Overview

Weeks 8–9 · Co-culture experiments · Diffusion-mediated contact · Growth data analysis

  • Evaluate microbial interactions through co-culture of different isolates
  • Observe growth while maintaining fluidic contact across a membrane
  • Analyze inhibition or facilitation patterns

Learning Outcomes

  • List safety considerations for propagating microbes
  • Explain the purpose of Duet wells and co-culture assays
  • Discuss the significance of microbial fluid interactions
  • Practice diluting cultures to a target value
  • Seed wells consistently without contamination
  • Collect and interpret co-culture growth data
  • Revise the draft for the individual and group projects

Skills & Knowledge

Skills

  • Follow lab safety and PPE protocols
  • Use Cerillo vertical membrane co-culture systems correctly
  • Analyze co-culture growth data

Knowledge

  • PPE requirements for microbial work
  • Co-culture design and growth assessment
  • Microbial interaction concepts

Background: Cerillo Duet System

Adjacent chambers separated by a membrane allow exchange of metabolites without direct mixing of cells.

Questions this module addresses:

  • Does Delftia acidovorans SPH-1 support, suppress, or reshape your isolate’s growth?
  • What metabolites or resources might explain the observed pattern?

Lab Safety

  • Wear required PPE; clean benches and pipettors with 70% ethanol
  • Treat all tips as biohazards
  • Decontaminate liquids, glass, and work surfaces after lab

Methods: Day of the Lab

Diagram of a Cerillo Duet co-culture plate with paired chambers for controls and co-culture conditions.

Cerillo Duet co-culture layout with isolate-only, control, and co-culture conditions.
  1. Dilute overnight culture 1:1000 into fresh TSB
  2. Add 800 µL diluted culture to triplicate isolate-only wells
  3. Add 800 µL diluted Delftia SPH-1 to triplicate co-culture wells
  4. Add 800 µL fresh TSB to triplicate negative-control wells
  5. Assemble Duet inserts; seat them securely in the co-culture wells
  6. Seal the plate with a breathable membrane seal
  7. Load into the plate reader at 30 °C with continuous shaking; read OD600 every 30 min for 48 h

Results & Discussion

  • Export raw OD600 readings and apply blank correction
  • Plot growth curves for isolate-only, SPH-1-only, and co-culture conditions
  • Identify whether the interaction is neutral, mutualistic, or inhibitory
  • Connect the observed pattern to possible metabolite exchange or competition

Reflection Questions

  1. What is the purpose of the membrane in the Cerillo Duet system?
  2. How would you distinguish a mutualistic interaction from a commensal one in these data?
  3. What follow-up experiment would you design to test your hypothesis about the interaction?