Microbiology Laboratory Course January 20-21, 2025 PDF

MICROBIOLOGY LABORATORY COURSE JANUARY 20-21, 2025 Contents Microbiology Practices and Lab rules Basic requirements of a microbiology laboratory – common glassware's Tools in microbiology The Microscope Instructions Care and Handling of the microscope Handling and exami...

MICROBIOLOGY LABORATORY COURSE JANUARY 20-21, 2025 Contents Microbiology Practices and Lab rules Basic requirements of a microbiology laboratory – common glassware's Tools in microbiology The Microscope Instructions Care and Handling of the microscope Handling and examining cultures Disposal of Laboratory Wastes and Cultures DURHAM TUBES, ETC. DURHAM TUBES PART – 2 MICROSCOPE MICROSCOPE - INSTRUCTIONS Observe that a flat platform, or stage as it is called, extends between the upper lens system and the lower set of devices for providing light. The stage has a hole in the center that permits light from below to pass upward into the lenses above. The object to be viewed is positioned on the stage over this opening so that it is brightly illuminated from below. Note the adjustment knobs at the side of the stage, which are used to move the slide in vertical and horizontal directions on the stage. This type of stage is referred to as a mechanical stage. MICROSCOPE - INSTRUCTIONS A built-in illuminator at the base is the source of light. Light is directed upward through the abbe condenser. The condenser contains lenses that collect and concentrate the light, directing it upward through any object on the stage. It also has a shutter or iris diaphragm which can be used to adjust the amount of light admitted. A lever is provided on the condenser for operating the diaphragm. MICROSCOPE – INSTRUCTIONS The condenser can be lowered or raised by an adjustment knob. Lowering the condenser decreases the amount of light that reaches the object. This is usually a disadvantage in microbiological work. It is best to keep the condenser fully raised and to adjust light intensity with the iris diaphragm. MICROSCOPE - INSTRUCTIONS Above the stage, attached to the arm, a tube holds the magnifying lenses through which the object is viewed. The lower end of the tube is fitted with a rotating nosepiece holding three or four objective lenses. As the nosepiece is rotated any one of the objectives can be brought into position above the stage opening. The upper end of the tube holds the ocular lens, or eyepiece (a monocular scope has one; a binocular scope permits viewing with both eyes through two oculars). MICROSCOPE - INSTRUCTIONS Depending on the brand of microscope used, either the rotating nosepiece or the stage can be raised or lowered by coarse and fine adjustment knobs. These are located either above or below the stage. On some microscopes they are mounted as two separate knobs; on others they may be placed in tandem with the smaller fine adjustment extending from the larger coarse wheel. MICROSCOPE - INSTRUCTIONS Locate the coarse adjustment on your microscope and rotate it gently, noting the upward or downward movement of the nosepiece or stage. The coarse adjustment is used to bring the objective down into position over any object on the stage, while looking at it from the side to avoid striking the object and thus damaging the expensive objective lens. The fine adjustment knob moves the tube to such a slight degree that movement cannot be observed from the side. It is used when one is viewing the object through the lenses to make the small adjustments necessary for a sharp, clear image. Care and Handling of the Microscope ✓ Always use both hands to carry the microscope, one holding the arm, other under the base. ✓ Before each use, examine the microscope carefully and report any unusual condition or damage. ✓ Keep the oculars, objectives, and condenser lens clean. Use dry lens paper only. ✓ At the end of each laboratory period in which the microscope is used, remove the slide from the stage, wipe away the oil on the oil-immersion objective, and place the low-power objective in vertical position. ✓ Replace the dust cover, if available, and return the microscope to its box. Part 3 – Handling and Examining Cultures Handling and Examining Cultures Microscopic examination of microorganisms provides important information about their morphology but does not tell us much about their biological characteristics. To obtain such information, we need to observe microorganisms in culture. If we are to cultivate them successfully in the laboratory, we must provide them with suitable nutrients, such as protein components, carbohydrates, minerals, vitamins, and moisture in the right composition. This mixture is called a culture medium. It may be prepared in liquid form, as a broth, or solidified with agar, a nonnutritive solidifying agent extracted from seaweed. Agar media may be used in tubes as a solid column or as slants, which have a greater surface area. They are also commonly used in petri dishes, or plates.  Solid media are essential for isolating and separating bacteria growing together in a specimen collected from a patient, for example, urine or sputum.  Solid media When a mixture of bacteria is streaked across the surface of an agar plate, it is diluted out so that single bacterial cells are deposited at certain areas on the plate. These single cells multiply at those sites until a visible aggregate called a colony is formed. Solid media - colony Each colony represents the growth of one bacterial species. A single, separated colony can be transferred to another medium, where it will grow as a pure culture. Pure culture Evaluation: Each colony represents the growth of one bacterial species. A single, separated colony can be transferred to another medium, where it will grow as a pure culture. Colonies of several different species are regularly present on the same agar plate when certain patient specimens are inoculated onto them. Working with pure cultures permits the microbiologist to study the properties of individual species without interference from other species. Colonial morphology The appearance of colonial growth on agar media can be very distinctive for individual species. Observation of the noticeable, gross features of colonies, that is, of their colonial morphology, is therefore very important The color, density, consistency, surface texture, shape, and size of colonies all should be observed, for these features can provide clues as to the identity of an organism, although final identification cannot be made by morphology alone. Example: TSA Tryptic Soy Agar Liquid media – Broth some bacteria grow diffusely, producing uniform clouding, whereas others look very granular. Liquid media – Broth Layering of growth at the top, center, or bottom of a broth tube reveals something of the organisms’ oxygen requirements. Liquid media – Broth Sometimes colonial aggregates are formed and the bacterial growth appears as small puff balls floating in the broth. Observation of such features can also be helpful in recognizing types of organisms. Part 3-Disposal waste Disposal of Laboratory Wastes and Cultures During laboratory practices, it has been noticed that the untreated waste is generally disposed off by the laboratory staff. It happens due to their unskilled work culture. Most of the laboratories situated in the rural area used discarded hospital material for land filling purposes. Any material which contains microorganisms should be treated first and thereafter, with the proper treatment should be thrown properly. Disposal of Laboratory Wastes and Cultures The treatment is necessary due to the reasons: If it contains pathogenic microorganisms, the disease may transmit or spread to the healthy persons. It may contaminate soil and causes soil, water and air-pollution. Hence, to check from such hazards, proper treatment is required to kill microorganisms. The infected material is generally the solid or liquid culture media used for cultivation of microorganisms, or it may also contain cotton plugs, paper, cotton or cotton swabs, gloves, pins, PCR tubes, gel material etc. Cont. Some of the materials such as cotton plugs, paper, napkins, swabs etc. should be autoclaved first and then it is incinerated. But the microbial contaminants containing materials should be treated with some disinfectant and thereafter autoclaved by putting them in suitable containers. The molten material should be discarded. Sometimes, HCl is also added to hydrolyze the agar, if present in the medium. This is added before their safe disposal. All such laboratory materials should be disposed of after autoclaving. Thanks for listening! Quiz next meeting.