Observing Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens. Compound Light Micros

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1 PHARMACEUTICAL MICROBIOLOGY JIGAR SHAH INSTITUTE OF PHARMACY NIRMA UNIVERSITY

2 Observing Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens. Compound Light Microscopy: It has a series of lenses, uses visible light as source of illumination Parts of microscope: 1) Illuminator, light source, passes through a 2) Condenser, which has lenses that direct the light rays through the specimen. Light rays pass into the 3) Objective lenses, lenses closer to the specimen. The image of specimen is magnified again by the 4) Ocular lens or eyepiece. Total magnification of specimen: objective lens magnification power (10X, 45X, 100X) multiply by ocular lense magnification power (10). 100X (low power), 450X (high power), 1000X (oil immersion). Resolution: Ability of the lenses to distinguish fine detail and structure between two points a specified distance apart.

4 Microscope A general principle of microscopy is that the shorter the wavelength of light used in the instrument, greater the resolution. White light used in this microscope has relatively long wavelength and can not resolve structures smaller than about 0.2µm. To obtain a clear, finely detailed image under this microscope, specimens must be made to contrast sharply with their medium. To attain such contrast, we must change the refractive index of specimens from that of their medium. The refractive index is a measure of the light-bending ability of a medium. We change the refractive index of specimens by staining them. With different refractive indexes, the rays change directions (refract) from a straight path by bending an angle at the boundary between the materials and increase the image s contrast between specimen and medium.

6 Microscope As the light rays travel away from the specimen, they spread out and enter the objective lens, and the image is magnified. To achieve high magnification (100X) with good resolution, the objective lens must be small. Use of oil immersion objective lens: To preserve the direction of light rays at the highest magnification, immersion oil is placed between the glass slide and the oil immersion objective lens. The immersion oil has the same refractive index as glass. In absence of immersion oil, light rays are refracted as they enter the air from slide, and the objective lens would have to be increased in diameter to capture them, & the image becomes fuzzy, with poor resolution. Under usual operating conditions, the field of vision in a compound light microscope is brightly illuminated, & produces brightfield illumination.

8 Darkfield Microscopy: Microscope A darkfield microscope is used for examining live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics can not be identified. Instead of normal condenser, a darkfield microscope uses a darkfield condenser that contains an opaque disc. The disc blocks light that would enter the objective lens directly. Only light that is reflected off the specimen enters the objective lens. Because there is no direct background light, the specimen appears light against black background the dark field. This technique is used to examine unstained microorganisms suspended in liquid, examination of very thin spirochetes, such as Treponema pallidum (syphilis).

9 Phase-Contrast Microscopy: Microscope It permits detailed examination of internal structures in living microorganisms. Here, it is not necessary to fix or stain the specimen-procedures procedures that could distort or kill the microorganisms. The principle is based on the wave nature of light rays, and the fact that light rays can be in phase (their peaks and valleys match) or out of phase. If the wave peak of light rays from one source coincides with the wave peak of light rays from another source, the rays interact to produce reinforcement (relatively brightness). However, wave peak coincides with wave trough, rays produce interference (relative darkness). One set of light rays comes directly from the light source.

10 Microscope The other set comes from light that is reflected or diffracted from a particular structure in specimen. These two type of light rays form an image of the specimen on the ocular lens, containing area that are relatively light (in phase), through shades of gray, to black (out of phase). Differential Interference Contrast (DIC) Microscopy: Similar to phase-contrast microscopy, uses differences in refractive indexes. Uses two beams of light instead of one. Prisms split each light beam, adding contrasting colors to the specimen. So, resolution of a DIC microscope is higher than that of a standard phase-contrast microscope. The image is brightly colored and appears nearly three-dimensional.

11 Fluorescence Microscopy: Microscope It takes the advantage of fluorescence, the ability of substance to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some organisms fluoresce naturally under UV light. If specimen does not naturally fluoresce, it is stained with one of a group of fluorescent dyes called fluorochromes. E.g. Fluorochrome auramine O glows yellow when exposed to UV light, is strongly absorbed by M. Tuberculosis. Fluorescein isothiocyanate gives apple green with B. anthracis. Its principally used as a diagnostic technique called the fluorescent- antibody (FA) technique, or immunofluorescence. This technique can detect bacteria or other pathogenic microorganisms, even within cells, tissues or other clinical specimens. It is especially useful in diagnosing syphilis and rabies.

13 Confocal Microscopy: Microscope Here, one plane of a small region of specimen is illuminated with a laser, which passes the returned light through an aperture aligned with the illuminated region. Successive planes and regions are illuminated until the entire specimen has been scanned. Exceptionally clear three-dimensional images of entire cells and cellular components can be obtained, with improved resolution up to 40%, also to evaluate cellular physiology by monitoring the distributions and concentrations of substances such as ATP and calcium ions.

14 Electron Microscopy: Microscope Objects smaller than about 0.2µm can be examined. E.g. viruses. Use beam of free wave electrons as source of light. The resolving power is far greater than that of the other microscopes. Better resolution is due to the shorter wavelengths of electrons. Electromagnetic lenses are used to focus a beam of electrons onto a specimen and to control illumination, and magnification. Two types: 1) Transmission Electron Microscope (TEM), 2) Scanning Electron Microscope (SEM). Transmission Electron Microscope (TEM): Electron gun finely focused beam of electrons electromagnetic condenser lens specimen electromagnetic objective lens (magnifies the image) electromagnetic projector lens onto a fluorescent screen or photographic plate.

15 The specimen is usually placed on a copper Resolves objects close together as 2.5nm Magnified 10, ,000X. copper mesh grid. Microscope Shadow Casting technique: A heavy metal such as platinum or gold is sprayed at an angle of about 45º so that it strikes the microbe from one side, metal piles up on one side of specimen, and the uncoated area on the opposite side of specimen leaves a clear area behind it as a shadow. It provides a general idea of size and shape of specimen and gives three dimensional effect to the specimen. DISADVANTAGE: 1) electrons have limited penetrating power, 2) specimen viewed under a high vacuum to prevent electron scattering. These treatments kill the specimen, and also cause some shrinkage and distortion.

17 Microscope Scanning Electron Microscope (SEM): Provides striking 3 dimensional views of specimens. Electron gun finely focused beam of electrons (primary electron beam) electromagnetic condenser lens specimen primary electron beam knocks electrons out of surface and secondary electrons thus produced are transmitted electron collector amplified produce an image on a viewing screen or photographic plate. Useful in studying surface structures of intact cells and viruses. Resolves objects close together as 20 nm Magnified ,000X. Scanned-Probe Microscopy: Use various kinds of probes to examine the surface of specimen at very close range. Used to map atomic and molecular shapes, to characterize magnetic and chemical properties, to determine temperature variations inside cells. Two types: Scanning tunneling microscope, atomic force microscope.

18 Scanning Tunneling Microscopy: Microscope Uses a thin metal (tungsten) probe that scans a specimen and produces an image revealing bumps and depressions of the atoms on surface of specimen. The resolving power is much greater than other electron microscope. Used to provide detailed views of molecules such as DNA. Atomic Force Microscopy: A metal and diamond probe is forced down onto a specimen. As probe moves along the surface of the specimen, its movements are recorded and a three-dimensional image is produced. Doesn t require special specimen preparation. Used to image both biological substances (atomic detail) and molecular processes (a component of blood clot).

Observing Microorganisms through a Microscope

2016/2/19 PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College CHAPTER 3 Observing Microorganisms through a Microscope 1 Figure 3.2 Microscopes and Magnification.