Microscopy methods and laboratory techniques used in microbiology:
Microscopy: Methods, Parts, and Functions
Parts and Functions of a Microscope
1. Optical Components:
○ Eyepiece (Ocular Lens): Magnifies the image (typically 10x).
○ Objective Lenses: Primary magnification; usually 4x, 10x, 40x, 100x (oil
immersion).
○ Condenser: Focuses light on the specimen.
○ Iris Diaphragm: Adjusts light intensity and contrast.
2. Mechanical Components:
○ Stage: Platform for holding the slide.
○ Stage Clips: Hold the slide in place.
○ Coarse Adjustment Knob: Moves the stage for focusing with low-power
objectives.
○ Fine Adjustment Knob: Fine-tunes focus under high-power objectives.
3. Illumination System:
○ Light Source: Provides light for viewing the specimen.
○ Mirror (if applicable): Reflects light to the condenser.
Types of Microscopes
1. Light Microscopy:
○ Bright-Field Microscopy: Standard microscope; specimen appears dark against
a bright background.
○ Dark-Field Microscopy: Enhances contrast; specimen appears bright against a
dark background.
○ Phase-Contrast Microscopy: Highlights differences in refractive index; ideal for
observing live, unstained cells.
○ Fluorescence Microscopy:
■ Uses fluorescent dyes or proteins.
■ Excitation light causes the specimen to emit light (fluorescence).
2. Electron Microscopy:
○ Transmission Electron Microscope (TEM):
■ Produces high-resolution 2D images of internal structures.
■ Uses electrons passing through the specimen.
○ Scanning Electron Microscope (SEM):
■ Produces 3D images of surface structures.
■ Electrons are reflected off the specimen's surface.
3. Confocal Microscopy:
○ Uses lasers and fluorescence to create sharp, high-resolution 3D images.
○ Suitable for thick specimens or biofilms.
4. Atomic Force Microscopy (AFM):
○ Provides high-resolution surface images by scanning with a fine probe.
Preparation of Specimens for Microscopy
● Wet Mount:
○ For observing live specimens (e.g., protozoa).
● Smear Preparation:
○ Spread a thin layer of specimen on a slide, air-dry, and fix with heat or chemicals.
● Staining Techniques:
○ Simple Stain: Uses one dye (e.g., methylene blue).
○ Differential Stain:
■ Gram Stain:
■ Differentiates bacteria into Gram-positive (purple) and
Gram-negative (pink).
■ Steps: Crystal violet → Iodine → Alcohol (decolorizer) → Safranin
(counterstain).
■ Acid-Fast Stain: Identifies acid-fast bacteria (e.g., Mycobacterium).
○ Special Stains:
■ Endospore stain (e.g., Malachite Green).
■ Capsule stain (e.g., India ink).
Microbial Laboratory Techniques
Sterilization Techniques
1. Heat Sterilization:
○ Autoclaving (121°C, 15 psi): Sterilizes media, tools, and waste.
○ Dry heat (e.g., oven): Sterilizes glassware.
2. Filtration:
○ Filters microbes from heat-sensitive liquids.
3. Chemical Sterilization:
○ Uses agents like ethylene oxide or bleach.
4. Radiation:
○ UV light: Sterilizes surfaces.
○ Gamma rays: Sterilizes disposable medical equipment.
Culturing Techniques
1. Media Types:
○ Defined Media: Exact chemical composition is known.
○ Complex Media: Contains undefined components like yeast extract.
○ Selective Media: Inhibits unwanted organisms, allows specific microbes to grow
(e.g., MacConkey agar).
○ Differential Media: Distinguishes between organisms based on biochemical
reactions (e.g., Blood agar).
2. Techniques to Obtain Pure Cultures:
○ Streak Plate Method: Isolates single colonies using a loop.
○ Spread Plate Method: Spreads diluted samples on agar.
○ Pour Plate Method: Diluted samples mixed with molten agar.
Quantification of Microbial Growth
1. Direct Methods:
○ Microscopic Count: Counting cells under a microscope using a counting
chamber.
○ Viable Count:
■ Serial dilution followed by plating and colony counting.
■ Expressed as colony-forming units (CFUs).
2. Indirect Methods:
○ Turbidity Measurement: Uses a spectrophotometer to measure optical density
(OD).
○ Dry Weight: Measures biomass after drying the sample.
Preservation of Microbial Cultures
● Short-term: Refrigeration (4°C).
● Long-term:
○ Deep-freezing (-80°C) with cryoprotectants like glycerol.
○ Lyophilization (freeze-drying).
Techniques for Studying Microbial Metabolism
1. Enzyme Assays:
○ Detects enzyme activity in microbial cultures.
2. Metabolic Profiling:
○ Biochemical tests (e.g., sugar fermentation, catalase test, urease test).
○ Automated systems like API strips for multiple tests.
Other Important Techniques
1. Molecular Methods:
○ PCR (Polymerase Chain Reaction): Amplifies DNA sequences for identification
or genetic studies.
○ Gel Electrophoresis: Separates DNA/RNA/proteins based on size.
○ Next-Generation Sequencing: Provides comprehensive microbial genome
information.
2. Microbial Identification:
○ Biochemical Tests: Identifies metabolic capabilities.
○ 16S rRNA Sequencing: Identifies prokaryotes at the genetic level.
3. Immunological Techniques:
○ ELISA and Western blot for detecting microbial antigens or antibodies.
4. Antimicrobial Sensitivity Testing:
○ Kirby-Bauer disk diffusion method: Determines antibiotic susceptibility.
