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MICROSCOPY AND STAINING
OUTLINE
Introduction Types of microscope Parts and functions of microscop Types of microscopy Staining Cultivation of microbes -pure c culture media
INTRODUCTION
Microbes are small living organisms to be seen with the naked eye, hence the aid to see them with a devices called microscopes. The process of viewing the very tiny living things or objects with the aid of a microscope is called microscopy.
MICROSCOPY AND STAINING
The first microscope was made in 1590 by Hans and Zacharias Jansen. In 1665, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Antoni van Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells, including bacteria, Microscopy is used for many purposes such as: Diagnosis of infectious diseases, Identification of microorganisms in environmental samples Determination of the effect of pathogens on human cells
TYPES OF MICROSCOPE
- Compound Microscope: Compound microscopes are light illuminated. The image seen with this type of microscope is two dimensional. This microscope is the most commonly used especially hospitals and clinical research centers. Here individual cells even living ones can be viewed It has a high magnification but with low resolution. Visible light is the source of illumination
- Dissecting Microscope or Stereoscope A dissecting microscope is also light illuminated. The image that appears is three dimensional. It is used for dissection to get a better look at the larger specimen You cannot see individual cells because it has a low magnification Visible light is the source of illumination.
- Confocal Microscope This microscope uses a laser light. This light is used because of a wavelength. Laser light scan is used across the specimen with the aid of scanning mirrors, the specimen/results are then placed on a digital computer screen for analyzing. 4 Scanning Electron Microscope(SEM) SEM use electron illumination. The image is seen in 3- D. It has high magnification and a higher resolution The specimen is coated in gold and the electron bounce off to give you an exterior view of the specimen. Source of illumination or radiation for image formation are electron beams and the pictures are in black and white.
- Transmission Electron Microscope TEM is an electron illuminated. This is a 2-D view. Thin slices of specimen are obtained. The electron beams pass through this. It has high magnification and high resolution Source of illumination or radiation for image formation are electron beams. 6 Light Microscope: Light Microscope - found in most schools, use compound lenses and light to magnify objects. The lenses bend or refract the light, which makes the object beneath them appear closer. 7 Fluorescence Microscope: FM Utilizes fluorescence to view specimens. Commonly used in biological research to observe specific proteins or structures. 8 Phase-Contrast Microscope Enhances contrast in transparent specimen without staining. Very useful for living cells. 9 Polarizing Microscope use polarize light to examine materials. Commonly use geology and material science.
PARTS AND FUNCTIONS
Eyepiece The eyepiece or ocular Lens is a cylinder containing two or more lenses. Its function 1s to bring the image into focus to the eye.the Eyepiece are interchangeable. 1 Nose piece: This is the part that holds the set of objective lenses. It holds the objective lenses and allows the user to switch between them.
Bright-field microscopes (Compound) Series of lenses for magnification Light passes through specimen into objective lens Oil immersion lens increases resolution Have one or two ocular lenses Most have condenser lens (direct light through specimen Dark-field microscopes Best for observing pale objects Only light rays scattered by specimen enter objective lens Specimen appears light against dark background Increases contrast and enables observation of more details
PRINCIPLES OF LIGHT MICROSCOPY
Magnification- this is the apparent size of the object view directly by the eye, which depends on its distance from the eye. This means that, the closer an object is to the eye, the bigger it appears, that is apparent size. If however, the object is too close to the eye (distance of closest approach)the eye can no longer form a clear image. A light microscope allows the formation of a large image of a specimen. Light is refracted as it moves through a series of lenses. Light passes through objective and ocular lenses to produces an enlarge images. Light, typically from an incandescent bulb source is focused onto the specimen by a condenser lens The specimen is usually put on a clear glass slide. Light passes through the specimen and enter the objective lens which refract the light and begins the magnification process. Light then passes through ocular lenses which further refracts the lights to complete magnification process. he light enter the eye and the magnified image is seen
MAGNIFICATION
The extent to which magnification can be achieved with a compound microscope is the product of the individual magnifying powers of the ocular and objective lenses. => Total magnifying power- power objective lenses X power of the ocular. •Eg, the magnifying powers of 10x objective and 10x ocular lenses are 100 x the original size Note The most commonly used light microscopes have objective lenses with power of 10x,40x, and 100x and ocular lens of 10x so that one obtain magnifying powers of 100x,400x and
1000x with them. NOTE: the practical limit to magnification with a microscope is 1300x The bending of the light rays by glass lens is known a Refraction
RESOLUTION
A resolution is the ability to distinguish in detail the object that is viewed. In biological specimen, resolution is equated as the ability to see structures of organisms more clearly. In simple terms clarity of an image is called resolution
STAINING
•This is to increase the differences in intensity between a specimen and its background. Most microbes are transparent and thus cannot be seen easily without been stained. Most staining procedures begin with the transfer of a suspension of microbes onto a slide.
TYPES OF STAINING
Simple stains Differential stains Gram stain Acid-fast stain Endospore stain Special stains Negative (capsule) stain Flagellar stain NOTE The microbes are spread in a thin film across the slide that is allowed to dry in the air The slide is quickly passed through a flame to heat fixed the cells to the slide(This preserves the shape of the cells and prevent them from been wash off during staining) A stain is added and allowed to penetrate to react with the cells on the slide for a period of timeExcess stain is then washed from the slide and the specimen is ready for viewing under the microscope
SIMPLE STAINING
A stain is a salt which consist of a positively charged ion and a negatively charged ion, when one of the ion is a chromophore (the colored part). If the colored part of the stain is positively charged, it will be attracted to microbial cells which have negative charges. Simple staining procedures use only one single stain
other cells. Makes it easy to isolate the progeny of a single cell for the establishment of pure cultures. Also allowed observation of population single cell for the establishment of pure cultures Also observation of population of single species of microbes. Culture Media: to grow (culture) microorganisms, microbiologist must make sure that the culture medium that contains the variety of organic and inorganic nutrients are needed for microbial metabolisms. All organisms require carbon, nitrogen, oxygen, hydrogens, phosphorus, sulphur and various elements for growth. These elements must be available in usable chemicals form to meet the nutritional requirements of a particular microbes and to allowed that microbes to grow. Culture Media: not all microbes have the same nutritional requirement to grow. Commonly used culture media contains protein, has digested enzymes or acids, carbohydrate. are region to nestine odium neal no i order ions microorganism. Clinical microbiologist always add blood into the media for the growth some microbes. Some pathogens, called FASTIDIOUS MICROORGANISMS have specific demanding nutritional requirement. They are very difficult to in the laboratory SELECTIVE MEDIA are media that favor the growth of specific microorganisms whiles preventing the growth of others DIFFERENTIAL MEDIA are media that permits the recognition of specific types of microorganisms In some cases a media is both selective differential
