Cryosections (6 μm) were fixed in Acetone and stained for Gram stain (Sigma-Aldrich S.r.l., Milan, Italy) by procedures described by the manufacturer.
Gram stain
Manufactured by Merck Group
Sourced in Italy, United Kingdom, United States
The Gram stain is a laboratory technique used to classify bacteria based on their cell wall composition. It involves staining the bacteria with a series of dyes, which results in either a purple (Gram-positive) or pink (Gram-negative) color. This staining method allows for the differentiation and identification of different types of bacteria, which is an important step in diagnostic and research applications.
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Lab products found in correlation
Acetone, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Slide scanner, by Hamamatsu Photonics (1 mentions)
Nanozoomer software, by Hamamatsu Photonics (1 mentions)
Pellet pestle, by Merck Group (1 mentions)
Oxidase test, by bioMérieux (1 mentions)
Polymethylmethacrylate pmma, by McMaster-Carr (1 mentions)
Paramagnetic beads, by Spherotech (1 mentions)
Iron nanopowder, by Merck Group (1 mentions)
6 protocols using gram stain
1
Gram Staining of Cryosections
2
Histological Analysis of Macaque and Hamster Tissues
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Necropsy, tissue processing, and histology for macaques were performed by SNPRC and for hamsters by ISMMS. Samples were fixed in 10% neutral buffered formalin and processed routinely into paraffin blocks. Tissue blocks were sectioned to 5 μm and stained with hematoxylin and eosin (Cat. No. H9627; Sigma-Aldrich, St. Louis, MO) or Gram stain (Cat. No. AR175; Agilent, Santa Clara, CA) as per manufacturer’s protocol. Microscopic evaluation was performed blindly by SNPRC (macaques), ISMMS (hamsters), and Pfizer (both macaques and hamsters) pathologists and a consensus was reached among pathologists before unblinding.
3
Canine Nasal and Perineal Swabs for Staphylococcus
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One nasal swab and one perineal swab were collected from each dog (Copan Eswab LQ Amies Minitip Nylon Flocked Applicator, Appleton Woods, Birmingham, UK). A sterile swab was either inserted 5 mm into one nostril or rubbed on the skin of the perineum for 3–5 seconds before being placed in Amies transport media, stored at 4°C and processed within 36 hours. Swabs were incubated aerobically overnight at 37°C in nutrient broth with 6.5% sodium chloride. The broth was streaked onto mannitol salt agar (MSA), oxacillin resistance screening agar (ORSA) supplemented with 2 μg/ml of oxacillin and Columbia 5% horse blood agar (CAB), and incubated aerobically overnight at 37°C. Where present, isolates typical of staphylococci were selected from all plates, sub-cultured onto CAB and incubated aerobically overnight at 37°C. Fresh staphylococcal cultures on CAB were subject to Gram stain (Sigma-Aldrich Company Ltd., Gillingham, UK), tested for catalase (Sigma-Aldrich Company Ltd., Gillingham, UK) and free coagulase production (Rabbit plasma, Pro-Lab, Bromborough, UK) according the manufacturer’s instructions and stored at − 80°C in Microbank vials (Pro-Lab, Bromborough, UK). All media were obtained from LabM Ltd, Bury, UK.
4
Tissue Preparation and Staining Protocol
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For frozen sections, tissues were embedded in OCT (optimal cutting temperature compound), sectioned and post-fixed in 4% paraformaldehyde/PBS pH 7.4 for 10 min before staining. X-gal staining on frozen sections was performed as described54 (link). The tissues were sectioned and stained with haematoxylin and eosin (H&E) and with Gram stain (Sigma) by conventional methods. Images were acquired using a Hamamatsu slide scanner and analysed using NanoZoomer software (Hamamatsu).
5
Bacterial Enumeration from Tissue Biopsies
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Biopsies were collected in sterility and cut in small pieces (between 0.05 and 0.10 mm of maximum diameter). Next, they were macerated in 0.9%(w/v) NaCl (Merck®) at 100 mg/mL concentration for 5 min with a Pellet pestle (Sigma-Aldrich®). Serial dilutions were performed from the supernatant in 0.9%(w/v) NaCl (between 10−1 and 10−5) and were spread on LB dishes in duplicated. The dishes were incubated at 37 °C during 14 h followed by quantification of CFUs. The CFUs counted were verified by running a series of tests, namely, Gram stain (Merck®); MacConkey growth (Carl Roth®) and Oxidase test (BioMérieux®) according to the manufacturers’ instructions.
To certify the inoculation dose of P. aeruginosa, the effective average bacterial inoculation dose under the flap was determined by bacterial growth and found to be (9.735 ± 0.120) X 104 CFU, which was similar to the initial inoculation concentration of 105 CFU/ml. There were no statistically significant differences between the initial bacterial inoculum in the different experimental groups.
To certify the inoculation dose of P. aeruginosa, the effective average bacterial inoculation dose under the flap was determined by bacterial growth and found to be (9.735 ± 0.120) X 104 CFU, which was similar to the initial inoculation concentration of 105 CFU/ml. There were no statistically significant differences between the initial bacterial inoculum in the different experimental groups.
6
Magnetic Particle-Based Biosensor Protocol
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The permanent magnets 3×3 mm Neodymium N42 were purchased from a local hobby shop; Poly(methyl methacrylate) (PMMA) was purchased from McMaster-Carr (Elmhurst, IL, USA); ferromagnetic beads of 8, 4, and 2 μm in diameter were purchased from Spherotech (Lake Forest, IL, USA); paramagnetic beads of 4 μm in diameter were purchased from Spherotech; and superparamagnetic beads of 1 μm in diameter were purchased from chemicell (Berlin, Germany). Iron nanopowder of 60–80 nm in diameter was purchased from Sigma-Aldrich Co., (St Louis, MO, USA); gram stain was purchased from EMD Millipore (Billerica, MA, USA); gauss meter was purchased from Sypris/FW Bell (Orlando, FL, USA); and magnetic beads for capturing E. coli 0157:H7 were purchased from Lab M Ltd (Heywood, UK).
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