Staphylococcus aureus: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Measurement of Staphylococcus aureus Pigmentation by Methanol Extraction
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 2: Analysis of Murein Hydrolases and Proteases Through Zymography
	1 Introduction
	2 Materials
		2.1 Preparation of S. aureus Culture Supernatants
		2.2 Preparation of S. aureus Cell Wall Fractions
		2.3 Zymography
	3 Methods
		3.1 Preparation of S. aureus Culture Supernatants
		3.2 Preparation of S. aureus Cell Wall-Associated Proteins (See Note 5)
		3.3 Casting Zymogram Substrate  Gel
		3.4 SDS-PAGE and Zymography
	4 Notes
	References
Chapter 3: Detection and Quantification of Secreted Nuclease Activity in Staphylococcus aureus Culture Supernatants
	1 Introduction
	2 Materials
		2.1 Preparation of Cell-Free Supernatants
		2.2 Salmon Sperm DNA Degradation Assay
		2.3 Densitometry Analysis of Degraded Salmon Sperm DNA
		2.4 FRET Probe Nuclease Assay
	3 Methods
		3.1 Preparation of Cell-Free Supernatants
		3.2 Salmon Sperm DNA Degradation Assay
		3.3 Densitometry Analysis of Degraded Salmon Sperm DNA
		3.4 FRET Probe Nuclease Assay
	4 Notes
	References
Chapter 4: Quantitative Hemolysis Assays
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Blood Solution
		3.2 Prepare Microtiter Plate with Test Samples (Plate 1 in Fig. 1)
		3.3 Prepare Microtiter Plate with Blood (Plate 2 in Fig. 1)
	4 Notes
	References
Chapter 5: In Vitro Assay for Quantifying Clumping of Staphylococcus aureus
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 6: Measuring Staphylococcal Promoter Activities Using a Codon-Optimized β-Galactosidase Reporter
	1 Introduction
	2 Materials
		2.1 General Supplies
		2.2 Supplies Specific to High-Throughput Method
	3 Methods
		3.1 High-Throughput Screening Method
		3.2 Modified Traditional Method
	4 Notes
	References
Chapter 7: Evaluation of Staphylococcus aureus Antibiotic Tolerance Using Kill Curve Assays
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 8: Fluorescence Polarization (FP) Assay for Measuring Staphylococcus aureus Membrane Fluidity
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sample Preparation
			3.1.1 Whole Cell Suspension (Modified from)
			3.1.2 Protoplast Preparation (Modified from)
			3.1.3 Cell Membrane Extraction (Protocol Modified from)
		3.2 DPH Treatment
		3.3 Fluorometry
	4 Notes
	References
Chapter 9: Quantification of Staphylococcus aureus Biofilm Formation by Crystal Violet and Confocal Microscopy
	1 Introduction
	2 Materials
		2.1 Materials for Biofilm Growth
		2.2 Materials for Crystal Violet Biofilm Staining and Detection
		2.3 Materials for LIVE/DEAD Biofilm Staining and Detection
	3 Methods
		3.1 Biofilm Growth
		3.2 Crystal Violet Staining and Quantification
		3.3 LIVE/DEAD Staining and Confocal Microscopy
	4 Notes
	References
Chapter 10: Growth of Staphylococcus aureus Using a Rotary Cell Culture System
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparing Medium and Streaking S. aureus Strain
		3.2 Wetting HARVs and Inoculating Overnight Culture
		3.3 Inoculating HARVs and Starting Experiment
		3.4 Removing Samples from the HARV (See Note 14)
	4 Notes
	References
Chapter 11: Time-Resolved Fluorescence Assay for Measuring Oxygen Consumption Rates in Staphylococcus aureus
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparing Bacterial Cultures for MitoXpress Assay
		3.2 Typical Assay
		3.3 Experimental Example
		3.4 Analysis
	4 Notes
	References
Chapter 12: Quantifying Staphylococcus aureus Membrane Potential Using Flow Cytometry
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 13: Best Practices for Preparation of Staphylococcus aureus Metabolomics Samples
	1 Introduction
	2 Materials
		2.1 Materials for Acetone Washing Glassware
		2.2 Materials for Growth of S. aureus
		2.3 Materials for Culture Harvest and Sample Collection
		2.4 Material for Processing Samples for CFU, OD600, and  pH
		2.5 Materials for Lysing Cell Pellets for Protein Measurements
	3 Methods
		3.1 Acetone Washing Protocol for Preparing Glassware
		3.2 Growth of S. aureus Cultures
		3.3 Culture Harvest and Sample Collection
		3.4 Processing Samples for CFUs/ml, OD600, and  pH
		3.5 Lysing Cell Pellets for Protein Quantification
	4 Notes
	References
Chapter 14: A Rat Model of Orthopedic Implant-Associated Infection for Identification of Staphylococcal Biofilm Proteins
	1 Introduction
	2 Materials
		2.1 Bacterial Culture and Inoculum
		2.2 Preparation of Rat Implant Infection
		2.3 Harvesting of In Vivo Biofilm and Proteomics Analysis
	3 Methods
		3.1 Preparation of Bacterial Inoculum
		3.2 Preparation of Implants
		3.3 Orthopedic Implant-Associated Infection
		3.4 Harvesting Biofilm Materials from Implants
	4 Notes
	References
Chapter 15: An Ex Vivo Model for Assessing Growth and Survivability of Staphylococcus aureus in Whole Human Blood
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 16: Bone Marrow-Derived Macrophage Infection Assay
	1 Introduction
	2 Materials
		2.1 Generation of L929 Supernatants from Frozen Cell  Line
		2.2 Generation of Bone Marrow-Derived Macrophages
		2.3 Infection Assay
	3 Methods
		3.1 Generation of L929 Supernatants from Frozen Cell  Line
		3.2 Generation of Bone Marrow-Derived Macrophages
			3.2.1 Day 1: Isolating Bone Marrow
			3.2.2 Generating Macrophages for Infection Assay
		3.3 Infection Assay
	4 Notes
	References
Chapter 17: Measurement of Osteoblast Cytotoxicity Induced by S. aureus Secreted Toxins
	1 Introduction
	2 Materials
		2.1 Bacterial Supernatant Preparation
		2.2 Isolation of Primary Murine Osteoblasts
		2.3 Supernatant Cytotoxicity Assay with Primary Murine Osteoblasts
	3 Methods
		3.1 Bacterial Supernatant Preparation
		3.2 Isolation of Primary Murine Osteoblasts
		3.3 Supernatant Cytotoxicity Assay with Primary Murine Osteoblasts
		3.4 Analysis of  Data
	4 Notes
	References
Chapter 18: Human Skin In Vitro Colonization Model for a Skin Wound Infected by Staphylococcus aureus Biofilm
	1 Introduction
	2 Materials
	3 Methods
		3.1 Day 1: Preparation of Bacterial Culture and EpiDermFT Tissues
		3.2 Day 2: Preparation of Wound on Reconstructed Human Skin Model (EpiDermFT)
		3.3 Day 3: Harvesting and Enumeration of Wound Biofilm Growth by Colony Forming Unit (CFU) Plating
	4 Notes
	References
Index