Overview
This lecture covers essential microbiology laboratory tools and techniques, focusing on microscopy, specimen handling, and staining used to observe and identify microorganisms.
The Six I's of Microbiology Lab
- Specimen collection involves obtaining samples from environments, animals, or humans (e.g., blood, urine, swabs).
- Inoculation means transferring the sample to a suitable growth medium like agar plates or live systems.
- Incubation allows microorganisms to grow over time in controlled conditions.
- Isolation separates individual colonies or species, often working with pure cultures.
- Inspection involves examining microorganisms using microscopes and stains.
- Information gathering includes biochemical tests, drug sensitivity testing, and DNA analysis for identification.
Microscopy Basics
- Microscopes must provide magnification (enlarging objects) and resolution (clarity and detail).
- Magnification = objective lens power × ocular lens power (ocular lens is typically 10x).
- Resolution is calculated as wavelength of light ÷ (numerical apertures of objective and ocular lens combined).
- Oil immersion is used with the 100x objective lens to increase resolution by allowing more light to enter.
- Bright field microscopes (used in lab) show specimens darker than the bright background.
Types of Microscopes
- Bright field: for live or stained specimens, most commonly used in labs.
- Dark field: bright specimens on a dark background, useful for live and unstained samples.
- Phase contrast: enhances inside structures of cells using light phase changes.
- Fluorescent: uses dyes that glow under UV light for diagnostic purposes.
- Confocal: laser-based, provides 3D views by scanning through specimen layers.
- Electron microscopes: use electron beams; Transmission Electron Microscope (TEM) gives 2D internal images, Scanning Electron Microscope (SEM) gives 3D external images.
Specimen Preparation and Staining
- Wet mount: live bacteria mixed with water for immediate observation.
- Hanging drop mount: another method to view living, motile bacteria.
- Fixed mount (smear/heat-fixed): bacteria are killed and attached to slide by heat before staining.
- Positive staining: uses basic dyes, stains the cells themselves.
- Negative staining: uses acidic dyes, stains background leaving cells clear.
- Simple stains: one dye to reveal shape, size, and arrangement (e.g., crystal violet, methylene blue, safranin).
- Differential stains: multiple steps to distinguish cell types or structures (e.g., Gram stain, acid-fast stain).
- Structural stains: highlight particular cell structures (e.g., capsule stain, flagella stain).
Key Terms & Definitions
- Magnification — enlarging the image of a specimen.
- Resolution — ability to distinguish two points as separate entities.
- Numerical aperture (NA) — a measure of a lens’s ability to gather light.
- Bright field microscope — shows dark specimens on a light background.
- Gram stain — differential stain distinguishing Gram-positive (purple) and Gram-negative (pink/red) bacteria.
- Acid-fast stain — identifies acid-fast bacteria like Mycobacterium.
- Capsule stain — highlights protective layers around certain bacteria.
- Flagella stain — visualizes bacterial flagella for motility studies.
Action Items / Next Steps
- Practice using microscopes, focusing on magnification, resolution, and oil immersion techniques.
- Complete simple, differential, and structural staining procedures in lab.
- Review lab manual protocols for specimen preparation and staining methods.