Home › Microscopy & Imaging › Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

DOI: 10.3791/3145-v
What you'll learn
  • ✓ Prepare bacterial cultures for live-cell fluorescence imaging
  • ✓ Set up and configure automated time-lapse microscopy protocols
  • ✓ Analyze single-cell bacterial behavior from time-lapse image data
Protocol

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Difficulty
intermediate
Total time
~4–6 hours (culture prep + microscopy acquisition)
Model organism
Bacillus subtilis, Streptococcus pneumoniae
Biosafety
BSL-1

Steps

1
Prepare B. subtilis bacterial cultures

Grow Bacillus subtilis to log phase under defined conditions for single-cell imaging. Culture preparation is essential for obtaining healthy, uniform cell populations.

▶ 01:10
2
Prepare microscope sample slide

Mount bacteria onto a glass slide or chamber with appropriate medium and cover preparation. Proper sample preparation ensures optical clarity and minimal phototoxicity during live-cell imaging.

▶ 02:18
3
Configure and execute time-lapse imaging

Set up automated fluorescence microscopy acquisition parameters including interval timing, focal plane maintenance, and multi-position imaging. Execute the time-lapse protocol to capture single-cell dynamics over time.

▶ 05:14
4
Analyze microscopy image time-series data

Process and quantify single-cell behavior from acquired time-lapse images using image analysis software. Extract metrics such as cell growth rate, division timing, and morphological changes.

▶ 06:36
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