Lect 3- Examination of M.o. 2.pdf

Examination of Microorganisms  I. Microscopy The technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye).  The Compound Microscope A light microscope Generally, light...

Examination of Microorganisms  I. Microscopy The technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye).  The Compound Microscope A light microscope Generally, light is passed from a source through a condenser to focus it on the sample to have maximum brightness then it goes through the objective lens to magnify the image of the sample & then to the oculars, where the enlarged image is viewed.  Ocular lens – further magnifies the object and forms a real image Magnification: 10X Microscope objective lenses Initially magnifies object-form real image Magnifications; Scanning objective –4x Low Power Objective (LPO) –10x High Power Objective (HPO)–40x Oil immersion objective (OIO) – 100x  Total Magnification of the microscope: Product of the magnifying powers of the objective and ocular lenses When using the scanning objective: 4 x 10 = 40 (meaning object is enlarged 40x) LPO –10 x 10 = 100 times HPO–40 x 10 = 400 times OIO – 100 x 10 = 1000 times A good image is dependent on magnification and resolution Magnification is the product of the magnifying powers of the ocular (eyepiece) and the objective lens. Resolution -the shortest distance between two points on a specimen that can still be distinguished by observer, camera or lens of the microscope  magnification can be increased without limit but not resolution because it is dictated by the physical properties of light (i.e. wavelength) It is necessary to have high resolution to view the details of a specimen; without sufficient resolution a magnified specimen will appear blurry. The resolution of a specimen viewed through a microscope can be increased by changing the objective lens to a higher magnification (eg. from LPO to HPO).  Shorter wavelength light (eg. Ultraviolet light) affords/gives higher resolution In light microscopy, oil immersion is a technique used to increase the resolving power of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of higher refractive index, thereby increasing the numerical aperture of the objective lens.  The numerical aperture of a microscope objective lens is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance. The higher the numerical aperture, the higher the resolving power of a lens Numerical aperture values are marked as N.A. in the objective lenses  Refractive index- Light bending ability of a medium. Ordinary light has lower refractive index while cedarwood oil has higher refractive index. Light may bend in air such that it misses the small high- magnification lens Immersion oil is used to keep the light from bending. Types of Microscopes: A. Light microscopes – use visible light to illuminate cell structures/specimens a.simple – has a single lens (e.g. a hand lens) b.compound – more than 1 lens  Types of light compound microscopes based on type of illumination bright-field microscopes – specimen appear dark against a light background dark-field microscopes- specimen appears bright against a dark background LMBorines 1st sem 2021-2022 3. Stereo, stereoscopic or dissecting microscope is an optical microscope variant designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. 4. Phase contrast microscopes- enhances contrasts of transparent and colorless objects by influencing the optical path of light 5. Fluorescence microscopes- are used to study specimens, which can be made to fluoresce. 6.Atomic force microscopes (AFM)- AFM is a very high- resolution type of scanning probe microscope AFM can investigate the topological surfaces of biological objects, from whole cells to protein particulates and other materials 7.Confocal Scanning Laser Microscopes (CSLM)- obtains high-resolution optical images with depth selectivity) B. Electron microscopes – employ a beam of electrons instead of a beam of light; - afford a resolution as much as 1000 fold higher than the light microscopes - Magnification as high as 100,000 to 200,000 as compared to 1000x to 1500x of light microscopes Types: 1. transmission electron microscopes (TEM) - used to study internal cell structures or ultramicroscopic entities such as viruses and viroids 2. scanning electron microscopes (SEM) -used if only external features of cells need to be observed