The Biolog GN2 plates (Biolog, Inc., Hayward, Calif.) were used for metabolic characterization and preliminary identification of GN calcifying bacteria. A pre-inoculum of the selected strains was prepared on Biolog Universal Growth Agar (BUG) and incubated at the optimal growth conditions of the strains. Inoculation and reading (at 590 nm) of microplates were performed according to the manufacturer's instructions using a Biolog Microstation with OmniLog. Pseudomonas fluorescens DSMZ 50090 was used as a reference strain. The Biolog method is based on oxidation tests of 95 substrates in a 96-well microtiter plate. Each well contains a redox dye, tetrazolium violet, allowing the colorimetric determination when microbial cells oxidize a carbon substrate. The reactions were compared to the GN database based on the similarity index, which must be at least 0.99 for acceptable species identification after 4 h of incubation and 0.75 after 16–24 h of incubation.
Universal growth agar
Manufactured by Biolog
Sourced in United States
Universal Growth agar is a general-purpose culture medium that supports the growth of a wide range of microorganisms, including bacteria, fungi, and some types of viruses. It provides the necessary nutrients and growth factors for the cultivation of a diverse range of microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Microstation, by Biolog (2 mentions)
Turbidimeter, by Biolog (2 mentions)
Gn2 plates, by Biolog (1 mentions)
Gen 3 omnilog system, by Biolog (1 mentions)
Phenotype microarray, by Biolog (1 mentions)
Redox dye mix a, by Biolog (1 mentions)
Inoculating fluid a, by Biolog (1 mentions)
Omnilog instrument, by Biolog (1 mentions)
6 protocols using universal growth agar
1
Metabolic Profiling of Calcifying Bacteria
2
Carbon Substrate Utilization of DDT-1 Isolate
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Carbon substrate utilization for the isolate DDT-1 was assessed using BIOLOG GN system (BIOLOG Inc., Hayward, CA, USA). The isolate DDT-1 was incubated on BUG (BIOLOG Universal Growth Agar) at 30 °C for 24 h prior to assay. The fresh colonies were removed from BUG media with a long cotton swab and suspended in 15 ml of inoculation fluid with turbidity equivalent to 72% transmittance as measured by BIOLOG turbidimeter. Each well in the BIOLOG GN2 microplateTM was inoculated with 150 μl of bacterial suspension using an 8-channel pipette, and then incubated in a biochemical incubator at 25 ± 1 °C. Color development in the wells was monitored at 6 h and 24 h, respectively, using a microplate reader with a 590 nm filter (BIO-TEK Instruments, Winooski, VT, USA). The output data were recorded by the automatic threshold option using BIOLOG software (BIOLOG Gen III database v2.7).
3
Bacterial Identification via Metabolic Fingerprinting
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Metabolic fingerprint analysis was conducted for bacterial identification using the Biolog Omnilog GEN III system (Biolog, Hayward, CA, USA). The Biolog Omnilog GEN III system is primarily used for soil and water microorganism identification (Avidano et al., 2005 (link)). It tests the ability of microorganisms to utilize or oxidize different carbon sources that can be detected by a redox dye (tetrazolium violet). In brief, the isolated bacteria were grown on Biolog Universal Growth agar at 37 °C for 24 h. The pure colonies were suspended in a 0.4% saline solution, and then their inoculum density was adjusted to the specified turbidity range required. Then, the cells were inoculated in a Biolog GP Microplate and incubated at 37 °C. After 24 h, the result was manually read, and the purple well pattern was analyzed by Biolog_Microlog 2 software. Fungal identification was performed by microscopically examining colony characteristics and their micro- and macromorphological characteristics using standard taxonomic keys (Von Arx, 1981 ) and by using a Biolog MicroStation (Biolog, Hayward, CA, USA).
4
Salmonella Growth and Antimicrobial Assay
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All bacterial strains, plasmids, and primers used in this study are listed in Tables 1 –3 , respectively. All Salmonella strains used in this work were derived from the Salmonella enterica serovar Typhimurium 4/74. Unless stated otherwise, bacterial cultures were routinely grown at 37°C for 16 h under dynamic or static conditions in Lysogeny Broth (LB) or on agar plates, respectively. According to manufacturer’s recommendations, Biolog Universal Growth agar with 5% sheep blood was used to grow bacteria for the Biolog Phenotype Microarray. Mueller Hinton Broth (MHB) was used to measure antimicrobial activity. When necessary, ampicillin (Amp, 100 μg/mL) or kanamycin (Km, 50 μg/mL) was added. For lac promoter induction, isopropylthio-β-galactoside was added to a final concentration of 0.5 mM. Cell growth was monitored by measuring the optical density (OD) at 600 nm.
5
Comparative Phenotypic Analysis of P. chlororaphis Strains
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The biosynthetic pathways of strain P. chlororaphis GP72(rpeA-), MDS10 and MDS22 were compared using phenotype microarray (PM) technology in plate PM5 containing 94 different nutrients (BIOLOG Inc. CA, USA). All procedures were performed as described by Bochner [86 (link)]. The tested strains were grown overnight at 28 °C using Biolog Universal Growth agar (BIOLOG Inc. CA, USA) plates. The single colony was swabbed from the plate and suspended using IF-0 GN base inoculating fluid (BIOLOG Inc. CA, USA) to a density of 85% transmittance in the Biolog turbidimeter. Suspensions were added to each well of the PM5 microplates at a volume of 150 μl and incubated at 28 °C for 72 h. Plates were placed in the OmniLog instrument, and cell growth was recorded by the respiration-dependent color change of tetrazolium violet every 15 min. Finally, the readouts were analyzed based on the different kinetic curves reflecting the phenotypic changes. The added nutrient for each well is listed in Additional file 6 . The PM assay was performed twice.
6
High-Throughput Microbial Phenotyping
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Cells were streaked on Biolog Universal Growth Agar (Biolog) plates and grown overnight at 37 °C. Then, cells were resuspended and diluted with Inoculating Fluid A (80% IF-0a GN/GP Base in sterile water; Biolog) to 42% transmittance (T) measured using a Turbidimeter (Biolog). A 42% T cell resuspension was diluted with Inoculating Fluid B (83.33% IF-0a GN/GP Base and 1.2% Biolog Redox Dye mix A in sterile water) to generate 85% T cell resuspension. For PM plate 3B and 4A, 19.8 mM of sodium succinate and 1.98 nM of ferric citrate were added to Inoculating Fluid B as carbon sources. Finally, 100 μl of the 85% T cell resuspension was inoculated onto PM plates and cellular respiration was measured using an Omnilog instrument (Biolog).
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