Basic techniques of microbiology
Dr Fairol give some briefing and explanation about the experiment 16 and experiment 17. On tuesday, we did the experiment 16 using nutrient broth cultures, Sabouraud broth cultures and thioglycollate broth culture and experiment 17 using nutrient broth cultures and thioglycollate broth cultures. Encik Hussain also give some demo and explanation about the both experiment because this both experiments are difficult and need more careful and concentration while we did the experiments.
Physical Factors: Atmospheric Oxygen Requirement
Microorganisms exhibit great diversity in their ability to use free oxygen for cellular respiration. These variations in oxygen requirements reflect differences in biooxidative enzyme systems present in the various species. Microorganisms can be classified into one of five major groups according to their oxygen needs:
Aerobes requires the presence of atmospheric oxygen for growth. Their enzyme system necessitates use of oxygen as the final hydrogen (electron) acceptor in the complete oxidative degradation of high-energy molecules, such as glucose. Microaerophiles require the absence of free oxygen for growth because their oxidative enzyme system requires the presence of molecules other than oxygen to act as the final hydrogen (electron) acceptor. In these organisms, as in aerobes, the presence of atmospheric oxygen result in the formation of toxic metabolic end products, such as superoxide, oxygen , a free radical of oxygen. Aerotolerant anaerobes are fermentative organisms, and therefore they do not use oxygen as a final electron acceptor. Unlike the obligate anaerobes, they produce catalase and/or superoxide dismutase, and thus they are not killed by the presence of oxygen. Facultative anaerobes can grow in the presence or absence of free oxygen. They preferentially use oxygen for aerobic respiration. However, in an oxygen-poor environment, cellular respiration may occur anaerobically, utilizing such compunds as nitrates or sulfates as final hydrogen acceptors, or via a fermentative pathway.
The oxygen needs of microroganisms can be determined by nothing their growth distributions following a shake-tube inoculation. This procedure requires introduction of the inoculum into a melted agar medium, shaking of the test tube to disperse the microorganisms throughout the agar, and rapid solidification of the medium to ensure that the cells remain dispersed.
Techniques for the Cultivation of Anaerobic Microorganisms
Microorganisms differ in their abilities to use oxygen for cellular respiration. Respiration involves the oxidation of substrates for energy necessary to life. A substrate is oxidized when it loses hydrogen ion and its electron. Since the loses hydrogen ion and its electron cannot remain free in the cell, it must immediately be picked up by an electron acceptor, which becomes reduced. Therefore, reduction means gaining the loses hydrogen ion and its electron. These are termed oxidation-reduction reactions. Some microorganisms have enzyme systems in which oxygen can serve as an electron acceptor, thereby being reduced to water. This discussion is limited to cultivation of the strict anaerobes, which cannot be cultivated in the presence of atmospheric oxygen. The procedure is somewhat more difficult because it involves sophisticated equipment and media enriched with substances that lower the redox potential. This experiment uses fluid thioglycollate medium and the GasPak anaerobic system.
Experimet 16: nutrient broth cultures of Enterococcus faecalis
Experiment 16 : Sabouraud broth cultures of Saccharomyces cerevisiae
Experiment 17: nutrient agar plate of aerobic of Bacillus cereus and Escherichia coli
Experiment 17: thioglycollate broth culture of Clostridium sporogenes
Microbiology
Electron microscope
An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode[1] and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x. Electron microscopes have electron optical lens systems that are analogous to the glass lenses of an optical light microscope. Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the image.
The Scanning Electron Microscope (SEM) produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). When the electron beam interacts with the specimen, it loses energy by a variety of mechanisms. The lost energy is converted into alternative forms such as heat, emission of low-energy secondary electrons and high-energy backscattered electrons, light emission (cathodoluminescence) or X-ray emission, all of which provide signals carrying information about the properties of the specimen surface, such as its topography and composition. The image displayed by an SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown below and to the right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs. Generally, the image resolution of an SEM is lower than that of a Transmission Electron Microscope (TEM). However, because the SEM images the surface of a sample rather than its interior, the electrons do not have to travel through the sample. This reduces the need for extensive sample preparation to thin the specimen to electron transparency. The SEM is able to image bulk samples that can fit on its stage and still be maneuvered, including a height less than the working distance being used, often 4 millimeters for high resolution images. The SEM also has a great depth of field, and so can produce images that are good representations of the three-dimensional surface shape of the sample. Another advantage of SEMs comes with environmental scanning electron microscopes (ESEM) that can produce images of good quality and resolution with hydrated samples or in low, rather than high, vacuum or under chamber gases. This facilitates imaging unfixed biological samples that are unstable in the high vacuum of conventional electron microscopes.
The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. The image detected by the digital camera may be displayed on a monitor or computer. The resolution of TEMs is limited primarily by spherical aberration, but a new generation of aberration correctors have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical aberration for the high-resolution transmission electron microscopy (HRTEM) has allowed the production of images with resolution below 0.5 angstrom (50 picometres) and magnifications above 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.
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