5  Module 5: Microbial Interactions

5.1 Overview

Weeks 8 and 9 focus on co-culture experiments that examine how isolates interact through diffusion-mediated contact.

5.2 Purpose

  • Evaluate microbial interactions through co-culture growth of different isolates.
  • Observe growth while maintaining fluidic contact across a membrane.
  • Analyze possible inhibition or facilitation patterns.
  • Use pipettes to seed co-culture wells for growth assays.
  • Apply the principles of the Cerillo Duet system to your research question.

5.3 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.
  • Describe the purpose, methods, and preliminary results of co-culture experiments.
  • Collect and interpret co-culture growth data.
  • Revise the draft for the individual and group projects.
  • Explain how the experiment contributes to the overall research goal.

5.4 Skills and Knowledge

5.4.1 Skills

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

5.4.2 Knowledge

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

5.5 Task

Review the background and procedure information before lab and work with your partner to set up the co-culture assays and document results.

5.6 Criteria for Success

Successful completion requires participation in assay setup, collection of analyzable data, and a complete ELN record.

5.7 Background

After characterizing individual growth and metabolism, this module asks how isolates behave in the presence of other organisms. The Cerillo Duet co-culture platform allows isolates to grow in adjacent chambers separated by a membrane that permits exchange of metabolites without direct mixing of cells.

5.8 Procedures

5.8.1 Lab Safety

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

5.8.2 Methods

5.8.2.1 What Was Prepared in Advance

  • Overnight cultures of each isolate were started in 2.5 mL TSB and incubated at 30 C with shaking.

5.8.2.2 Day of the Lab

Figure Figure 5.1 maps the isolate-only, control, and co-culture conditions across the Cerillo Duet platform.

Diagram of a Cerillo Duet co-culture plate with paired chambers for controls and co-culture conditions.
Figure 5.1: Cerillo Duet co-culture layout with isolate-only, control, and co-culture conditions.
  1. Dilute the overnight culture 1:1000 into fresh TSB.
  2. Add 800 µL of diluted culture to triplicate wells for the isolate-only condition.
  3. Add 800 µL of TSB to the adjacent control wells.
  4. Add 800 µL of diluted SPH-1 to the paired experimental wells for co-culture.
  5. Seal the plate carefully.
  6. Incubate with shaking at 30 C for 24 to 48 hours.
  7. Analyze OD600 measurements collected every 5 minutes.

5.8.3 Protocol Notes

Record any mistakes, deviations, or strain-specific observations.

5.9 Results

Add images of the co-culture plate and any figures generated from the growth data.

5.10 Result Analysis

Compare isolate-only growth to co-culture growth. Explain whether the second organism appeared to support, suppress, or otherwise reshape your isolate’s growth profile.

5.11 Discussion Questions

  1. How can co-culture systems help researchers understand microbial behavior?
  2. What kinds of metabolites or resources might explain improved or reduced growth in co-culture?