Bacterial sugar utilization growth assay (Orbaceae)

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Method synopsis

Growth assays testing bacterial ability to utilize different monosaccharides as carbon sources, with spectrophotometric monitoring of growth and HPLC quantification of residual sugars.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Metabolism of toxic sugars by the bee gut symbiont <i>Gilliamella apicola</i> (2017), mBio. Implementations in other papers (listed below) may differ.

1. Sugar utilization growth assays
   Tested ability of G. apicola strains to grow on different substrates by cultivating in BYI medium (pH 7.0) containing BSS (10 mM MOP-morpholinopropanesulfonic acid), supplemented with 5 mM each of glucose, fructose, and/or mannose as carbon substrate. Cultures were established by inoculating medium with single colonies and incubated at 35°C under 5% CO2. Growth monitored spectrophotometrically by following increase in optical density at 600 nm over 24 hours.
   Quantities: 5 mM concentration for each sugar substrate testedDuration: 24 hours growth monitoringConditions: 35°C, 5% CO2, pH 7.0
   Equipment: BYI medium, BSS, MOP buffer, spectrophotometer
2. Sugar concentration measurement
   After 24 hours of incubation, 1-ml aliquots of culture medium were sampled and centrifuged at 12,000 × g for 5 min. The supernatant was diluted 1:100 with water and analyzed by high-performance liquid chromatography (HPLC grade, Fisher Chemical). Concentration of residual glucose, fructose, or mannose in culture medium determined using D-mannose/D-fructose/D-glucose assay kit (Megazyme, Inc.).
   Quantities: 1 ml samples, 1:100 dilution, centrifuged at 12,000 × g for 5 minDuration: 5 min centrifugationConditions: Room temperature processing
   Equipment: centrifuge, HPLC grade chemicals, Fisher Chemical, Megazyme D-mannose/D-fructose/D-glucose assay kit

Typical Equipment

• BYI medium
• spectrophotometer
• HPLC
• Megazyme assay kit

Output Measurements

• growth curves
• sugar consumption rates
• metabolic capabilities

Papers Using This Protocol (1)

Species Studied With This Protocol

Places Where Used