R2A Broth — Two-Cycle Growth Protocol

Timeline Overview

1
Growth in R2A ▼
⏱ 24–72 hours Until visible turbidity
📋 Inoculum Preparation
  • Select an individual colony from R2A agar plates using a sterile inoculating loop
  • Gently suspend the colony in 10 mL liquid R2A medium in a 50 mL Falcon tube
🌡️ Incubation Conditions
Temperature 15°C
Shaking 150–200 rpm
Duration 24–72 hours
Endpoint Visible turbidity
✓ Growth Assessment
Monitor growth by visual inspection or OD600 measurements. Proceed to Cycle 2 when adequate growth is achieved (uniformly turbid medium).
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Media Preparation & Transition
Prepare 10% Urea Stock Solution
  1. Dissolve 10 g urea in 100 mL Milli-Q water by stirring at room temperature
  2. Do not heat — urea degrades at high temperature
  3. Filter-sterilize through a 0.22 µm syringe filter in the Biosafety Cabinet
  4. Store at 4°C until use
Prepare R2A + Urea Medium (100 mL total)
Sterile R2A medium 80 mL
10% urea stock 20 mL
Final volume @ 2% urea 100 mL
Mix gently by swirling. Prepare aseptically in the Biosafety Cabinet.
2
Urea Conditioning ▼
⏱ 24–72 hours With OD monitoring every 12–24 hours
🧪 Subculture Preparation
  • Inoculate at 1:10 dilution from Cycle 1 culture
  • Add 1 mL of Cycle 1 culture to 9 mL fresh R2A + urea medium
  • Mix gently
🌡️ Incubation Conditions
Temperature 15°C
Shaking 150–200 rpm
Duration 24–72 hours
Monitoring Every 12–24 hours
📊 Monitoring & Endpoint
  • Check optical density every 12–24 hours
  • Note any color changes (R2A may shift with pH changes from urea hydrolysis)
  • Harvest cells when turbid for downstream applications
Initial Volume 10 mL (Cycle 1) in 50 mL tube
Temperature 15°C (both cycles)
Shaking Speed 150–200 rpm
Urea Concentration 2% final (Cycle 2)
Dilution Factor 1:10 (Cycle 1 → Cycle 2)
Growth Indicator Visible turbidity