Staining Techniques Lecture 2 PDF

Microscopy ▪ The technology of making very small things visible to the naked eye. The word microscope is derived from the Latin word micro (small) and the word scope (to look at). The human eye can resolve objects down to about 0.2 mm The Microscope is used to magnify small objects, those be...

Microscopy ▪ The technology of making very small things visible to the naked eye. The word microscope is derived from the Latin word micro (small) and the word scope (to look at). The human eye can resolve objects down to about 0.2 mm The Microscope is used to magnify small objects, those below the 0.2 mm range Bacteria and cells are measured in µm or 1 x 10-6 meters Viruses are even smaller, measured in nm or 1 x 10-9 meters Microscope Microscopes depending on illumination source, can be divided into ❑ light microscope and ❑ electron microscope ◦ Light microscopes are classified into several types: 1. Simple Microscopes Leeuwenhoeck’s simple microscopes consisted of Only one lens which allowed him to magnify images from 100 to 300 X. His simple microscopes enabled him to be the first person to see bacteria. 2. Compound Light (CL) Microscopy: Joseph Jackson Lister (Lister’s father) developed improved compound light microscope in 1830s Types of Microscopes Compound Light Microscopy (CM) Characterized by: ▪ Have several lenses : objective and ocular lenses ▪ Light originates from an illuminator and passes through condenser lenses, which direct light onto the specimen. ▪ Light then enters the objective lenses, which magnify the image. These are the closest lenses to the specimen: o Scanning objective lens: 4 X o Low power objective lens: 10 X o High power objective lens: 40-45 X o Oil immersion lens: 95-100 X ▪ The image of the specimen is magnified once again by the ocular lens or eyepiece (10 X). ▪ Total magnification: Obtained by multiplying objective lens power by ocular lens power Factors affect the efficiency of compound microscope 1- Resolution (Resolving power): Ability of microscope to see two items as separate and discrete units. o The smaller the distance between objects at which they can be distinguished as separate, the greater the resolving power. o Depends on light wavelength. If wavelength is too long to pass between objects, they will appear as one. ❖White light has a relatively long wavelength (550 nm), and cannot resolve structures less than 220 nm (0.2 um) apart. ❖ Ultraviolet (UV) light has a shorter wavelength (100 to 400 nm), and can resolve distances as small as 110 nm. The shorter the wavelength, the greater the resolution Factors affect the efficiency of compound microscope 2. Refraction: (‫)االنكسار‬Bending of light as it passes from one medium to another of different density. ▪ Refraction Index : A measure of the speed at which light passes through a material.(light bending ability) ❑ Can be improved by staining, which increases contrast between specimen and surrounding medium. ▪ As light passes through different media such as a glass slide, air, and the objective lens, it bends each time, causing loss of light and a blurred image. ▪ Immersion oil has the same index of refraction as glass slide, preventing light loss from refraction. 3. Contrast power: the ability to distinguish between two adjacent point related to their light intensity. > Improved by staining Darkfield Microscopy (DM) Useful to examine live or unstained specimens. Light sensitive organisms Specimens that lack contrast with their background. o Spirochetes (Treponema pallidum) which cause syphilis. ❑ Image: Light specimen against dark background. Phase Contrast Microscopy (PM) Useful to examine live specimens: o Doesn’t require fixing or staining, which usually kill and/or distort microorganisms. ❑ Permits detailed examination of internal structures Fluorescence Microscopy Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). For example, Trepnema when stained by the fluorochrome , appears green when exposed to ultraviolet light, Confocal Microscopy Shows fluorochromes stained specimen in three- dimensional images. Limitations of light microscopy Magnification: Up to 2000 X. u Resolving Power: Up to 0.2 um. Because of the limits of magnification and resolving power, viruses and most internal structures of cells cannot be seen with a light microscope.  Electron Microscopy Use a beam of electrons instead of a beam of light Used to examine structures too small to be resolved with a light microscope It has the ability to magnify specimen by millions times Two modes of electron microscope 1. Transmission Electron Microscopy ((TEM) In the transmission electron microscope shows intracellular details 2. Scanning Electron Microscope (SEM) shows details of the surface of specimen