Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Summary

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Abstract

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Introduction

There is a dynamic interaction and co-evolution between bacteria and the hosts in which they reside. Bacteria have evolved adherence organelles, secretion systems, and/or the ability to produce toxins that enable their productive infection of host phagocytic and non-phagocytic cells. The bacteria must also contend with recognition and antimicrobial activities of the host immune system. The host immune system is comprised of innate and adaptive components including physical and chemical barriers, immune cells, the complement system, and other components of humoral immunity. While many bacteria are susceptible to killing and clearance by the multilayered host immune res....

Protocol

1. Assessing Bacterial Viability with Propidium Iodide and SYTO9

1. Infect cells that are adherent to 12 mm diameter circular glass coverslips in 24-well plates with bacteria of interest. Do NOT fix the cells with aldehydes or organic solvents.
2. Rinse cells once gently in 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.2, containing 1 mM MgCl₂ (MOPS/MgCl₂).
3. Incubate cells for 10 min in the dark at room temperature with Alexa Fluor 647-coupled antibody or lectin that binds to the bacterial species of interest, in MOPS/MgCl₂, to detect external bacteria.

Results

The protocols outlined were used to examine survival of N. gonorrhoeae after exposure to primary human neutrophils ^5,26. Neutrophils were infected with N. gonorrhoeae and processed with protocol 1, using the green-fluorescent viability dye SYTO9 and the red-fluorescent propidium iodide (Figure 4A). The dyes were added in the presence of saponin, which sequesters cholesterol to preferentially permeabilize host cell plasma membranes, not N. gonorrhoeae membranes. Other.......

Discussion

Presented here are two protocols that use DNA binding and viability dyes in conjunction with a fluorescent lectin to identify live and dead bacteria attached to and inside human cells. Since both protocols effectively discriminate live from dead bacteria, the choice of which protocol to use depends upon the goal of the experiment. The first protocol uses propidium iodide to detect nonviable bacteria and SYTO9 to detect intact bacteria. Shown in Figure 4A is a representative image of protocol 1 applied to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
21 G 3/4 butterfly needles for blood collection	Becton Dickinson	367251
Blood collection tubes with Sodium Heparin 10 ml	Becton Dickinson	366480
Sterile water for irrigation	Baxter	07-09-64-070
Dextran 500	Sigma	31392
Sodium Chloride	Fisher Scientific	S641
Dextrose	Ricca Chemical Company	RDCD0200
Dulbecco's PBS no Ca2+ or Mg2+	Thermo Scientific	SH3002802
Ficoll solution	GE Healthcare	17-1440-03
Acetic Acid	Fisher Scientific	BP2401
12 mm circular glass coverslips	Fisher Scientific	12-545-80 12CIR-1
24-well plates	Corning Incorporated	3524
Pooled Human Serum	Sigma	S7023
RPMI	Mediatech	15-040-CV
Fetal Bovine Serum	Thermo Scientific	SH3007103
Human interleukin-8	R&D Systems	208-IL/CF
MOPS	Sigma	M3183
MgCl2	Fisher Scientific	BP214
Propidium Iodide	Life Technologies	L7007 or L7012
SYTO9	Life Technologies	L7007 or L7012
Saponin	Fluka Analytical	47036
Alexa Fluor 647-coupled soybean lectin	Life Technologies	L-32463
DAPI	Sigma	D8417
SYTOX Green	Life Technologies	S7020
Mouse anti-CD63	Developmental Studies Hybridoma Bank	H5C6
Alexa Fluor 555 Antibody Labeling Kit	Life Technologies	A20187
Hemacytometer Bright Line	Hausser Scientific	1492
Forceps	EMS	78320
Sorvall Legend RT + Centrifuge	Thermo Scientific	75004377