Bright Microscope Practical Tips, History, Types of Microscope, Tissue Processing all these things are going to be discussed in this post. If you are studying Microbiology then you must know about Bright Microscope and their types and how tissue processing works.
Before we move to Bright Microscope Practical Tips we need to understand some basic so if you are not a student of Microbiology then you can also understand it
What is Microbiology?
Microbiology is a branch of biology in which we study microorganisms (also known as microbes), which are microscopic unicellular or cell cluster organisms and infectious agents.
The different types of microbes studied by microbiologists include bacteria, archeology, viruses, eukaryotes, fungi, prunes, protozoa, and algae. These microbes can vary dramatically in size and characteristics.
Although microbes often carry a negative connotation due to the association of some microbes with diseases, many other microbes have many benefits. For example, microbes underperform processes such as industrial yeast (which is used to make useful products such as alcohol, vinegar, and dairy products) and produce antibiotics. They also act as molecular vehicles for the transfer of DNA to complex organisms, such as plants and animals.
Microbiology is a broad discipline, and microbiologists study microbes at the level of proteins and genes, cellular levels (cell biology and physiology), and community levels (public health, environment, and epidemiology). ۔ Branches of microbiology include virology, pathology, mycology, microbial genetics, and bacteria.
Now let’s move to our topics Bright Microscope Practical Tips, Types & Tissue Processing
Introduction of Microscope
The microscope is an instrument used to see the structures that are very small to be visualized by naked eyes. Microscopy is the science which deals with the study of small objects using a microscope.
The use of a hand lens to observe small object dates back to the 13th century, but it was Robert Hooke who first used the microscope which could provide magnification up to 42 times. An improved version with a magnification of up to 300 times was developed by Antony van Leeuwenhoek (Father of Microscopy). Bonnannus added a source of light, condenser, rack, and pinion for better focusing.
Principle Of Microscopy
The objective lens of the microscope forms an enlarged image of the object under observation, this image is called a primary image ( and the magnification caused is called primary or initial magnification which is engraved on the objective. The primary image acts as an object for the eyepiece and a secondary image is formed within the barrel by first lens (field lens). The eye lens, which is above the field lens, forms a smaller image (of millimeter diameter) of the secondary image called Ramsden’s disk. The lens of the observer’s eye converts this image into an actual image that is formed on the retina of the observer.
Practical tips in Using A Bright-Field Microscope
1. Microscope should be placed on the level surface.
2. Switch on the power supply for the light source. If there is no inbuilt light source, then natural light has to be focused, so that light strikes the center of the mirror and directed toward the condenser.
3. Adjust the condenser at the optimal position. Remember only a plane mirror has to be used with the condenser. Open the aperture of the iris diaphragm fully.
4. Focusing starts at lower magnification in order to center the specimen by the user on the stage. Place the slide on the center of the stage just below the objective, ensure that the coverslip faces upward.
5. Move the stage using coarse adjustment until the slide comes close to the objective and focus the section sharp. Care should be taken not to strike the objective on the slide. So, it is always better to keep the objective as near as possible to the slide and then moving it away until the specimen is focused.
6. Finally use fine adjustment for optimal focusing and better view.
Types of Microscopes
Now we cam to the last point of the article Bright Microscope Practical Tips and Types.
Following are the types of Microscopes:
Microscopes are mainly classified based on the source of light they use. Following are some varieties of microscopes other than bright-field microscope:
1. Dark-field microscope: In this microscope, only the objects are illuminated by strong oblique peripheral light, whereas the central beam of light is prevented from entering the objective lens by a central patch stop fitted into the condenser. It is used to study extremely tiny particles (colloid suspension) and transparent objects like crystals, protozoa, etc.
2. Phase-contrast microscope: It mainly works on the difference in the refractive indices of different media and objects. With the difference in the refractive indices, there occurs phase difference and image will be formed due to interference between direct light (not passed through an object) and diffracted light (passed through the object). The phase differences are converted into differences of amplitude, hence objects which causes more difference in the phase leads to more difference in amplitude thus results in the brighter image without staining.
3. Interference microscope: In this microscope, beam of light from single source is split and passed through the object and outside. Later, they are recombined by beam combiner at the image plane. After recombination, the difference in retardation caused by interference is used to measure the refractive index, thereby measuring the thickness of the object under observation.
4. Polarization microscope: It is a conventional microscope having a rotating stage with 2 polarizing elements and balsam. A polarizer interferes below the condenser and the analyzer is placed above the objective lens. The polarizing microscope is mainly used to distinguish between amorphous and crystalline biological specimens like bones, lipid droplets, etc.
5. Fluorescence microscope: In this microscope, ultraviolet light is incident on an object and made to emit visible light. Tissues treated with a fluorescent dye are examined with ultraviolet light, by emitting light of a longer wavelength. They are seen as bright objects against dark backgrounds.
6. Ultraviolet microscope: In this microscope, ultraviolet light is used as a source. The amount of light absorbed is recorded photographically and the molecules in the specimen are detected. Mainly, nucleic acids, purine, and pyrimidine are detected by this kind of microscope.
7. Electron microscope: This is a refracting microscope where a stream of high-velocity electrons is used instead of light. Since it provides resolution around 3–5 Å finer details of the structure can studied. As electrons are used, the image is focused by the magnetic coils on the fluorescent screen. There are two types of electron microscopes— transmission electron microscope and scanning electron microscope.
Credit: Inderbir Singh’s Textbook of Human Histology 9th edition
1. Always carry the microscope in both hands. Hold the arm in one hand and support the base of the microscope with another hand.
2. After the tissue is procured, it has to be properly fixed in an ideal fixative. Fixation is done in order to enable the tissue to withstand further steps of tissue processing. An ideal fixative acts rapidly, causes immediate death of the tissue, and preserves life-like appearance. It also prevents autolysis, putrefaction neither shrinks nor swells the tissue, cheap, non-toxic and provides a wide range of staining options. Commonly used are alcohol, acetone, formalin, Zenker’s fluid, etc.
3. Once the tissue is properly “fixed”, it is later dehydrated using increasing strengths of alcohol ending with absolute alcohol.
4. After the tissue is dehydrated, excess alcohol is removed by a process called “dealcoholization” or “clearing”. Clearing agents to raise the refractive index of the tissue and make it transparent. Commonly used are xylene, cedarwood oil, chloroform, etc.
5. Cleared tissue is impregnated both externally and internally by embedding media (paraffin), hence blocks are prepared. Embedded tissue is sectioned with microtome into very thin slices without distortion and attached to a glass slide.
6. Mounted paraffin sections are deparaffinized before staining. In a routine histology laboratory, thin sections are stained using hematoxylin and eosin stains. Hematoxylin is a basic dye, hence it combines with acid components of the cell-like DNA, RNA, and renders them purple/blue color. Hematoxylin usually stains cell nuclei, myelin, elastic fibers, fibrin, neuroglia, and muscle striations. Whereas eosin is an acidic dye, hence it combines with basic components of cytoplasm like proteins (amino acids) and stains it pink.
7. After hematoxylin and eosin (H & E) staining, the slide is cleared, mounted using DPX, and covered with a coverslip for future use. 8. Special stains are used when particular components have to be studied. For example, collagen is stained by the Van Gieson’s method, elastic fibers by Verhoeff’s method, and so on.
Read More: Histology