This book discusses principles, methodology, and applications of microbiological laboratory techniques . It lays special emphasis on the use of various automated machines that are essential for medical microbiology and diagnostic labs. The book contains eleven major chapters. The first chapter describes the good lab practices which should be followed by the students in all biological, chemistry or microbiology laboratories. The next chapter describes manual and automated characterization of antibiotic resistant microbes, followed by a chapter on genomics based tools and techniques that are integral to research. Further chapters deal with other important techniques like immunology based techniques, spectrophotometry and its various types, MALDI-TOFF and microarrays, each with illustrations and detailed description of the protocols and applications. The book also gives certain important guidelines to the students about the planning the experiment and interpreting results.
The book is highly informative and provides latest techniques. It is a handy compendium for graduate and post graduate students, as well as more advanced researchers.
Author(s): Santi M. Mandal, Debarati Paul
Publisher: Springer
Year: 2022
Language: English
Pages: 212
City: New York
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
