A good microscope should have three characteristics:
Good Resolution: Resolution capability refers to the ability to produce separate images of closely placed objects so that they can be distinguished as two separate entities. Resolution:
Approximately 0.2 mm (200μm) to the naked eye
Optical microscope is about 0.2μm
Electron microscopy is about 0.5 nm
. The resolution depends on the refractive index. Oil has a higher refractive index than air.
Good contrast: can be further improved by staining the sample.
Good magnification: This is achieved by using concave lenses.
Brightfield or light microscopy
Brightfield or light microscopy produces dark images against a lighter background.
dark field microscopy
Principle: In darkfield microscopy, objects appear bright against a dark background. This can be achieved by using special darkfield condensers.
Applications: Used to identify viable, unstained cells and thin bacteria such as Spirochaetes that cannot be observed by light microscopy.
phase contrast microscope
It visualizes living cells by creating contrast differences between cells and water. It converts subtle differences in refractive index and cell density into easily detectable changes in light intensity.
Useful for studying:
microbial movement
Determine the shape of living cells
Detect bacterial components such as endospores and inclusion bodies.
Fluorescence microscope
How it works: When fluorescent dyes are exposed to ultraviolet (UV) light, they are excited and fluoresce, i.e. they convert this invisible, short-wavelength ray into longer wavelength light (visible light).
Applications: Epifluorescence microscopy has the following applications:
Autofluoresces when placed under UV light, such as cyclosporine
Microorganisms coated with fluorescent dyes, such as atriol orange for malaria parasites (QBC) and aurinol for Mycobacterium tuberculosis.
Immunofluorescence: It uses antibodies labeled with fluorescent dyes to detect cell surface antigens or antibodies that bind to cell surface antigens. There are three types: direct IF, indirect IF, and flow cytometry.
electron microscope
It was invented by Ernst Ruska in 1931. It differs from light microscopy in a different way.
There are two types of EM:
Transmission EM (MC type, examines internal structure, resolution 0.5 nm, gives a two-dimensional view)
Scanning EM (examines the surface with a resolution of 7 nm, providing a 3D view)
Principles of transmission electron microscopy
Sample Preparation: Perform the following steps on cells to prepare very thin samples (20 to 100 nm thick)
Fixation: Fix cells using glutaraldehyde or tetroxide to stabilize them.
Dehydration: The sample is then dehydrated using an organic solvent such as acetone or ethanol.
Embedding: The specimen is embedded in a plastic polymer, which then hardens to form a solid mass. Most plastic polymers are water-insoluble. Therefore, samples must be completely dehydrated before embedding.
Sectioning: The specimen is then cut into thin sections with an ultramicrotome and the sections are mounted on metal slides (copper).
Freeze-etching technique: This is an alternative method for visualizing organelles inside cells for specimen preparation.
The cells are quickly frozen, then heated → ruptured using a knife to expose the internal organelles → sublimated → covered with platinum and carbon coatings.
Measures to improve EM contrast include:
Staining with solutions of heavy metal salts such as lead citrate and uranyl acetate
Negative staining with heavy metals such as phosphotungstic acid or uranyl acetate.
Shading: The specimen is coated with a thin film of platinum or other heavy metal at a 45° angle so that the metal hits the microorganisms on only one side.
