Microscope, Simple and Compound: Introduction, Principle, Parts, Terminology, Size of Microorganisms, Care and Maintenance
Introduction of Microscope
The microscope is the instrument, the most characteristic of the microbiology laboratories. The magnification, it provides, enables us to see microorganisms and their structures otherwise invisible to the naked eye. The magnitudes attainable by microscopes range from 100 X-10,000,000X. It may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making enlarged or magnified images of minute objects. (Micro: Small; scope: to view)
Antony Von Leuwenhoek is considered to be the first person who has seen a microorganism through a simple microscope made by him with a magnification of 270-480 times.
He described the size, shape, movement of bacteria, protozoa, and algae. These findings were confirmed after the development of a compound microscope by Robert Hooke.
The characteristic morphological studies enabled by the discovery of powerful microscopes, helped scientists to classify microorganisms. Later improvements in the compound microscopes were made and Amici discovered an oil immersion lens, which enabled the scientists to study the characteristics more minutely. Microscopes are continuously improved to enable us to have higher magnifications and better resolutions.
The light microscope, in which the magnification is obtained by a system of optical lenses uses light waves. Whereas the electron microscope uses a beam of electrons in place of light waves for visualization of objects.
Compound Microscope: It is most commonly used in microbiology laboratories. In compound microscopy, the microscopic field or area observed is brightly lighted and the objects being studied appear dark because they absorb some of the light. Ordinarily, microorganisms do not absorb much light but staining them with a dye greatly increases their light-absorbing ability resulting in greater contrast and color differentiation.
Generally, microscopes of this type produce a useful magnification of about 1000X to 2000X.
Principle of Simple/ Dissecting Microscopes:
The dissecting microscope consists of a biconvex lens which is moved up and down by an adjustment screw to bring the object in sharp focus. The object is placed on the platform and light is focused with the help of a concave mirror fitted below
In simple microscopes, a convex lens of short focal length is used to see magnified images of a small object. The object is placed between the optical Centre and the focus of a convex lens, its image is virtual, erect and magnified, and on the same side as the object. The position of the object is so adjusted that the image is formed at the least distance of distinct vision.
Principle of Compound Microscopes
A compound microscope consists of two sets of convex lenses. A lens of short aperture and short focal length facing the object is called objective. Another set of the lens of relatively moderate focal length and large aperture facing the eye is called the eyepiece. The objective and the eyepiece are placed coaxially at the two ends of a tube.
The object is placed between the center of curvature and focus of the objective – it forms a real, inverted, and magnified image on the other side of the objective. This image acts as an object for the eyepiece which then acts as a simple microscope to produce a virtual, erect, and magnified image.
Parts of Compound Microscope
It consists of
It is the main framework of the microscope and consists-
An arm, which supports the main tube, body, and the stage
A substage, and
A foot or base upon which the whole instrument rests.
Main tube: The main tube primarily holds the objective and eyepieces. The eyepiece, also known as an ocular piece, is present at the top of the main tube.
Body and arm: The tube is attached to the microscope by the component of the microscope called the body. The body of the microscope and the tube attached to it are supported at the correct height by a firm arm, which may also provide a lifting handle for the microscope.
Substage: The sub-stage lies immediately below the stage. This holds a condenser lens with an inbuilt diaphragm and a holder for light filter and stops.
Foot: The microscope rests firmly on the laboratory bench with the base called a foot. This may be U-shaped or rectangular.
A fixed platform with an opening in the Centre allows for the passage of the light from an illuminating source below to the lens system. It provides a surface for the placement of a slide over the central opening.
Mechanical Adjustment of a microscope:
It is being carried out to focus the specimen examined by the microscope. This adjustment includes coarse and fine focusing adjustments and condenser.
Coarse adjustment- required when focusing the specimen with low power (10X) objectives.
Fine adjustment-It is carried out when finer focusing is required by using high power (40X) objectives or oil immersion objectives.
Condenser adjustment-It is classified depending on its uses such as bright field, darkfield, phase contrast, etc.
The light source: A good source of light is needed to examine the specimen correctly. This may be daylight or electric light.
The purposes of microscopes are to produce an enlarged, well-defined image of objects too small to be observed with the naked eye.
Magnification: Objective lens× eyepiece
Three objectives most commonly used are
-10X, 40X and 100X
and eyepieces use 5X, 10X and 15X
Numerical Aperture: It may be defined as the ratio of the diameter of the lens to its focal length.
Resolution: Resolving power is the ability to reveal two closely adjacent structural details as separate and distinct.
The greatest resolution in the light microscope is obtained with the shortest wavelength of visible light and object with maximum numerical aperture.
Illumination: Effective illumination is required for efficient magnification and resolving power. Since the intensity of the daylight is an uncontrolled variable, artificial light from a tungsten lamp is the most commonly used light source in microscopy.
Difference between simple and compound microscopes
Sizes of Microorganism
Cocci 0.5-1.0 µm while bacilli 1-10 µm×3-10µm
Smallest-Parvo virus-20 nm while largest –Pox virus-300 nm
Most protozoa around 50µm in size except Balantidium coli ≥100µm
Helminthes sizes are variable-
Cestodes 1 mm to several meters in length
Nematodes vary in size from 5 mm to even 1 meter
Yeast like appearance 2-30µm
Mould appearance 2-5 µm
Spherical like appearance 5-300µm
Handle and care of microscopes
Handle with care
Most microscope problems occur as a result of improper handling. When carrying it, hold it by the base and the metal support arm. Do not pick it up by the stage, as this can cause misalignment. When transporting it, use its own bag.
Examination of the slide should always begin with a low power objective (10X).
Keep lenses clear of slides
When using microscopes and adjusting the focus you will need to lower the objective lens down as far as it will go. However, you should never allow the lens to touch the slide you are looking at. Dirty lenses can be difficult to clean.
Clean after using immersion oil
If using immersion oil, always ensure the objectives are cleaned immediately after use. Objective, eyepieces, and condenser may be removed for cleaning. Use only lens paper and lens cleaner. Do not use solvents.
Cover when not in use
All microscopes are sold with dust covers. Always keep it covered when not in use even if it is stored in a cabinet. Eye tubes also need to be kept free of dust so do not store a microscope without the eyepieces. If the microscope eyepieces must be removed, cover the tubes with caps or a plastic bag with a rubber band around the eye tube.
Look after the bulb
After using the microscope, turn off the illuminator and wait for it to cool for several minutes before putting it away. By allowing the bulb to cool you will extend its life. When turning the microscope on and off, use the switch, not the PowerPoint. Do not switch it on while using full light intensity. Never touch the bulb with your fingers as the body oils can burn into the bulb and reduce its life. Use a tissue. Keep a store of replacement bulbs and always use the correct bulb.
Store in a clean, dry place and away from direct sunlight
Make sure you do not store your microscope in an area that has corrosive chemical fumes that can destroy lenses or metal parts or beside solutions that may leak. Salt air and pervasive damp can also cause damage over time. Make sure your cabinet is ventilated.
Only use special lens paper or wipes for cleaning the lenses
Lenses can easily be scratched and should be treated with great care. Use an aspirator to remove dust. Sticky residue can be removed with lens paper moistened with distilled water or lens cleaning solution and rubbed gently using a circular motion. Never use sharp instruments or anything abrasive on the microscope lenses.
Keep your User’s Manual and wrenches in a safe place
Each microscope should come with a user’s manual and specialist wrenches as required. Always consult the User’s Manual before making any adjustments to your microscope and use the wrenches provided. Never over-tighten or use force when performing any maintenance on your microscope, or use inappropriate tools. This can damage the parts.
An attempt should never be made to repair the microscope by oneself.
On the basis of the lens, the microscope is of two types, simple and compound microscope.
Magnification based principle, the microscope is mainly two types-