Module 3: Microbial Growth Curves

MB 360: Scientific Inquiry in Microbiology At the Bench

Carlos C. Goller, Ph.D.

NC State University | Department of Biological Sciences

Camila Loyola, Graduate Teaching Assistant

2026-08-19

Module 3: Microbial Growth Curves

MB 360: Scientific Inquiry in Microbiology At the Bench

MB 360 Lab Workbook
NC State University | Department of Biological Sciences

Overview

Weeks 4–5 · Growth-curve experiments · Multichannel pipetting · Biofilm viability assays

  • Evaluate growth rates of different Delftia species
  • Analyze growth parameters from shaking cultures
  • Investigate biofilm formation differences among isolates

Learning Outcomes

  • List safety considerations for propagating microbes
  • Explain the differences between static and shaking growth
  • Describe the features of biofilms
  • Discuss the significance of growth curves
  • Practice multichannel pipetting and diluting overnight cultures
  • Collect and interpret growth and biofilm data
  • Create an annotated bibliography with at least five sources

Skills & Knowledge

Skills

  • Work safely with liquid cultures in 96-well plates
  • Document protocols and results clearly
  • Interpret growth-curve and biofilm data
  • Propagate bacteria in liquid culture

Knowledge

  • PPE requirements for microbial work
  • Propagation in liquid media
  • Seeding 96-well plates for growth and biofilm analyses

Lab Safety

  • Wear required PPE
  • Clean the bench and pipettors with 70% ethanol
  • Treat all tips as biohazards
  • Dispose of plates and gloves in the correct waste streams

Methods: Liquid Culture Setup

  1. Isolated colonies were transferred to liquid medium
  2. Inoculated tubes were grown overnight at 30 °C with shaking at 200–250 RPM

Methods: Growth Assay Setup

Blank 96-well plate diagram for organizing growth-curve and control conditions.

Blank 96-well plate layout for planning growth-curve wells and controls.

  • Prepare 1:1000 dilutions of overnight cultures
  • Use positive and negative controls
  • Add 100 µL per well with multichannel pipette
  • Seal with breathable seal; load into plate reader at 30 °C for 48 h

Methods: Biofilm Viability Assay

96-well plate diagram indicating wells used for biofilm staining with crystal violet.

Crystal violet staining layout for quantifying biofilm biomass in a 96-well plate.

  1. Remove medium from static-growth wells at 24 h and 48 h time points
  2. Wash each well gently with sterile PBS (×2) to remove planktonic cells
  3. Add 125 µL 0.1% crystal violet; incubate 15 min at room temperature
  4. Remove stain; wash wells ×3 with PBS until wash runs clear
  5. Solubilise retained stain with 125 µL 30% acetic acid
  6. Transfer to a clean plate; read absorbance at 590 nm

Results & Discussion

  • Plot OD600 growth curves for each isolate and species compared
  • Plot OD590 biofilm values at 24 h and 48 h
  • Identify which isolate forms the most and least biofilm
  • Discuss what the results suggest about isolate lifestyle (planktonic vs. sessile)

Reflection Questions

  1. Why are positive and negative controls essential for growth-curve experiments?
  2. What could cause a flat growth curve even when viable cells were added?
  3. How does biofilm formation relate to antibiotic resistance or environmental persistence?