Ch. 2 - Tools and Techniques

Page 1: Introduction

• Tools and techniques for studying microorganisms

• Importance of advanced study techniques in microbiology

• Metaphorical expressions denoting excitement or engagement in this field

Page 2: Modern Classification Systems

• Sequencing comparison: analyzes similarities across genes or conserved genes in various organisms

• Use of whole-genome sequencing (WST)

Page 3: rRNA and Modern Classification

• rRNA: nucleic acid sequences found universally across living organisms

  • Used for comparative studies since all organisms share ribosomal RNA (rRNA)

  • Constant regions of rRNA allow comparison of ancient relatives

  • Highly variable regions of rRNA useful for studying more recent relatives

• Three domains of life based on rRNA: Bacteria, Archaea, and Eukarya

Page 4: Photosynthesis and rRNA Phylogeny

• Consequences of rRNA phylogeny reveal evolutionary relationships

• Old bacterial tree indicated narrow distributions

• Important traits previously misunderstood

Page 5: Evolution of Photosynthesis

• Photosynthesis is now understood as being widely distributed and evolving multiple times

• Analysis using new rRNA bacterial branches (16S rRNA tree) illuminates these connections

Page 6: Consequences of rRNA Phylogeny

• Previous understandings suggested many unusual bacteria existed

• The old bacterial branch yielded a narrow view regarding these organisms

Page 7: Discovery of Archaea

• Identification of some unusual bacteria as belonging to the Archaea domain

• The emergence of Archaea was clarified through rRNA analysis

Page 8: Revised Tree of Life

• Transition from the 5 Kingdoms model to a simplified 3 Kingdom model

  • Old kingdoms: Bacteria, Protista, Planta, Fungi, Animalia

  • New classification: Bacteria, Archaea, Eukarya

• This shift entailed a complete revision of the Tree of Life, acknowledging parallelism and equality between Bacteria and Eukaryotes

Page 9: Biological Nomenclature

• Carl Woese proposed the 3 Domain system

• Detailed classification hierarchy:

  • Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species

• Illustrative examples included in classification

Page 10: Endosymbiotic Theory

• Mitochondria and chloroplasts possess bacterial rRNA

• Eukaryotic cells are characterized as containing ancestral bacterial forms

Page 11: Learning Objectives

• Challenges in studying microbes and methodologies to address them:

  1. Understanding obstacles to studying microbes

  2. Examining terminology for bacterial growth

  3. Discussing microscopy techniques including magnification, resolution, and contrast

  4. Methodological planning to identify infectious microbes starting from specimen collection

Page 12: Size of Microbes

• Differentiation between macroscopic and microscopic organisms

  • Example: Bacteria typically range from 1-2 μm in size

Page 13: Challenges in Microbial Studies

• Size and diversity of microbes complicate studies:

  • 500 million bacteria can fit in a single grain of sand

  • A teaspoon of soil may contain up to 1 billion cells from about 10,000 species

  • Only 1% of bacteria can be cultured in laboratory settings

  • Contamination by non-target microbes is a prominent issue

Page 14: The Five I's of Microbiology

• Overview of essential microbiology procedures:

  1. Inoculation: introducing a sample to a growth medium

  2. Incubation: creating optimal growth conditions

  3. Isolation: separating individual species from a sample

  4. Inspection: observing culture outcomes

  5. Identification: determining species identity of microbes

Page 15: Growth Terminology

• Definitions of key terms in bacterial growth:

  • Inoculation: sample introduction to nutrient medium

  • Incubation: growth conditions for microorganisms

  • Culture: observable growth of microbes on nutrient mediums

  • Relationship between inoculation, incubation, and culture

Page 16: Visualization of Bacterial Growth

• Bacillus cereus growth monitored over 85 hours through time-lapse

Page 17: Types of Growth Media

• Various media types for bacterial culture:

  • Liquid (broth): promotes uniform growth

  • Semisolid: used for motility studies

  • Solid (slant/plate): ideal for separating individual cells

Page 18: Components of Bacterial Growth Media

• Essential ingredients for growth media:

  • Carbon sources (sugars)

  • Nitrogen sources

  • Salts and water as core components

Page 19: Agar as a Growth Medium

• Agar: a solidifying agent for growth media

  • Derived from seaweed (Gelidium) and solidifies at room temperature

  • Functions solely as a medium solidifier; does not provide nutrients

Page 20: Purposes of Bacterial Growth Media

• Different purposes of various types of media:

  • General-purpose: fosters growth of most microbes

  • Selective: allows growth of specific microbes while inhibiting others

  • Differential: allows growth of multiple microorganisms with visible differentiation among species

Page 21: Further Classification of Bacterial Media

• Selective media grows only selected microbial species

• Differential media allows multiple species to grow while distinguishing them visibly based on reactions

Page 22: Isolation Techniques

• Definitions for pure and mixed cultures:

  • Pure culture: contains one species

  • Mixed culture: contains two or more species

  • Techniques to isolate individual species from mixed populations

Page 23: Isolation of Microbial Cells

• Separation of microbial cells to produce distinct colonies

• Definition of a colony: visible cluster of cells formed on solid media

Page 24: Isolation Methods

• Methods for isolating bacterial colonies:

  • Streak plate method: cells dragged across media for dilution

  • Pour plate method: spread progressively more diluted samples

Page 25: Counting Bacteria

• Method for counting bacteria through colony growth:

  • Assumes that each starting cell forms a colony: each colony equates to one colony-forming unit (CFU)

Page 26: Estimating Cell Numbers via Dilution

• Example question on predicting colony growth based on starting cells

  • If starting with 100 E. coli cells, expect to see 100 colonies

  • For unknown larger populations, dilution is necessary to extrapolate counts accurately

Page 27: Inspecting Microbes

• Inspection defined as observing microbes in the laboratory

• Microscopy is the most convenient method for quick direct inspection of bacterial samples

Page 28: Historical Microscopy

• Overview of Antony van Leeuwenhoek's early microscope designs

  • Year: 1670s; identified 'Animalcules' through magnification

  • Maximum magnification reached: 300X

Page 29: Modern Bright Field Microscopes

• Description and components of modern bright field microscopes

  • Maximum magnification of 1000X

  • Essential parts: ocular lens, objective lens, coarse and fine adjustments

Page 30: Electron Microscopes

• Modern electron microscopes provide significant magnification

  • Maximum magnification of 50,000,000X illustrating superior resolution capabilities

Page 31: Types of Microscopes

• Overview of different types of microscopes:

  • Bright-field: multipurpose

  • Phase-contrast: reliance on light trickery for contrast

  • Fluorescence: utilizes dyes that emit light for visualization

  • Electron: best resolution and magnification for detailed structures

Page 32: Principles of Microscopy

• Three core principles governing microscopy:

  1. Magnification: size appearance of an image

  2. Resolution: clarity to distinguish between separate specimens

  3. Contrast: ability to differentiate an image from its background

Page 33: Understanding Magnification

• Magnification defined and its impact on image size

  • Example: 100X magnification means an image appears 100 times larger than visible

Page 34: Understanding Resolution

• Clarity in distinguishing specimens is impacted by resolution

  • Examples of low and high resolution comparisons

Page 35: Importance of Resolution

• High resolution is critical to avoid misleading conclusions

  • A single dot may be misinterpreted as multiple cells in poor resolution

Page 36: Concept of Contrast

• Contrast defined as the ability to distinguish an image from its background

  • Examples of unstained (low contrast) vs. stained (high contrast) images

Page 37: Enhancing Contrast with Staining

• Discusses staining techniques:

  • Positive stains: dye stains bacteria

  • Negative stains: dye stains background, leaving bacteria clear

  • Phase contrast utilizes light interference to enhance contrast without killing microbes

Page 38: Staining Techniques

• Two basic staining techniques are identified:

  • Positive stain: directly stains the bacterial cells

  • Negative stain: stains the background, leaving cells clear

Page 39: Identification of Microbes

• Identification techniques leverage unique traits of microbes:

  • Utilizes selective and differential growth media

  • Application of biochemical tests and molecular techniques

  • Value of staining in identifying bacteria

Page 40: Differential Stains

• Overview of differential stains that utilize multiple dyes:

  • Utilized for distinguishing different cell types or structures for quick diagnosis

Page 41: Understanding Gram Stain

• Gram stain as a critical differential stain:

  • Categorizes bacteria based on cell wall composition (peptidoglycan)

  • Gram-positive: retains the stain due to thick cell walls

  • Gram-negative: easily loses stain due to thin positive wall

Page 42: Applications of Gram Staining

• Rapid diagnostic relevance of Gram staining for antibiotic treatment decisions:

  • Determines bacterial type, helping select appropriate initial antibiotic therapy

Page 43: Special Stains

• Special staining techniques are key for identifying unique cellular components:

  • These stains reveal structures not exposed by standard methods, e.g., capsules or flagella

Page 44: Case Study Scenario

• Exploration of a case study involving infection:

  • Patient's antibiotic treatment strategy and necessity for accurate microbial identification to tailor appropriate therapy.