Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

Summary

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

Abstract

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads.

For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Introduction

Bacillus subtilis is a rod-shaped, gram-positive bacterium that is able to produce quiescent spores when environmental conditions do not allow cell growth¹. Spores are extremely stable cell forms and those of several species, including B. subtilis, are widely used as probiotics for human and animal use². Due to its resistance and safety properties, the spore of B. subtilis, which displays heterologous proteins, has been proposed as a mucosal adjuvant, a vaccine delivery system, and an enzyme-immobilization platform^3,4.

Protocol

See the Table of Materials for details related to all materials, instruments, and software used in this protocol.

1. Flow cytometry setting

1. Alignment of optical parameters of flow cytometer coupled to a computer
   1. Log in to Cytometer software.
   2. From the software workspace, select Cytometer | Startup and wait a few minutes | Clean mode | Sit fluids.
      ​NOTE: Air bubbles and obstructio.......

Discussion

Traditional methods, such as plate counting of colonies, are not only time-consuming, but also need viable cells and do not allow for quantification of inactivated spores⁵. The Petroff-Hausser chamber is an alternative methodology, but it requires an experienced microscopist to perform it. Flow cytometry has proven to be a useful alternative for this purpose.

Genovese et al.¹² described the use of flow cytometry for the quantification of viable c.......

Materials

Name	Company	Catalog Number	Comments
((1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC)	Sigma	341006
(N-hydroxysuccinimide) (NHS)	Sigma	130672
Anti-human fluorescent antibody	BioLegend	501410	APC anti-human IL-10
Anti-mouse fluorescent antibody	Thermo Scientific	A32723	Alexa Fluor Plus 488
BD FACSCanto II	BD		Flow cytometer
BD FACSDiva Cytometer Setup & Tracking Beads Kit (use with BD FACSDiva software v 6.x)	BD	642412	Quality control reagent
BD FACSDiva Software v. 6.1.3	BD	643629	Software
Centrifuge MegaFuge 8R	Thermo Scientific	75007213
Counting Beads	BD	340334	TruCount Tubes
Eclipse 80i	Nikon		Fluorescent Microsope
Ethidium Bromide	Ludwig Biotec
Phosphate buffered saline	Sigma-Aldrich	A4503
Plastic Microtubes	Eppendorf
Polystyrene tube	Falcon	352008	5 mL polystyrene tube, 12 x 75 mm, without lid, non-sterile