb control and phage-treated group). D Before termination of the experiment, the mice were subjected to pulmonary function testing. The measurements of elastance, compliance, total lung resistance, and total lung volume were collected, and the statistics are shown ( n = 3 for Mtb control group, n = 4 for phage-treated group). E CT scans were performed for each mouse, and two representative figures for each each group show the Mtb -infected control mouse and phage-treated mouse lungs, respectively. The left panel of each mouse CT scan figure shows the 3D image, the white areas represent the high-density scan (e.g., tissues), while the black areas represent low density scan (e.g., air). The three small figures in each mouse scan show different angles of scan results. F The colonies from the plates that spread with lung or spleen homogenates of the H37Rv infected control or phage-treated mice were picked and mixed with 1 × 10 6 pfu of phage DS6A or a negative control phage (phage Chah) in 10 µl 7H9 media and incubated at 37 °C for 1 h. Then the infection cultures were pipetted onto the 7H10 agar plates OADC and incubated at 37 °C for determination of CFUs. The animal experiments were performed four times, and the 3rd and 4th experiments were shown. Except the pulmonary functions were tested once (3rd animal experiment, n = 3 for Mtb control group, and n = 4 for phage DS6A-treated group), the other data are shown as the combined results from the 3rd and 4th experiments ( n = 9 for each group). All the statistics are shown as mean ± SEM. Unpaired Student T-test was used to analyze the differences between groups. Statistical significance was defined as * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001.

Article Snippet: M. smegmatis was streaked on 7H10 agar plate supplemented with 10% Middlebrook Oleic Albumin Dextrose Catalase (OADC), 1 mM CaCl 2 , 10 μg/mL of carbenicillin, and 100 μg/mL of cycloheximide and cultured at 37 °C for about 3–4 days for single colony growth.

Techniques: Aerosol, Infection, Sterility, Control, Computed Tomography, Negative Control, Incubation