Module 2: Microbial Propagation

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

2026-09-20

Module 2: Microbial Propagation

MB 360: Scientific Inquiry in Microbiology At the Bench

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

Overview

Weeks 2–3 · Plate streaking · Colony observation · Liquid cultures

  • Recover and propagate Delftia acidovorans from colonies
  • Observe colony morphology and start liquid cultures
  • Review safety procedures for live microbial isolates

Learning Outcomes

  • List items needed to propagate microbes in liquid cultures and 96-well plates
  • Explain the stages of bacterial growth on a growth-curve graph
  • Describe defined, complex, selective, enriched, and differential media
  • Discuss the importance of controls
  • Practice inoculating and diluting liquid cultures
  • Collect and interpret colony morphology and growth data
  • Create and use a team data management plan

Skills & Knowledge

Skills

  • Work safely with bacterial growth on agar plates
  • Document protocols and results clearly
  • Interpret colony morphology and growth in TSB medium
  • Propagate bacteria in liquid culture

Knowledge

  • PPE for microbial work
  • Loop use, sterilization, and streaking methods
  • Propagation in solid and liquid media

Lab Safety

Before Lab

  • Wear lab coat, goggles, and gloves
  • Wipe the bench and pipettors with 70% ethanol

During Lab

  • Treat all tips as potential biohazards
  • Remove PPE when leaving the lab
  • Discard samples and disposables correctly

After Lab

  • Wipe the station with 70% ethanol
  • Decontaminate any personal items touched during lab
  • Dispose of gloves in the main biohazard container

Methods: Colony Morphology

Important: We are working with live microbial isolates today — follow all safety protocols.

  • Obtain a TSA plate containing colonies of your isolate
  • Observe colony appearance without opening the plate
  • Record: size, shape, colour, texture, any other visible features
  • Photograph the plate on the black background

Methods: Streak Plating

Diagram showing a sterile loop transferring a single colony onto a fresh agar plate for streaking.

Single-colony transfer onto a fresh TSA plate for streaking practice.

  1. Sterilise the inoculation loop in the flame until glowing red
  2. Cool the loop briefly on the unused edge of the agar
  3. Pick a single colony from the original plate
  4. Streak zone 1 (≈ 5 parallel lines) across one-quarter of a fresh TSA plate
  5. Sterilise the loop; rotate the plate 90°; streak zone 2 from the end of zone 1
  6. Repeat to create zone 3 and zone 4 for maximum dilution
  7. Invert and incubate at 30 °C for 48 h

Methods: Plate Labeling

Diagram showing how to label the bottom of an agar plate with isolate name, class section, and date.

Correct placement of labels on the bottom of a TSA plate.

  • Write on the bottom of the plate with a permanent marker
  • Include isolate name, class section, group number, and date
  • Keep labels near the edge so they do not obscure the agar surface

Methods: Liquid Culture Inoculation

  1. Label a 15 mL tube with isolate name, date, and group
  2. Add 5 mL sterile TSB
  3. Pick one well-isolated colony with a sterile loop
  4. Swirl the loop in the TSB to release the cells
  5. Cap loosely and incubate at 30 °C with shaking (200–250 RPM) for 24–48 h

Results & Discussion

  • Compare colony morphology on TSA to your initial description
  • Describe turbidity of the liquid culture (clear, slightly turbid, very turbid)
  • Record any unexpected observations or contamination events

Reflection Questions

  1. Why do we sterilise the loop between zones during streak plating?
  2. What is the difference between a pure culture and a mixed culture?
  3. How does liquid culture growth differ visually from growth on solid media?