Unit 2: Microscopy
General Microbiology notes · PTU syllabus (BMLS102-18)
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Unit summary
Microscopes make microbes visible. This unit covers the history and types of microscope — light, dark-ground illumination, fluorescence, phase contrast and electron (transmission and scanning) — with their principles and working.
After this unit you can
- Trace the history of microscopy
- Explain magnification and resolving power
- Explain the principles of light, dark-ground, fluorescence and phase contrast microscopes
- Compare transmission and scanning electron microscopes
PTU syllabus topics
- History and types of microscope — light
- DGI
- fluorescent
- phase contrast
- electron (transmission and scanning)
- principles and operational mechanisms of each
Bright-field light
Visible light through a stained sample
Routine stained smears
Phase contrast
Converts phase shifts into contrast
Living, unstained cells
Fluorescence
UV light excites fluorescent dyes
Auramine stain for TB, immunofluorescence
TEM
Electrons pass through thin sections
Internal ultrastructure
SEM
Electrons scan the surface
3D surface detail
Topic 1
History of the microscope
- Janssen (around 1590) made an early compound microscope; Robert Hooke (1665) described cells in cork; Leeuwenhoek made powerful single-lens microscopes (about 270×); Ernst Abbe improved lenses and condensers; Ruska and Knoll built the electron microscope (1931).
Topic 2
Magnification and resolving power
Total magnification
Objective × eyepiece (e.g., 100 × 10 = 1,000×)
Resolving power (limit of resolution)
d = 0.61 λ ÷ NA
Numerical aperture
NA = n sin θ; oil (n = 1.515) raises NA
- Light microscopes resolve about 0.2 µm; electron microscopes about 0.2 nm. Immersion oil has the same refractive index as glass, preventing light loss at the 100× objective.
Topic 3
Bright-field (light) microscope
- Eyepiece (ocular)
- Usually 10×
- Objectives
- 10×, 40×, 100× oil immersion
- Condenser and iris diaphragm
- Focus and control light
- Stage and mechanical stage
- Hold and move the slide
- Coarse and fine adjustment
- Focusing
- Light source
- Built-in lamp or mirror
- Principle: light passes through a stained specimen; objects appear dark on a bright background. Used for Gram and other stained smears.
Topic 4
Dark-ground (dark-field) illumination
- A special condenser with a central stop lets only oblique light hit the specimen; scattered light enters the objective, so organisms shine bright on a dark background. Used for unstained, thin organisms such as Treponema pallidum and Leptospira and for motility.
Topic 5
Fluorescence microscope
- Specimens stained with fluorochromes absorb short-wavelength (UV or blue) light and emit longer-wavelength visible light. An exciter filter, dichroic mirror and barrier filter separate the light.
- Uses: auramine-rhodamine stain for tuberculosis, immunofluorescence for antigens and antibodies (FTA-ABS, rabies).
Topic 6
Phase contrast microscope
- Converts small differences in refractive index (phase shifts) into differences in brightness using an annular diaphragm and a phase plate, so living, unstained cells and internal structures become visible — useful for motility, cell division and parasites.
Topic 7
Electron microscopes
Beam
Passes through ultra-thin sections
Scans the specimen surface
Image
2D internal detail
3D surface view
Resolution
About 0.1–0.2 nm
About 1–10 nm
Preparation
Fixation, embedding, ultra-thin sectioning, heavy-metal staining
Coating with gold
Use
Viral structure, organelles
Surface structures
- Principle: a beam of electrons (very short wavelength) is focused by electromagnetic lenses in a vacuum; images appear on a fluorescent screen or detector. Specimens must be dead and dry.
Source
Visible light
Electron beam
Lenses
Glass
Electromagnetic
Magnification
Up to about 1,500×
Up to 1,000,000× or more
Specimen
Living or dead
Dead, in vacuum
Cost
Low
Very high
Key terms
- Resolving power
- Ability to distinguish two close points as separate
- Numerical aperture
- Measure of a lens's light-gathering ability
- Dark-ground microscopy
- Bright objects on a dark background
- Fluorochrome
- Dye that fluoresces under UV light
- TEM
- Microscope transmitting electrons through thin sections
Quick revision
- History: Janssen, Hooke, Leeuwenhoek, Abbe, Ruska.
- Magnification; resolution 0.61 λ ÷ NA; oil immersion.
- Bright-field, dark-ground, fluorescence, phase contrast.
- TEM vs SEM; light vs electron microscope.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Why is oil used with the 100× objective?
- Q2.Define resolving power.
- Q3.Which microscope is used to see Treponema pallidum?
- Q4.What is a fluorochrome?
- Q5.What is the advantage of phase contrast microscopy?
- Q6.Distinguish TEM and SEM.
Long-answer questions
- Q1.Describe the parts and working of a compound microscope.
- Q2.Explain the principles of dark-ground and fluorescence microscopy.
- Q3.Explain phase contrast microscopy.
- Q4.Compare light and electron microscopes.
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