Automation and Basic Techniques in Medical Microbiology

Preface
Acknowledgements
Contents
About the Authors
1: Good Laboratory Practices
	1.1 Introduction
	1.2 Basic Record and Lab Note Book
	1.3 Laboratory Safety Equipment
	1.4 Biosafety Levels and Practices
2: Automation in Medical Microbiology
	2.1 Introduction
	2.2 Applications of Automation
	2.3 Advantages and Disadvantages
		2.3.1 Advantages of Using Auto-analysers
		2.3.2 Disadvantages of Automation
	2.4 Types of Auto-analysers
	2.5 History of Auto-analysers
	2.6 Laboratory Automation and Total Laboratory Automation
	2.7 Types and Applications of Auto-analysers in Microbiology
		2.7.1 Microbiological Specimen Processor
		2.7.2 Routine Biochemistry Analysers
		2.7.3 Immunology-Based Analysers
		2.7.4 Haematology Analysers
		2.7.5 Cell Counter
		2.7.6 Coagulometer(s)
		2.7.7 Additional Instrument for Haematology-Based Methods
		2.7.8 Other Miscellaneous Analysers
	References
3: Manual and Automated Characterization of Multi-antibiotic-Resistant (MAR) Bacteria
	3.1 Introduction
	3.2 Types of Antibiotic Sensitivity Tests
		3.2.1 Kirby-Bauer Disc Diffusion Method
		3.2.2 The Minimum Inhibitory Concentration (MIC) Method
		3.2.3 RAPD PCR Analysis
		3.2.4 Multiplex PCR
		3.2.5 Padlock PCR and Microarray Analysis
		3.2.6 Real-Time PCR for Quantitative Data
	References
4: Rapid Microbial Genome Sequencing Techniques and Applications
	4.1 Introduction
	4.2 WGS Techniques
	4.3 Data Analysis
		Protocol for WGS (adapted from Gautam et al. 2019)
	4.4 Applications
	4.5 Challenges
	References
5: Spectroscopy: Principle, Types and Microbiological Applications
	5.1 Introduction
	5.2 General Types of Spectra
		5.2.1 Continuous Spectra
		5.2.2 Discrete Spectra
			5.2.2.1 Emission Line Spectra
			5.2.2.2 Absorption Line Spectra
	5.3 Principle of Spectroscopy
	5.4 Optical Instruments in Spectroscopy
	5.5 Is Spectroscopy Different from Spectrometry?
	5.6 Uses of Spectroscopy
	5.7 Types of Spectroscopy
		5.7.1 Ultraviolet and Visible Spectroscopy
			5.7.1.1 Background
			5.7.1.2 Principle
			5.7.1.3 Applications of UV-Vis Spectroscopy
				5.7.1.3.1 Spectroscopy in Environmental Analysis
				5.7.1.3.2 UV-Vis Spectroscopy for Water Analysis and Environmental Applications
				5.7.1.3.3 Spectrophotometric Analysis of Bacterial Water Contaminants
				5.7.1.3.4 Spectrophotometers for Chlorine and Flouride Quantification
				5.7.1.3.5 UV-Vis Spectroscopy for Geological Studies Linked to Water Contamination
				5.7.1.3.6 Other Applications
		5.7.2 Infrared Spectroscopy
			5.7.2.1 Introduction
				5.7.2.1.1 Molecular Vibrations and Vibrational Frequency
					5.7.2.1.1.1 Vibration of Diatomic Molecules
					5.7.2.1.1.2 Vibrational Transitions
					5.7.2.1.1.3 Types of Vibrations (Sharma 2007)
			5.7.2.2 Instrumentation
				5.7.2.2.1 Source
				5.7.2.2.2 Sample Types and Preparation
				5.7.2.2.3 Various Types of Detectors Used
			5.7.2.3 FTIR (Fourier Transform IR Spectrometers)
			5.7.2.4 Advantages of FTIR
			5.7.2.5 Applications of IR Spectroscopy
		5.7.3 Mass Spectrometry
			5.7.3.1 The Mass Spectrometer
			5.7.3.2 The Nature of Mass Spectra
			5.7.3.3 The Working Principle of a Mass Spectrometer
			5.7.3.4 Applications of Mass Spectrometry
				5.7.3.4.1 Analysis of Biomolecules
				5.7.3.4.2 Analysis of Glycans
				5.7.3.4.3 Analysis of Lipids
				5.7.3.4.4 Analysis of Proteins and Peptides
				5.7.3.4.5 Analysis of Oligonucleotides
		5.7.4 Nuclear Magnetic Resonance (NMR) Spectroscopy
			5.7.4.1 NMR Spectrum
			5.7.4.2 NMR Spectrometers
			5.7.4.3 Applications of NMR
	5.8 Applications of Spectroscopy in Microbiology
	References
6: MALDI-TOF MS for Bacterial Identification
	6.1 Introduction
	6.2 MALDI: Sample Preparation and Analysis
		6.2.1 Sample Preparation
		6.2.2 Protein Digestion
		6.2.3 MALDI/MS Analysis
	6.3 Uses of MALDI-TOF
	6.4 MALDI-TOF MS-Based Antimicrobial Susceptibility Testing
		6.4.1 Detection of Antibiotic Degradation
		6.4.2 Identification of Biomarker for Detecting Antibiotic-Resistant Strains
		6.4.3 Phenotypic Antibiotic Resistance Analysis of Bacterial Strains
	6.5 Advantages and Limitations
	6.6 Challenges
	References
7: Enzyme-Linked Immunosorbent Assay (ELISA)
	7.1 Introduction
	7.2 Indirect ELISA
		7.2.1 Steps of Indirect ELISA
	7.3 Direct or Sandwich ELISA
		7.3.1 Steps of Double Antibody Sandwich (DAS) ELISA
		7.3.2 Steps of Triple Antibody Sandwich (TAS) ELISA
	7.4 Competitive ELISA
	7.5 Radioimmunoassay (RIA)
		7.5.1 Steps of RIA
	7.6 Automated ELISA
	References
8: Isolation of Normal Microbiota from the Human Body and Microbial Identification
	8.1 Introduction
	8.2 Collection of Samples from Various Parts of the Body
	8.3 Biochemical Tests for Identification of Bacteria
		8.3.1 Carbohydrate Fermentation
		8.3.2 Indole Production Test
		8.3.3 Methyl Red Test
		8.3.4 Voges-Proskauer Test
		8.3.5 Citrate Utilization
		8.3.6 Urease Test
		8.3.7 Catalase Test
		8.3.8 Coagulase Test
		8.3.9 Lactophenol Cotton Blue
	8.4 Rapid Multitest Systems
		8.4.1 Automated Validation of Every Result (VITEK) System for Microbial Identification
		8.4.2 Biolog: Phenotype Microarrays
		8.4.3 Electromigration Techniques
		8.4.4 MIDI Sherlock System for FAME Analysis
	8.5 Computer-Aided Gene Analysis for Identification of Microbes
		8.5.1 Ribosomal RNA Gene Sequencing
		8.5.2 Phylogenetic Analysis
		8.5.3 Generating Multiple Sequence Alignments
	8.6 Conclusion
	References
9: Microarrays and Its Application in Medical Microbiology
	9.1 Introduction
	9.2 Basic Principle
	9.3 Immobilization Strategies Used for Preparing Microarrays
	9.4 Manufacture of the Different Components of Microarrays
		9.4.1 Oligonucleotide Synthesis
	9.5 Properties of Fluorescence and Fluorophores
	9.6 Measuring Fluorescence
	9.7 Labelling Samples for Analysis of Gene Expressions
	9.8 Labelling Strategies
		9.8.1 Labelling Bacterial Transcripts
	9.9 Labelling Samples for Gene Expression Microarray
	9.10 Calculating Label Density in Probe
	9.11 Steps for Microarray Hybridization
	9.12 Different Slide Types for Microarray
	9.13 Comparing Automated and Manual Hybridization (Table 9.2)
	9.14 Imaging for Microarray System
	9.15 Optical System for Imaging in Microarray
	9.16 Detector System, Amplifier System and Digital Resolution for Imaging in Microarray
	9.17 Scanners and Excitation Light System for Microarray
	9.18 Data Analysis in Microarray
	9.19 Normalization of Data for Correcting Experimental Variation Between Slides
	9.20 Visualizing of Data and Clustering
	9.21 Troubleshooting During Microarray-Based Experiments
	9.22 Applications of Microarrays
	9.23 Limitations of Microarray Technique
	9.24 Conclusion and Future Direction
	References
10: Immunotechnology
	10.1 Introduction
		10.1.1 Monoclonal Antibodies: Purification and Concentrate
			10.1.1.1 Principle
			10.1.1.2 Method
		10.1.2 Concentrate the Purified Antibody
		10.1.3 Analysis and Quality Assurance
		10.1.4 Preparation of Separation Gel
		10.1.5 Preparation of Protein Sample and Loading
		10.1.6 Staining and Distaining of the Gel
		10.1.7 Quality Assurance
	10.2 Immunoelectrophoresis
		10.2.1 Protocol
	10.3 Western Blotting
		10.3.1 Required Material
		10.3.2 Protocol
		10.3.3 Blocking of Membrane
		10.3.4 Binding of Primary Antibody
		10.3.5 Binding of Secondary Antibody
	10.4 Determination of Cell Number
		10.4.1 Required Material
		10.4.2 Method
	10.5 Immunofluorescence Assay
		10.5.1 Principle
		10.5.2 Immunofluorescence Technique
		10.5.3 Labelling of Antibodies with Fluorochromes
		10.5.4 Detection of Fluorochrome-Labelled Reagent
		10.5.5 Selection of Fluorochrome
		10.5.6 Materials
		10.5.7 Blocking Buffer
		10.5.8 Dilution Buffer
		10.5.9 Fixative Solution
		10.5.10 Immunostaining
		10.5.11 Immunofluorescence Staining Method
		10.5.12 Uses
	References
11: Advances in Microscopy
	11.1 Introduction
	11.2 Light Microscopy
		11.2.1 Physical Properties of Light
		11.2.2 Reflection
		11.2.3 Transmission
		11.2.4 Absorption
		11.2.5 Refraction
		11.2.6 Diffraction
		11.2.7 The Human Eye
		11.2.8 Polarization
		11.2.9 Fluorescence
		11.2.10 Important Concepts in Microscopy
		11.2.11 Contrast
		11.2.12 Magnification
		11.2.13 Sensitivity
		11.2.14 Simple Theory of Microscopy
		11.2.15 Metric Units Used in Microscopy
		11.2.16 Light Microscopes
			11.2.16.1 The Compound Light Microscope
			11.2.16.2 Inverted Microscope
	11.3 Dark Field Microscope
	11.4 Phase Contrast Microscopy
	11.5 Differential Interference Contrast Microscopy (DIC)
	11.6 Fluorescence Microscopy
		11.6.1 Fluorescent Antibody Technique or Immunofluorescence
		11.6.2 Identification of Chromosome
			11.6.2.1 Fluorescence In Situ Hybridization (FISH)
	11.7 Polarization Microscopy
	11.8 Confocal Microscopy
	11.9 Electron Microscopy
		11.9.1 Introduction
		11.9.2 Transmission Electron Microscope (TEM)
		11.9.3 Scanning Electron Microscope
		11.9.4 Scanning Tunneling Microscope (STM)
		11.9.5 Atomic Force Microscope
		11.9.6 Sample Preparation for Light Microscope
			11.9.6.1 Wet Mount Method
		11.9.7 Histological Techniques
		11.9.8 Sample Preparation for Electron Microscope
		11.9.9 Sample Preparation of TEM and SEM
		11.9.10 Cryoelectron Microscopy
	References
Index