R2A Broth — Two-Cycle Growth Protocol

Two-cycle liquid culture protocol using R2A broth for bacterial isolate growth and urea conditioning.

R2A Broth — Two-Cycle Growth Protocol, v1.0 Kathryn E. Caruso · 0009-0003-2436-1791 Foreman Lab · Center for Biofilm Engineering, Montana State University Updated December 2025

How to cite this protocol Caruso, K.E. (2025). R2A Broth — Two-Cycle Growth Protocol, v1.0. Foreman Lab, Center for Biofilm Engineering, Montana State University. https://kathryncaruso.github.io/methods/r2a-broth/

Purpose

Two-cycle liquid culture protocol using R2A broth. Cycle 1 grows bacterial isolates from agar plates in liquid R2A medium. Cycle 2 subcultures into R2A supplemented with 2% urea to condition isolates to urea-containing media before downstream assays.

Safety

PPE:

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

Materials

Media/reagents:

  • R2A liquid medium (sterile)
  • R2A agar plates with bacterial cultures
  • Urea (for Cycle 2 stock solution)
  • Milli-Q or deionized water

Equipment:

  • 50 mL Falcon tubes
  • Sterile inoculating loops
  • Sterile pipettes and tips
  • 0.22 µm syringe filter and sterile syringe
  • Shaking incubator (set to 15°C)
  • Spectrophotometer (optional, for OD600 monitoring)

Growth Cycle 1 — Growing in liquid R2A

1.1 Inoculum prep

  1. Select an individual colony from R2A agar plates using a sterile inoculating loop
  2. Gently suspend the colony in 10 mL liquid R2A medium in a 50 mL Falcon tube

1.2 Incubation

  • Temperature: 15°C
  • Shaking: 150–200 rpm
  • Duration: 24–72 hours until visible turbidity develops
  • Monitor growth by visual inspection or OD600 measurements

1.3 Growth assessment

  • Target growth: uniformly turbid medium
  • Proceed to Cycle 2 when adequate growth is achieved

Growth Cycle 2 — Conditioning with urea

2.1 Prepare 10% urea stock solution

  1. Dissolve 10 g urea in 100 mL Milli-Q water by stirring at room temperature (do not heat — urea degrades at high temperature)
  2. Filter-sterilize through a 0.22 µm syringe filter in the Biosafety Cabinet
  3. Store at 4°C until use

2.2 Prepare R2A + urea medium (2% final concentration)

For 100 mL of complete medium:

  • C1V1 = C2V2
  • (10%)(V1) = (2%)(100 mL)
  • V1 = 20 mL of 10% urea stock
Component Volume
Sterile R2A medium 80 mL
10% urea stock 20 mL
Final volume 100 mL
  1. Prepare 80 mL of sterile R2A medium
  2. Aseptically add 20 mL of 10% urea stock
  3. Mix gently by swirling

2.3 Subculture preparation

  1. Inoculate at 1:10 dilution from Cycle 1 culture
  2. Add 1 mL of Cycle 1 culture to 9 mL fresh R2A + urea medium

2.4 Incubation

  • Same conditions as Cycle 1 (15°C, 150–200 rpm)
  • Duration: 24–72 hours
  • Monitor growth

2.5 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

Notes

  • Always work in a Biosafety Cabinet for inoculation and transfer steps
  • Use sterile pipettes and tubes throughout
  • Always include a negative control (uninoculated medium)
  • Document colony morphology before transfer
  • Record all observations (turbidity, color, odor, pH)