Part 1 – Microscopy
Microbiology deals with the study of microorganisms that cannot be seen distinctly with the unaided eye. Considering the nature of the objects to be studied, the microscope becomes an instrument of paramount importance. Modern microscopes produce images with great clarity, magnifications that range from ten to thousands of times.
Compound Light Microscopy
This is the most basic microscope used for studying microorganisms. It consists of a series of lenses that utilizes visible light as its source of illumination. Various small specimens can be studied to fine details with a compound light microscope.
Components of a Compound Microscope
The major components of a compound microscope are :
Framework: The basic frame structure is made up of metal, which includes the arm and base to which whole of the magnification and optical components are attached. The metallic arm is connected to a U shaped strong and heavy base that provides stability to the instrument.
Stage: this is the flat horizontal platform positioned at about halfway through the length of the microscope with a hole at the centre that allows the passage of light for illumination of the sample.
Focus knobs: Two pairs of knobs are attached to the arm that help in up and down movement of the stage and in adjustment and focusing of specimens of different thickness.
Lens Systems: All microscopes employ a set of different types of lens systems: the oculars, the objectives, and the condenser, that have different focusing power, and contribute to the complete magnification system.
Nose piece: A revolving nosepiece which holds the objectives is attached to the curved upper part of the arm of the microscope. The nosepiece can be rotated to position the objective with the required magnification in path of the magnification system, beneath the body assembly and the eye piece.
Eyepiece (ocular lens): The eyepiece or ocular lens is a set of lenses held in a cylindrical tube kept inserted in a tubular structure on the curved upper part of the arm, above the nose piece. It consists of two or more lenses which focus the image into the eye. The newest microscopes consist of a pair of eye pieces that allows the observer to use both the eyes to observe the specimen in the microscope. Such microscopes are called binocular microscopes. The normally used eye pieces have 2X, 50X and 10X magnifications.
Objective: The objectives are usually small cylindrical objects containing a single or a set of lenses attached to the nosepiece. The nosepiece holds three to five objectives, which contain lenses of varying magnifying power (2X-400 X). The total arrangement of the lenses is parfocal, which means that the sample stays in focus even when the lenses are changed from one to another in a microscope.
Condenser: A condenser is also a lens which is fixed below the stage and it focuses the beam of light coming from the light source onto the slide. The condenser is usually aided with diaphragm and/or filters, to control and manage the quality and intensity of the light passing through the sample.
Light Source: The light source is mounted at the base of the microscope. The source of light may be the day light, a halogen light, or even LEDs and lasers, as used in the latest microscopes. The microscopes have some provision for reducing light intensity with a neutral density filter.
Types of Compound Microscopes
- The Bright-Field Microscope
- Dark Field Microscope
- Phase-Contrast Microscope
- The Differential Interference Contrast Microscope
- The Fluorescence Microscope
- Confocal Microscope
- Two-Photon Microscope
All these types of microscopes yield a distinctive image and may be used to observe different aspects of microbial morphology.
Points to Remember:
- Microorganisms are too minute in size to be seen by unaided eyes, hence are observed and studied using microscopes.
- Compound microscopes are commonly used in research labs and institutes to study microorganisms. They use glass lenses to bend and focuses light rays and produce enlarged images of small objects.
- Microscopes are delicate and very expensive instruments in any academic or research institutes. Even the most basic compound microsopes require a lot of investment. They, hence, need critical care in usage and handling.