CH 1 pt 2 Microscopy, Microbial Cultivation, and the Molecular Basis of Microbial Diversity

II. Microscopy and the Origins of Microbiology

1.7 Light Microscopy and the Discovery of Microorganisms

Microbiology as a scientific discipline began with the invention and use of the microscope, which allowed scientists to observe microorganisms for the first time.

• Robert Hooke (1635–1703): First to describe microbes, illustrated the fruiting structures of molds, and coined the term "cells."

• Antoni van Leeuwenhoek (1632–1723): First to describe bacteria using a set of simple lenses (light microscope).

• Light microscope: Uses visible light to illuminate samples.

• Magnification: The ability to make an object appear larger.

• Resolution: The ability to distinguish two adjacent objects as distinct and separate. The limit of resolution for light microscopes is about 0.2 μm.

• Types of light microscopy:

  • Bright-field

  • Phase-contrast

  • Differential interference contrast

  • Dark-field

  • Fluorescence

• Compound light microscope: Uses two sets of lenses (objective and ocular) to form an image. Total magnification is calculated as: $\text{Total magnification} = \text{Objective magnification} \times \text{Ocular magnification}$ Typically, a magnification of 1,000x is needed to resolve objects of 0.2 μm diameter.

• Bright-field scope: Visualizes specimens based on differences in contrast (density) between the specimen and its surroundings. Pigmented microbes add contrast.

1.8 Improving Contrast in Light Microscopy

Contrast in light microscopy can be improved by staining, which enhances the visibility of cells and their components.

• Staining: Uses dyes (organic compounds) that bind to specific cellular materials.

• Basic dyes: Positively charged; bind strongly to negatively charged cell components (e.g., nucleic acids, acidic polysaccharides, cell surfaces). Examples include methylene blue, crystal violet, and safranin.

• Simple stain: Uses dried cells and a single dye to increase contrast.

• Differential stains: Differentiate between types of cells based on staining properties. The most common is the Gram stain:

  • Bacteria are divided into two groups based on cell wall structure:

  • Gram-positive bacteria: Appear purple-violet.

  • Gram-negative bacteria: Appear pink.

Stain Type	Purpose	Example
Simple stain	Increase contrast of all cells	Methylene blue
Differential stain	Distinguish cell types	Gram stain

1.10 Probing Cell Structure: Electron Microscopy

Electron microscopy uses electrons instead of visible light to achieve much higher resolution, allowing visualization of cellular ultrastructure.

• Electromagnets function as lenses.

• Operates in a vacuum; images are captured as electron micrographs.

• Two main types:

  • Transmission Electron Microscope (TEM): Electrons pass through thin sections of specimens, revealing internal structures.

  • Scanning Electron Microscope (SEM): Electrons scan the surface, producing detailed 3D images of cell surfaces.

III. Microbial Cultivation Expands the Horizon of Microbiology

1.11 Pasteur and Spontaneous Generation

Key experiments in the 19th century disproved the theory of spontaneous generation and established the biological basis of fermentation and disease.

• Louis Pasteur: Demonstrated that living organisms discriminate between optical isomers and that fermentation is a biological process.

• Disproved spontaneous generation using the swan-necked flask experiment, showing that life does not arise from nonliving material.

• Developed sterilization methods, food preservation techniques, and vaccines for anthrax, fowl cholera, and rabies.

1.12 Koch, Infectious Disease, and Pure Cultures

Robert Koch established the link between specific microbes and infectious diseases, laying the foundation for medical microbiology.

• Robert Koch (1843–1910): Demonstrated the germ theory of infectious disease.

• Identified causative agents of anthrax, tuberculosis, and cholera.

• Formulated Koch's postulates for proving the cause of infectious diseases:

Koch's Postulate	Description
1	The suspected pathogen must be present in all cases of the disease and absent from healthy animals.
2	The suspected pathogen must be grown in pure culture.
3	Cells from a pure culture must cause disease in a healthy animal.
4	The suspected pathogen must be reisolated and shown to be the same as the original.

• Developed solid media for obtaining pure cultures and observed that colonies have distinct shapes, colors, and sizes.

• Awarded the Nobel Prize for Physiology and Medicine in 1905.

1.13 Discovery of Microbial Diversity

Microbial diversity research explores the metabolic and ecological roles of microbes beyond medical contexts.

• Sergei Winogradsky (1856–1953): Introduced the concept of chemolithotrophy (energy from oxidation of inorganic compounds).

• Demonstrated that specific bacteria are linked to specific biogeochemical transformations (e.g., nitrogen and sulfur cycles).

• Showed that chemolithotrophs use CO2 as a carbon source (autotrophy).

• First to demonstrate nitrogen fixation (Clostridium pasteurianum) and nitrification.

IV. Molecular Biology and the Unity and Diversity of Life

1.14 Molecular Basis of Life

Understanding the molecular basis of heredity was crucial for modern microbiology.

• Frederick Griffith: Demonstrated genetic transfer in bacteria (Streptococcus pneumoniae) through transformation.

• Watson, Crick, Franklin: Elucidated the structure of DNA, confirming it as the genetic material.

1.15 Woese and the Tree of Life

Advances in molecular biology led to the discovery of evolutionary relationships among all forms of life.

• Ribosomal RNA (rRNA): Present in all cells, used to infer evolutionary relationships.

• Carl Woese (1928–2012): Used rRNA sequences to discover a new domain of life, the Archaea, distinct from Bacteria and Eukarya.

• Phylogenetic tree: Depicts evolutionary history (phylogeny) of all cells, showing three domains: Bacteria, Archaea, and Eukarya. The root is LUCA (Last Universal Common Ancestor).

• Most microbes have not been cultured; cultivation-independent methods (e.g., rRNA gene analysis) reveal extensive microbial diversity.

• DNA sequencing technology: Enables sequencing of entire genomes and metagenomics (recovery of microbial genomes from environmental DNA samples).

Domain	Key Features
Bacteria	Prokaryotic, diverse metabolic pathways
Archaea	Prokaryotic, distinct rRNA, often extremophiles
Eukarya	Eukaryotic, includes plants, animals, fungi, protists

Additional info: Modern microbiology relies heavily on molecular techniques for classification, identification, and understanding of microbial ecology and evolution.