research methods

Autoclaving uses pressurized steam to sterilize equipment and media and to decontaminate biological waste. Different load types (dry equipment, liquid media, biological waste) require different cycle parameters to ensure effective sterilization without damaging materials. This protocol consolidates three previously separate procedures into a single reference covering all routine autoclave uses, with guidance on cycle selection, safety precautions, and troubleshooting common issues.

Streak plating from −80°C glycerol stocks produces isolated colonies that can be used to start fresh liquid cultures, confirm strain identity, or assess colony morphology. This protocol covers both the frozen scrape method (which preserves the stock for repeated use) and the thawed pipette method, with guidance on medium selection for different organism types including cold-adapted environmental isolates.

Optical density at 600 nm (OD₆₀₀) provides a quick, non-destructive estimate of cell concentration in liquid culture. This protocol describes a general-purpose method for standardizing bacterial inocula using dilution-based OD measurements and C₁V₁ = C₂V₂ calculations, applicable to any liquid culture experiment where a target starting OD is specified.

Combustion sterilization eliminates all organic residues from glassware by heating to 450°C for five hours. Unlike autoclaving, which sterilizes but does not remove organics, combustion ensures glassware is both sterile and organically clean. This is used for culture tubes in optical density measurements, where trace organic contaminants could interfere with growth readings or introduce unwanted carbon sources.

A 5% nitric acid solution denatures enzymes like urease on contact, locking in the urea concentration at the moment of sampling. This same solution serves as the matrix for preparing urea standards in colorimetric assays, ensuring that standards and samples share identical chemical backgrounds for accurate quantification.

Stock solutions of 1 M hydrochloric acid and 1 M sodium hydroxide are used for fine pH adjustments during media preparation. This protocol covers the preparation of small-volume (50 mL) working stocks suitable for bench-scale media preparation.

Ureolytic bacteria require both a carbon source for growth and urea as a substrate for urease activity. Different carbon sources can significantly affect growth rates and metabolic activity, particularly in cold-adapted organisms whose metabolic preferences may differ from mesophilic model strains. This assay provides a standardized framework for comparing bacterial growth across carbon sources using optical density measurements over time, paired with endpoint pH readings as an indicator of urease activity. It can be adapted for any combination of carbon sources, bacterial isolates, and incubation temperatures.

Quantifying ureolytic activity — the rate at which bacteria hydrolyze urea — is central to studying microbially-induced calcium carbonate precipitation (MICP). The Jung assay uses a colorimetric two-reagent system (o-phthalaldehyde and NED) to measure urea concentration at 505 nm, providing a direct, quantitative measure of urea hydrolysis over time. The serial transfer design in this protocol allows tracking of ureolytic activity across multiple growth cycles, which is useful for understanding how bacteria adapt to urea-containing media over extended culture periods. This approach can be scaled to screen panels of isolates under standardized conditions.

Brain Heart Infusion (BHI) agar is a nutrient-rich, general-purpose medium widely used for cultivating fastidious organisms. In this research, BHI agar plates are used specifically for growing up Sporosarcina pasteurii from freezer stock to serve as a positive control in ureolytic activity assays. S. pasteurii is a well-characterized ureolytic bacterium that grows optimally at 30°C on rich media, and BHI provides the nutritional complexity needed to reliably recover it from cryopreserved stocks. Having a standardized BHI plate preparation protocol ensures consistency across experiments and supports reproducibility when onboarding new lab members.

Brain Heart Infusion (BHI) broth is a commercially available, nutrient-rich liquid medium widely used for cultivating fastidious organisms. In this context, it serves as the growth medium for Sporosarcina pasteurii, a well-characterized ureolytic bacterium used as a positive control in urea hydrolysis experiments. BHI supports rapid growth of S. pasteurii at both its optimal temperature (30°C) and at lower experimental temperatures.

BHI Urea Agar combines a nutrient-rich base (Brain Heart Infusion) with 2% urea to support growth of ureolytic bacteria on solid medium. The urea is added via a filter-sterilized stock after autoclaving to prevent thermal degradation. This medium is used for plating and screening ureolytic organisms, where urea hydrolysis can be detected through downstream assays or pH-based indicators.

Sporosarcina pasteurii is a well-characterized ureolytic bacterium commonly used as a positive control in urea hydrolysis experiments. This protocol covers revival from frozen glycerol stocks and preparation of starter cultures at both 15°C and 30°C incubation temperatures. Standardized positive control preparation supports consistent reference performance across assay runs.

This defined medium uses sodium succinate as the sole carbon source, paired with 2% urea as a nitrogen source and urease substrate. The succinate concentration is matched on a molar carbon basis to a 0.5 g/L glucose reference formulation (16.67 mmol C/L). The medium can be used for growth experiments, ureolytic activity assays, or any application requiring a defined carbon source with urea supplementation.

R2A is a low-nutrient medium originally developed for enumerating heterotrophic bacteria in treated drinking water. Its minimal formulation makes it suitable for culturing slow-growing environmental isolates that may be inhibited by richer media. This two-cycle protocol first establishes growth in standard R2A liquid, then subcultures into R2A supplemented with urea to condition bacteria to urea-containing media before use in downstream assays.