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Theme 1. Microbiological laboratory. Methods for studying microorganisms.The morphology of microorganisms. Simple staining methodsThe learning objectives for this lesson are that:o you will know about rules in the microbiological laboratory; o you will capture microscopic research method using an immersion system;o you will understand how to investigate the morphological characteristics ofdifferent microorganisms; o you will also apply the technique of simple staining methods for coloring of smearpreparations.Outlines1. Microbiological laboratory. Equipment, workplace organization. Safety rules forworking with microbial cultures.2. Methods for studying microorganisms. Microscopic study method. Types of modernmicroscopes. Immersion microscopy.3. The principles of microorganisms classification. The organisms classification based onmorphological and tinctorial characteristics.4. Stages of preparing smears, their fixation. Simple staining methods.Materials and EquipmentMicroscopes. A set of readymade solutions, paints in bottles. Black mascara. Flasks andneedles. The slides, cover slips, glass bridge in the bath for staining, distilled water. Disinfectant, cotton wool, flannel cloth, pencil on glass, immersion oil, filter paper, matches, alcohol burners (spirit lamps). Pure cultures of microorganisms grown on agar medium.Microbiology Lab Practices and Safety Rules1. Wash your hands with disinfectant soap when you arrive at the lab and again beforeyou leave.2. Absolutely no food, drinks, chewing gum, or smoking is allowed in the laboratory. Donot put anything in your mouth such as pencils, pens, labels, or fingers. Do not store food in areas where microorganisms are stored.3. Bring to class and use a lab coat and safety glasses. This garment must cover your armsand be able to be removed without pulling it over your head. Leave protective clothing in the lab and do not wear it to other nonlab areas.4. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals are not allowed)in the laboratory. Use only indoor shoes. Long hair must be tied back in such a manner as to not be in danger of catching fire.1
5. Keep your workspace free of all unnecessary materials. Backpacks, purses, and coatsshould be placed in the cubbyholes by the front door of the lab. Place needed items on the floor near your feet, but not in the aisle.6. Disinfect work areas before and after use with 70% ethanol or fresh 10% bleach.Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, and especially after spills, splashes, or other contamination..7. Replace caps on reagents, solution bottles, and bacterial cultures. Do not open Petridishes in the lab unless absolutely necessary.8. Inoculating loops and needles should be flame sterilized in an alcohol burner beforeyou lay them down.9. Turn off alcohol burners when they are not used. Long hair must be restrained ifalcohol burners are in use.10. When you sterilize in flame with alcohol, be sure that you do not have any papersunder you.11. Treat all microorganisms as potential pathogens. Use appropriate care and do not takecultures out of the laboratory.12. Wear disposable gloves when working with potentially infectious microbes orsamples (e.g., sewage). If you are working with a sample that may contain a pathogen, then be extremely careful to use good bacteriological technique.13. Sterilize equipment and materials. 14. Never pipette by mouth. Use a pipetting aid or adjustable volume pipettors. In thedistant past, some lab personnel were taught to mouth pipette. This practice has been known to result in many laboratoryacquired infections. With the availability of mechanical pipetting devices, mouth pipetting is strictly prohibited.15. Consider everything a biohazard. Do not pour anything down the sink. Autoclaveliquids and broth cultures to sterilize them before discarding.16. Dispose of all solid waste material in a biohazard bag and autoclave it beforediscarding in the regular trash.17. Dispose of broken glass in the broken glass container. 18. Dispose of razor blades, syringe needles, and sharp metal objects in the “sharps”container.19. Report spills and accidents immediately to your teacher. Clean small spills with care.Seek help for large spills.20. Report all injuries or accidents immediately to the teacher, no matter how small theyseem.Biological Safety CabinetA biological safety cabinet (BSC) is used as a primary barrier against exposure toinfectious biological agents. A BSC has High Efficiency Particulate Air (HEPA) filters. The airflow in a BSC is laminar, i.e. the air moves with uniform velocity in one direction along parallel flow lines. Depending on the design, a BSC may be vented to the outside or the air may be exhausted into the room. BSCs are not chemical fume hoods. A percentage of the air is2
recirculated in most types of BSCs. HEPA filters only trap particulates, allowing any contaminant in nonparticulate form to pass through the filter.Proper Use of Biological Safety Cabinets1. Operate the cabinet for five minutes before and after performing any work in it in orderto purge airborne contaminants. 2. Before and after use, wipe the surface of the BSC with a suitable disinfectant, e.g., 70alcohol or a 10% bleach solution.3. Place everything you will need inside the cabinet before beginning work, including awaste container. You should not have to penetrate the air barrier of the cabinet once work has begun.4. Do not place anything on the air intake grills, as this will block the air supply. 5. You should prevent unnecessary opening and closing of door because this will disruptthe airflow of the cabinet.6. Always wear a lab coat while using the cabinet and conduct your work at least fourinches inside the cabinet.7. Place burners to the rear of the cabinet to reduce air turbulence. 8. Do not work in the BSC while the ultraviolet light is on. Ultraviolet light can quicklyinjure the eye.9. When finished with your work procedure, decontaminate the surfaces of anyequipment.10. Remove the equipment from the cabinet and decontaminate the work surface. 11. Thoroughly wash your hands and arms.Principles of Light MicroscopyThe light microscope, so called because it employs visible light to detect small objects, isprobably the most wellknown and wellused research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments.A beginner tends to think that the challenge of viewing small objects lies in gettingenough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order,obtaining sufficient contrastfinding the focal planeobtaining good resolutionrecognizing the subject when one sees itThe smallest objects that are considered to be living are the bacteria. The smallestbacteria can be observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field microscopes, though. These pages will describe types of optics that are3
used to obtain contrast, suggestions for finding specimens and focusing on them, and advice on using measurement devices with a light microscope.The BrightField MicroscopeIn light microscopy, light typically passes through a specimen and then through a seriesof magnifying lenses. The most common type of light microscope, and the easiest to use, is the brightfield microscope, which evenly illuminates the field of view.Magnification4
The modern light microscope has two magnifying lenses – an objective lens and anocular lens – and is called a compound microscope. These lenses in combination visually enlarge an object by a factor equal to the product of each lens’ magnification. For example, an object is magnified 1,000fold when it is viewed through a 10μ ocular lens in conjunction with a 100μ objective lens. Most compound microscopes have a selection of objective lenses that are of different powers – typically 4μ, 10μ, 40μ, and 100μ. This makes a choice of different magnifications possible with the same instrument.The condenser lens does not affect the magnification but, positioned between the lightsource and the specimen, is used to focus the light on the specimen.ResolutionThe usefulness of a microscope depends both on its degree of magnification, and itsability to clearly separate, or resolve, two objects that are very close together. The resolving power is defined as the minimum distance existing between two objects when those objects can still be observed as separate entities. The resolving power therefore determines how much detail actually can be seen. The resolving power of a microscope depends on the quality and type of lens, wavelengthof the light, magnification, and how the specimen under observation has been prepared. The maximum resolving power of the best light microscope is 0.2 μm. This is sufficient to observe the general morphology of a prokaryotic cell but too low to distinguish a particle the size of most viruses. To obtain maximum resolution when using certain highpower objectives such as the 100μlens, oil must be used to displace the air between the lens and the specimen. This avoids the bending of light rays, or refraction, that occurs when light passes from glass to air. Refractioncan prevent those rays from entering the relatively small openings of higherpower objective lenses. The oil has nearly the same refractive index as glass. Refractive index is a measure of the relative velocity of light as it passes through a medium. As light travels from a medium of one refractive index to another, those rays are bent. When oil displaces air at the interface of the glass slide and glass lens, light rays pass with little refraction occurring.The PhaseContrast MicroscopeThe phasecontrast microscope amplifies the slight difference between the refractiveindex of cells and the surrounding medium, resulting in a darker appearance of the denser material. As light passes through cells, it is refracted slightly differently than when it passesthrough its surroundings. Special optical devices boost those differences, thereby increasing the contrast.The Interference MicroscopeThe interference microscope causes the specimen to appear as a threedimensional image.This microscope, like the phasecontrast microscope, depends on differences in refractive index as light passes through different materials. The most frequently used microscope of this type is the Nomarski differential interference contrast (DIC) microscope, which has a device for separating light into two beams that pass through the specimen and then recombine. The light5
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School: St Petersburg College
Term: Summer 2016
Tags: microbiology lab
Name: Microbiology lab theory about microorganisms
Description: It's covers the microbiology lab work.
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