S. pasteurii Starter Culture

Revival and starter culture preparation of Sporosarcina pasteurii from freezer stock for use as a positive control in ureolytic activity assays

S. pasteurii Starter Culture, v1.0 Kathryn E. Caruso · 0009-0003-2436-1791 Foreman Lab · Center for Biofilm Engineering, Montana State University Updated March 2026

How to cite this protocol Caruso, K.E. (2026). S. pasteurii Starter Culture, v1.0. Foreman Lab, Center for Biofilm Engineering, Montana State University. https://kathryncaruso.github.io/methods/s-pasteurii-starter/

Sporosarcina pasteurii Starter Culture

Standard Operating Procedure — Positive Control Preparation

Purpose: Revival and starter culture preparation of *Sporosarcina pasteurii* from freezer stock for use as a positive control in ureolytic activity assays. *S. pasteurii* is a highly ureolytic organism and serves as a reference for urea hydrolysis experiments.

1 Safety

PPE:

  • Lab coat
  • Nitrile gloves
  • Safety glasses
  • Work in Biosafety Cabinet for all inoculation steps

2 Equipment and materials

  • Sterile inoculating loops (for plating)
  • Micropipette and sterile tips (for liquid inoculation from freezer stock)
  • 50 mL Falcon tubes
  • Shaking incubator (set to experimental temperature)
  • BHI Broth
  • BHI Agar
  • Access to -80°C freezer

3 Retrieve and thaw freezer stock

  • Location: Retrieve from lab −80°C freezer stock
  • Organism: Sporosarcina pasteurii
  • Stock type: Glycerol freezer stock (liquid upon thawing)
  • Thaw at room temperature or on ice
  • Gently vortex or invert the thawed vial to resuspend cells before pipetting

4 Starter culture inoculation

4.1 For experiments at 15°C (e.g., Jung assay serial transfer)

Medium: Brain Heart Infusion (BHI) broth

Inoculation:

  • Pipette 100 µL of thawed freezer stock into a 50 mL Falcon tube containing 10 mL sterile BHI broth (1:100 dilution)

Growth conditions:

  • Temperature: 15°C
  • Agitation: 150 RPM (shaking incubator)
  • Duration: S. pasteurii grows quickly even at 15°C; check for turbidity after overnight–2 days
Note: *S. pasteurii* grows optimally at 30°C, so growth at 15°C will be slower than at its optimum. However, it is a fast grower and does not require the full 4-day lead time that cold-adapted test isolates need.

4.2 For experiments at 30°C (standard revival)

Medium: Brain Heart Infusion (BHI) broth

Inoculation:

  • Pipette 1 mL of thawed freezer stock into 100 mL BHI (1:100 dilution)
  • Flask size: ≥250 mL (minimum 50% headspace required for adequate aeration)

Growth conditions:

  • Temperature: 30°C
  • Agitation: 150 RPM
  • Duration: 24 hours

5 Working culture (subculture for assay)

  • Transfer ratio: 1:100 (e.g., 1 mL starter culture → 100 mL fresh growth medium, or 30 µL → 3 mL)
  • Medium: Same as experimental medium (e.g., succinate-urea medium for Jung assay)
  • Timing: Begin assay sampling immediately upon transfer (T0)