The metabolic repertoire of KH365_2T, KH569_7, and B. uniformis ATCC 8492 T was studied by performing a series of assays using Biolog AN MicroPlates™ (Biolog, Hayward, CA), following the protocol provided by the manufacturer with minor modifications. The strains were grown on CM agar under anaerobic conditions at 37 °C for 5 days to obtain colonies of sufficient size. Individual colonies were picked and inoculated in AN Inoculating Fluid (Biolog, Hayward, CA) such that the turbidity (600 nm) of 0.05–0.1 was achieved. The Inoculating Fluid was then pipetted into Biolog AN MicroPlates™ (100 μl per well), and the initial turbidity (590 nm) was measured using a Spark® microplate reader (TECAN, Männedorf, Switzerland). The plates were then incubated in anaerobic jars at 37 °C for 5 days, after which the final turbidity (590 nm) was measured. The difference between the final and initial turbidity, ΔT (590 nm), was used to determine the extent to which each of the metabolites were utilized by the strains. The metabolites differentially utilized by the strains were identified by testing whether the difference in ΔT for each metabolite was significantly different between each pair of strains (KH365_2T vs. B. uniformis ATCC 8492 T, KH569_7 vs. B. uniformis ATCC 8492 T, and KH365_2T vs. KH569_7) using the Kruskal–Wallis test.
An microplate
Manufactured by Biolog
Sourced in Switzerland, United States
The AN MicroPlate is a laboratory equipment designed for microbial identification and characterization. The core function of the AN MicroPlate is to facilitate the biochemical testing of microorganisms through the use of a standardized panel of substrates. This allows for the rapid and reliable identification of various microbial species based on their metabolic profiles.
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Lab products found in correlation
9 protocols using an microplate
1
Metabolic Profiling of Bacteroides Strains
2
Anaerobic Carbon Utilization of Gardnerella
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Bacterial isolates from freezer stocks were streaked on 5% sheep blood agar plates and were grown for 48 h anaerobically, prior to the inoculation of AN microplates (Biolog Inc, Hayward, CA). Each plate contained 95 carbon sources and one blank well. Colonies of Gardnerella isolates were harvested using a sterile swab and suspended in 14 mL of inoculating fluid supplied by the manufacturer. The cell density was adjusted to 55% T (OD595 approximately 0.25) using a turbidimeter. Each well was filled with 100 μL of culture suspension and was incubated at 35 °C anaerobically for 48 h. All inoculations and incubations were performed in an anaerobic chamber containing 10% CO2, 5% hydrogen and 85% nitrogen. All plates were read visually after 48 h of incubation. If there was no carbon source utilization, the wells remained colourless. A visual change from colourless to purple indicated carbon source utilization. To avoid bias in interpretation, a subset of the plates was read by a second observer who was blinded to the identity of the isolates. There was no disagreement between independent observers. The entire experiment was performed in two biological replicates.
3
Anaerobic Gardnerella Phenotyping by Biolog
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Bacterial isolates from freezer stocks were streaked on 5% sheep blood agar plates and were grown for 48 h anaerobically, prior to inoculation of AN Microplates (Biolog Inc, Hayward, CA). Each plate contained 95 carbon sources and one blank well. Colonies of Gardnerella isolates were harvested using a sterile swab and suspended in 14 mL of inoculating fluid supplied by the manufacturer. The cell density was adjusted to 55%T (OD595 approximately 0.25) using a turbidimeter. Each well was filled with 100 μl of culture suspension and was incubated at 35˚ C anaerobically for 48 h. All inoculations and incubations were performed in an anaerobic chamber containing 10 % CO2, 5% hydrogen, and 85% nitrogen. All plates were read visually after 48h of incubation. If there was no carbon source utilization, the wells remained colourless. A visual change from colourless to purple indicated carbon source utilization. To avoid bias in interpretation, a subset of the plates was read by a second observer who was blinded to the identity of the isolates. There was no disagreement between independent observers. The entire experiment was performed in two biological replicates.
4
Anaerobic Carbon Source Utilization
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Bacteria resuspended in inoculating fluid (Biolog) were added to AN MicroPlate (Biolog) with 95 distinct carbon sources as per manufacturer’s instruction. Plates were incubated in GasPak EZ anaerobic pouch system (BD) at 37°C. Growth was measured colorimetrically by microplate reader (BMG LABTECH) after 48 h incubation.
5
Bacterial Carbon Source Utilization
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Freshly cultured bacteria were first resuspended in inoculating fluid (Biolog) and then added to AN MicroPlate (Biolog) with 95 distinct carbon sources as per manufacturer’s instruction. Plates were incubated in GasPak EZ anaerobic pouch system (BD) at 37°C. Growth was measured colorimetrically by microplate reader (BMG LABTECH) after 24 h incubation. Data shown here is the average of two independent runs.
6
Metabolic Profile of Fusobacterium necrophorum
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We used the AN MicroPlate (Biolog) containing 95 carbon sources to determine metabolic activity of F. necrophorum. The assay was carried out according to the manufacturer’s instructions. Briefly, F. necrophorum KG34 (GenBank accession no. SRX5402669) was grown on Wilkins-Chalgren agar medium (Sigma-Aldrich) and suspended in 14 mL of AN inoculating fluid (Biolog) to make bacterial suspension with a transmittance level of about 65%. A 100 μL of bacterial suspension was quickly plated into each well of the AN MicroPlate and incubated at 37 °C in the GasPak EZ anaerobic pouch system (BD). Utilization of the carbon sources by F. necrophorum was indicated by tetrazolium violet forming a blue color, which was measured at 3, 6, 9, 12, 18, and 24 h post inoculation at OD590 using SmartSpec 3000 spectrophotometer (Bio-Rad).
7
Anaerobic Substrate Utilization Profiling
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Substrate utilization profiling of strain TM1 was performed under anaerobic conditions with an AN MicroPlate (Biolog, Hayward, CA, USA) according to the manufacturer’s recommendations, except that incubation was set to 25 °C for 72 h in an anoxic glove box. Staining of spores was carried out as described [26 (link)], except that a fuchsine solution (basic fuchsine 10 g·L−1, phenol 50 g·L−1, ethanol 100 mL·L−1) was used for staining instead of 1% aqueous safranin.
8
Pairwise Co-culture of Subgroup Isolates
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Representative isolates (VN003 of subgroup A, VN002 of subgroup B, NR001 of subgroup C and WP012 of subgroup D) from the four subgroups were co-cultured in the Biolog AN Microplate in a pairwise fashion (n = 6, AB, AC, AD, BC, BD, CD), by combining 50 μL of each isolate suspended in inoculation fluid in each well. The co-cultured AN Microplates were incubated at 35 °C for 48 h before being assessed visually for colour change. The experiment was repeated on separate days.
9
Pairwise Interactions of Microbial Isolates
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Representative isolates (VN003 of subgroup A, VN002 of subgroup B, NR001 of subgroup C, and WP012 of subgroup D) from the four subgroups were co-cultured in the Biolog AN Microplate in a pairwise fashion (n =6, AB, AC, AD, BC, BD, CD), by combining 50 µL of each isolate suspended in inoculation fluid in each well. The co-cultured AN Microplates were incubated at 35˚C for 48h before being assessed visually for colour change. The experiment was repeated on separate days.
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