Cell viability was determined with Acridine Orange/Propidium Iodide (AO/PI) Staining Solution on Nexcelom cellometer (Nexcelom Biosciences, CS-01060). Mycoplasma (by Lonza MycoAlert™ PLUS Mycoplasma Detection System at cGMP standard) and pyrogen (by limulus amebocyte lysate assay at USP standard) were tested in CTCEF while sterility was tested by BD gram stain kit (BD biosciences) and 14-day growth assessment in the SCRF at MSKCC. Adventitious virus tests consisted of in vitro tests on MRC-5, Vero, A549, and NIH-3T3, and in vivo tests for the presence of inapparent viruses in suckling and adult mice and embryonated hen’s eggs. MAP was performed by testing the sera for the presence of antibodies to murine virus four weeks after injecting MSK-DA01 into viral free mice under GLP regulation. Adventitious virus and MAP were tested by Bio Reliance (Rockville, Maryland, USA) and Indiana University vector production facility (Indianapolis, IN, USA).
Gram stain kit
Manufactured by BD
Sourced in United States
The Gram stain kit is a laboratory tool used to classify bacteria based on their cell wall structure. It provides the necessary reagents and instructions to perform the Gram staining procedure, which differentiates bacteria into Gram-positive and Gram-negative groups.
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Lab products found in correlation
Polyvar microscope, by Reichert Technologies (1 mentions)
Staphylase test kit, by Thermo Fisher Scientific (1 mentions)
Api staph, by bioMérieux (1 mentions)
Anaeropack sachet, by Mitsubishi (1 mentions)
Sheep blood, by BD (1 mentions)
Trypticase soy agar, by BD (1 mentions)
Endotoxin, by Associates of Cape Cod (1 mentions)
9 protocols using gram stain kit
1
Comprehensive Cell Characterization Protocol
2
Bacterial Morphological Characterization
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For morphological characterization, all 40 bacterial isolates were cultivated on Marine Agar 2216 plates as pure cultures. The colors of the bacterial colonies were recorded, and shape and Gram reaction of the bacterial cells were evaluated using a Polyvar microscope (Reichert-Jung, Wien, Austria) with 800× magnification. The Gram stain reaction of the bacteria was determined using a BD Gram Stain Kit (BD Diagnostics, Sparks, MD) according to the manufacturer's instructions. For the oxidase test a piece of filter paper was soaked in aqueous 1% N,N,N′,N′-tetramethyl-p-phenylenediaminedihydrochloride solution. Freshly grown bacteria were scraped from the plate and rubbed onto the filter paper. Development of blue color within 10 s was an indication of oxidase-positive isolates.
3
Gram Staining of Fecal Samples
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Freshly evacuated fecal samples were handled using sterile forceps and rolled across an unused glass microscope slide. Following brief heat fixation over an open flame, staining was performed using a commercially available Gram stain kit (Becton Dickinson), according to the manufacturer's instructions. Briefly, slides were first saturated with crystal violet followed by iodide. After decolorization with acetone, samples were counterstained with safranin and allowed to air dry. Slides were examined via light microscopy to determine whether feces of antibiotic-treated mice still contained bacterial forms.
4
Gram Staining Bacterial Cells
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The bacteria collected from the selected colony were suspended on the glass slide coated with 100 μl of 0.45% NaCl solution. The slide was then allowed to dry at the room temperature. The dried slide was then fixed over the flame of an alcohol burner and stained with a Gram stain kit from Becton Dickinson (Sparks, Maryland, USA).
The stained slide was assessed under a ×100 objective and ×20 eyepiece immersion microscope Olympus CH20 (Olympus Optical Co, Ltd., Japan). Based on the obtained image, the staining (gram-positive or gram-negative), shape (cocci, bacilli), and arrangement (clusters, chains, pairs, etc.) were determined.
The stained slide was assessed under a ×100 objective and ×20 eyepiece immersion microscope Olympus CH20 (Olympus Optical Co, Ltd., Japan). Based on the obtained image, the staining (gram-positive or gram-negative), shape (cocci, bacilli), and arrangement (clusters, chains, pairs, etc.) were determined.
5
Vaginal Flora Assessment in Macaques
6
Milk Bacteriological Examination Protocol
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Milk samples collection and bacteriological examination of milk were carried out following standard procedures, according to the standards of the National Mastitis Council (1999), as previously described (Watts, 1990; Addis et al., 2016a,b) . Briefly, 10 μL of milk were streaked onto 5% sheep blood agar plates and aerobically incubated at 37 °C. After 24 h and 48 h plates were examined and colony forming units (CFU) were counted. Isolated bacteria were classified for colony morphology, hemolytic activity, and Gram staining (Gram stain kit, Becton-Dickinson Co., Franklin Lakes, NJ).
Gram-positive cocci were screened for catalase activity. BBL Coagulase Plasma Rabbit (with EDTA, BD), Staphylase Test kit (Oxoid, Thermo Scientific), and API Staph (bioMérieux) were used to identify suspected staphylococci. All commercial tests were performed according to the manufacturers' instructions. For M. agalactiae isolation, milk was plated onto blood agar base supplemented with 20% heat-inactivated horse serum and 500 μg/ mL ampicillin and plates were controlled up to 14 days, as previously described (Cacciotto et al., 2010 (Cacciotto et al., , 2013)) (link). Mycoplasma species was confirmed by means of specific FS1/FS2 PCR, as previously described (Tola et al., 1996) (link).
Gram-positive cocci were screened for catalase activity. BBL Coagulase Plasma Rabbit (with EDTA, BD), Staphylase Test kit (Oxoid, Thermo Scientific), and API Staph (bioMérieux) were used to identify suspected staphylococci. All commercial tests were performed according to the manufacturers' instructions. For M. agalactiae isolation, milk was plated onto blood agar base supplemented with 20% heat-inactivated horse serum and 500 μg/ mL ampicillin and plates were controlled up to 14 days, as previously described (Cacciotto et al., 2010 (Cacciotto et al., , 2013)) (link). Mycoplasma species was confirmed by means of specific FS1/FS2 PCR, as previously described (Tola et al., 1996) (link).
7
Anaerobic Bacterial Culture and Gram Staining
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The strains used in this study were grown on trypticase soy agar supplemented with 5% sheep blood (BD, Franklin Lakes, NJ, USA) for 5 days at 37 °C under anaerobic conditions created in an anaerobic jar with an AnaeroPack sachet (Mitsubishi Gas Chemical, Tokyo, Japan). The colonies were observed, and Gram stain was performed using Gram Stain Kit (Stabilized) (BD).
8
Adipose-Derived Stem Cell Isolation
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Subcutaneous adipose tissue samples were obtained through tumescent liposuction from the gluteal regions of patients 1 day before SVF cell implantation. We collected 140 mL of adipose tissue, and this was suspended in phosphate-buffered saline solution, placed in a sterile box, and transported to the laboratory. A 120-mL aliquot of the tissue was used for implantation. Mature adipocytes and connective tissues were separated from the SVF by centrifugation (Hanil Scientific Inc., Gyeonggi-do, South Korea).16 (link) Before implantation, bacteriologic tests including mycoplasma (iNtRON, Gyeonggi-do, South Korea), endotoxin (Associates of Cape Cod, MA), and gram stain kit (BD Biociences, Franklin Lakes, NJ) were performed to ensure that the samples were not contaminated, and cell viability was assessed using the methylene blue dye exclusion test (NanoEntek, Seoul, South Korea). The remaining 20 mL of adipose tissue was processed similarly and used for laboratory analysis to examine the plastic-adherent cells that form colony-forming unit fibroblasts (CFU-Fs) and to confirm the multilineage differentiation of adipose-derived stem cells.
9
Characterization of Strain JC4 Bacterium
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The cell shape and size of strain JC4T were examined via a transmission electron microscope (CM-20, Philips). Motility was assessed by the hanging-drop method. The Gram reaction was performed using a Gram Stain kit (BD, USA). The ability of strain JC4T to grow on various media, including tryptone soy agar (TSA, Difco), nutrient agar (NA, Difco), and Luria-Bertani agar (LBA, Difco) was recorded for up to five days of incubation at 25°C. Growth was observed in R2A broth (Difco) at different pH values (4.0–12.0 at 0.5 pH unit intervals) and temperatures (4, 10, 15, 20, 25, 30, 37, and 40°C) for five days [13 (link)]. Catalase and oxidase activity were detected as described previously [2 (link)]. Metabolic activity was determined by commercial API kits (bioMérieux, France). The hydrolysis of lipid, casein, and skim milk was investigated following the methods of Smibert and Krieg [33 ]. Positive hydrolysis was indicated by a clear zone around colonies. Antibiotic susceptibility of strain JC4T was examined by a Kirby-Bauer disc diffusion method [34 (link)] on R2A agar medium [16 (link)].
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