Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Summary

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Abstract

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

Introduction

The field of bacterial cell biology has been significantly enhanced by recent advancements in microscopy techniques^1,2. Among other instruments, microscopes that are capable of conducting timelapse fluorescence microscopy experiments remain a valuable tool. Investigators can monitor various physiological events in real-time using fluorescent proteins such as, green fluorescent protein (GFP)-based transcriptional and translational reporter fusions, fluorescent D-amino acids (FDAA)³, or use other stains for labeling the cell wall, membrane and DNA. It is therefore of no surprise that fluo....

Protocol

1. General growth conditions

1. Inoculate 2 mL of appropriate growth medium supplemented with antibiotics (where required) with a single colony of the strain(s) to be imaged. Incubate these seed cultures overnight at 22 °C in a shaking incubator.
   NOTE: The specific bacterial growth conditions used in this article are provided under the representative results section.
2. Dilute overnight cultures 1:20 in fresh media in a 125 mL flask, supplement with antibiotics (where required).
3. Grow.......

Discussion

Microscopy has remained a mainstay in studies pertaining to microbial organisms. Given their micron-scale cell size, single-cell level studies have traditionally relied on electron microscopy (EM). Although EM has become quite a powerful technique in recent years, it has its own intrinsic limitations in addition to limited user access¹⁶. Improvements in fluorescence microscopy techniques and development of different fluorescent probes, such as FDAA³, have provided microbial.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Fisher BioReagents	BP160-100	Molecular Biology Grade - Low EEO
DAPI	Invitrogen	D3571	Microscopy
FM4-64	Invitrogen	T3166	Microscopy
Glass bottom dish	MatTek	P35G-1.5-14-C	Microscopy
IPTG	Fisher BioReagents	BP1755-10	Dioxane-free
Microscope	GE	DeltaVision Elite	Customized Olympus IX-71 Inverted Microscope Stand; Custom Illumination Tower and Transmitted Light Illuminator Module. Objectives: PLAPON 60X (N.A. 1.42, WD 0.15 mm); OLY 100X OIL (N.A. 1.4, WD 0.12 mm); DIC Prism Nomarski for 100X Objective; CoolSnap HQ2 camera; SSI Assembly 7-color; Environmental control chamber - opaque.
PC190723	MilliporeSigma	3445805MG	FtsZ inhibitor
SoftWorx	GE		Manufacturer-supplied imaging software