MICROSCOPES AND ITS APPLICATION

MICROSCOPES AND ITS APPLICATION. TUTORIAL-MBBS 2025.

Microscopy: The Window to the Microbial World. What is Microscopy? Microscopy is the technique of using a microscope to magnify and visualize microorganisms, cells, and their structures that are too small to be seen with the naked eye..

HISTORY OF MICROSCOPE. Antonie van Leeuwenhoek(1670) – Father of Microbiology developed powerful SINGLE lens simple microscopes with up to 270x magnification. First to observe bacteria, protozoa, sperm cells, red blood cells, etc. Robert Hooke- Compound Microscope Year: 1665 Magnification: ~30x Optical System: Two-lens system (objective + eyepiece) Light Source: Reflected external light (mirror, candle/sunlight.

Different types of Microscopes. Bright-field or light microscope Dark field (or dark ground) microscope Phase contrast microscope Fluorescence microscope Electron microscope..

MICROSCOPE. Microscopy: The science or technique of using a microscope to view tiny objects Microscope: Actual instrument used to magnify small objects not visible to the naked eye. Helps to observe cells, microorganisms, and fine structures in detail..

PROPERTIES OF A MICROSCOPE. 1. Good resolution: ability to produce separate images of closely placed objects - distinguished as two separate entities. Refractive Index – Oil>Air (oil enhances the resolution power) 2. Good contrast: Improved by staining the specimen. When stains bind to the cells, the contrast is increased.

3. Good magnification: Achieved by use of lenses. Ocular lens - magnification power of 10x Objective lens-scanning (4x), low power (10x), high power (40x) and oil immersion (100x) Total magnification = magnification of objective lens * ocular lens..

BRIGHT-FIELD OR LIGHT MICROSCOPE. Forms a dark image against a brighter background. Structure The parts are divided into three groups: Mechanical Parts Magnifying Parts Illuminating Parts.

Mechanical Parts. Base: Holds various parts of the microscope C-shaped arm: Holds the microscope, connects - ocular lens to objective lens Mechanical stage: Hold the slides..

Magnifying Parts Illuminating parts. Ocular lens Objective lens.

Working Principle. Rays from the light source pass through the iris and fall on the specimen Light rays are gathered by the objective and a magnified image is formed.

DARK FIELD MICROSCOPE. Working principle: Object appears bright against a dark background - by use of a special dark field condenser. Central opaque area (blocks light from entering the objective lens) Peripheral annular hollow area (allows the light to pass through and focus on the specimen obliquely).

Light - reflected by specimen enters the objective lens Unreflected light does not enter the objective. So the specimen is bright; and the background appears dark. Application- Used to identify the living, unstained cells and thin bacteria like spirochetes which cannot be visualized by light microscopy..

PHASE CONTRAST MICROSCOPE. Contrast is enhanced. Visualizes the unstained living cells by creating difference in contrast between cells and water..

Working principle. Condenser is similar to dark field microscope. Cone of light passes through a cell some light rays are bent due to variations in density and refractive index within the specimen and are retarded by 1/4th of a wavelength Undeviated light rays strike a phase ring Deviated rays miss the ring and pass through the rest of the plate Background is bright, while the unstained object appears dark.

Application. For studying: Microbial motility Determining the shape of living cells Detecting microbial internal cellular components.

FLUORESCENCE MICROSCOPE. Working principle: Fluorescent dyes - exposed to UV rays, become excited and fluoresce i.e. they convert short wavelength rays into longer wavelengths Mercury lamp emits rays that pass through an excitation filter Epifluorescence microscope: Simplest form of fluorescence microscope. Has following applications: Auto fluorescence (eg. Cyclospora) Microbes coated with fluorescent dye (eg. Acridine orange dye) Immunofluorescence.

Introduction to Fluorescence microscope. Figure 2. Commercial auramine kit, auramine/acridine orange.

ELECTRON MICROSCOPE. Invented by German physicist - Ernst Ruska in 1931. Source of illumination - Accelerated electrons Wavelength of electrons 100,000 times shorter than visible light photons - better resolving power than a light microscope. It is of two types: Transmission electron microscope (TEM, most common type) Scanning electron microscope (SEM).

Features Light microscope Electron microscope Highest practical magnification About 1,000–1,500 Over 100,000 Best resolution 0.2 µm 0.5 nm Radiation source Visible light Electron beam Medium of travel Air High vacuum Specimen mount Glass slide Metal grid (usually copper) Type of lens Glass Electromagnet.

Electron Microscope. TEM – Specimen Preparation. Very thin specimens (20–100 nm thickness) - suitable for EM. Steps to prepare thin specimen: Fixation: Cells are fixed by using glutaraldehyde or osmium tetroxide for stabilization Dehydration: Specimen is then dehydrated with acetone or ethanol. 3. Embedding: Specimen is embedded in plastic polymer. 4. Slicing: Specimen is then cut into thin slices by an ultramicrotome knife. Such thin slices of the specimen are mounted on a metal slide (copper)..

Electron Pathway. Electrons - generated by electron gun-travel in high speed. Medium of travel - fully vacuum path - because in air path - electrons get deflected by collisions with air molecules. Applications- Virus detection The contrast of EM - increased by — (i) Negative staining with heavy metals (phosphotungstic acid) (ii) Shadowing..

Scanning Electron Microscope. Examine the surfaces of microorganisms in detail. Resolution of 7 nm or less. Produces image from electrons emitted by object’s surface..

tem vs sem.

Clinical Applications of Microscopy. Initial identification of organisms by directly examining clinical specimens. Quick and definitive identification of certain organisms directly from samples. Detection of mixed infections—more than one organism in the same specimen. Assessment of patient samples for signs of inflammation or contamination (e.g., presence of white blood cells or epithelial cells). Helps determine clinical relevance—true pathogens are usually present in large numbers (e.g., >10⁵ cells/mL) unlike contaminants. Supports early initiation of appropriate antibiotic therapy based on presumptive identification..