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Methods for studying MB

What are the 6 main techniques used by microbiologists to locate, grow, observe, and characterize organisms? -Specimen collection •Inoculation •Incubation •Isolation •Inspection •Identification
What are the 3 unique problems associated with studying microbes that has to do with their size? 1. often exist in complex associations (mixed together) so first need to separate one species from another 2.often have to be grown under artificial conditions which can distort things 3. invisible and unwanted microbes can contaminate an experiment
Describe the specimen collection Microbes can be collected from almost any object or material (EX: blood, urine, food, water, soil)•Collection is accomplished using a swab, syringe, or other transport system•Goal is to hold, maintain, and preserve the microbes in the sample
Define inoculation and describe the ways it can be done. •Inoculation: the process of introducing a sample of microorganisms into a container of culture/nutrient medium •Inoculum: substance used for inoculation•Inoculation involves adding sample to flask or tube of medium, spreading sample on a solid medium, or
Define incubation. Incubation: use of a controlled environment (temperature, air content, etc) to promote growth and multiplication of microbes
Incubation creates .. Culture- (Noun)Microorganism that grow and multiple in a container (Verb) The process by which bacteria/cells are grown in an experiment.
Isolation: One result of incubation; a way of separating species of bacteria from a mixture
What is a colony? Isolated cells will become: a colony: mound of cells on solid media
How to get pure colonies? Pure colonies=to get pure cultures is the streak plate method
Define subculturing. Subculturing: further isolating by taking growth from an isolated colony and inoculating it onto new medium
How is identification of microorganisms accomplished? One major goal of characterization is to to determine the type of microbe at the species level•Use information from inspection stage (macroscopic and microscopic) and additional tests•Biochemical tests•Immunologic tests•Genetic analysis (DNA)
What is media? What are the main components of media? Media (singular = medium): provides the environment for the microbes to grow and multiply in outside of their normal habitat. Media must have a sufficient moisture, adjusted pH, and a suitable level of O2 or none at all. Must also be sterile.
Describe chemically defined media and what it would be used for. composition is standardized and chemically defined•Know exact chemical composition Contains pure organic and inorganic compounds Easily reproducible Varies little from one source to another reserved for the growth of autotrophic bacteria
Describe complex media and what it would be used for. Contains at lease one ingredient that is not chemically definable Most heterotrophic bacteria and fungi, grown on complex media made up of nutrients including extracts from yeasts, meat, or plants, or digests of proteins from these and other sources.
What is an enrichment culture? soil/fecal. usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others.The bacteria are given time to grow in the medium between transfers; this is the enrichment stage
Define fastidious. Organisms that require many growth factors.
Describe selective media and what it would be used for designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes.bismuth sulfite agar is one medium used to isolate the typhoid bacterium, the gram-negative Salmonella Typhi (Tī-fē), from feces.
Describe differential media and what it would be used for make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. Blood agar (which has red blood cells) is a medium that microbiologists often use to identify bacterial species that destroy red blood cells.
Describe reducing media and what it would be used for Reducing media: Contains ingredients like sodium thioglycolate, that chemically combines with dissolved oxygen and deplete the O2 in the culture medium. Prevents the loss of anaerobic bacteria. Needs to be O2 free.
Describe carbohydrate fermentation media and what it would be used for it is used to determine whether a bacteria can use a certain carbohydrate. It tests for acid or gas produced from process. used to identify Gram-negative enteric bacteria, all of which are glucose fermenters but only some of which produce gas
What three states can media be in? Liquid, solid, and semisolid
Define liquid media Water-based solutions, free flowing•Made by dissolving solutes in distilled water•Don’t solidify above freezing•Called “broths”, “milks”, or “infusions”
Define solid media gives a firm surface for cells to divide and form colonies•Used for isolating and culturing bacteria and fungi, holds moisture and nutrients•Contains agar, from marine red algae•Liquify at 100 ºC, remains liquid until cooled to 40 ºC,flexible and moldable
What is agar and what is it used for? A complex polysaccharide derived from a marine alga,Few microbes can degrade agar, so it remains solid. Also, agar liquefies at about 100°C. It can be incubated at 100°C before it again liquefies; useful when growing thermophilic bacteria.
Define Semisolid media Have a clotlike appearance at room temperature•Contain solidifying agent that thickens them but doesn’t make fully firm•Ex: agar, gelatin•Used to determine motility of bacteria
What are pure, mixed, and contaminated cultures? Pure- only has a single know species; Mixed- has 2 or more identifies species; Contaminated- has contaminants and was one a pure or mixed culture.
What is a contaminant? Unwanted microbes, may be of uncertain identity, may cause disruption.
What is monomorphic and pleomorphic? Monomorphic- (most)Bacteria maintains a single shape. Pleomorphic- When bacteria can have many shapes, not just one.
What are the 3 basic bacterial shapes? Vibrio-frown; Spirillium-fat worm/spiral; Spirochete-skinny worm.
Describe a coccobacilli shaped bacteria. short oval shaped rods that look like cocci;
Describe a coccus shaped bacteria. spherical shaped (meaning berries)
Describe a bacillus shaped bacteria and what are the two meanings? rod shaped; Bacterial shape and Specific genus
What are some of the more uncommonly shaped bacteria? Star-shaped(Stella) and rectangular prokaryotes(Harloarcule).
What are the bacterial cell arrangements? Diplo-Pair after division of 1 plane Strepto-in chains after division of 1 plane Staphyl- in clusters after division Tetrads- in groups of 4 after division of 2 planes Sarcinae- in groups of 8 after division of 3 planes
Define Magnification. Magnification: enlargement of image of a specimen•The magnification results from interaction between visible light rays and lens curvature and involves refraction
Define refraction and refractive index refraction: bending of light rays•Refractive index: measure of light bending ability
What is a compound microscope? Light microscope; uses multiple lenses and a lamp for artificial light.
What do the illuminator, condenser, objective lens, and ocular lens of a microscope do? Condenser- focuses light through specimen; Ocular lens-remagnifies the image formed by the objective lens; illuminator-light source; Objective lens is the lenses closest to the specimen
Describe brightfield microscopy and what the specimen would like look when using it •Type of illumination produced by regular compound light microscopes•image is formed when light is transmitted through the specimen•Dense regions of the specimen absorb light, so the specimen appears dark and the field around it is bright
What is staining and why is it done? Staining: coloring the microorganism with a dye that emphasizes certain structures•Bacterial stains apply contrast to bacteria so that they can be visualized by light microscopy •Separates light intensity of specimen and background
Define simple stain and give examples Simple Stain: staining procedures that uses only one stain (dye) to color the whole microorganism•Used to observe cellular shapes and simple structures Examples- Safranin, methylene blue and malachite green
Define differential stain and give examples Differential stains: react differently with different bacteria•Used to distinguish bacteria/classify bacteria Example is gram staining and acid-fast staining
Define special stain and give examples Special stains: used to color or isolate specific parts of microorganisms Example of uses- chemical, biological, pathological characters.
Describe darkfield microscopy. When is it used? Used when microorganisms is not visible with ordinary light microscope or specimen cannot be stained•Uses an opaque disk for the condenser•Block light that would enter the objective lens directly•No backlight specimen appears light against dark background
Describe phase contrast microscopy and what it would be used for Small differences in refractivity of objects turnedinto variations in brightness Light goes through a ring-shaped diaphragm 2 sets of light rays are created : Direct rays and rays through specimen lets you view internal structures
Define fluorescence Takes advantage of fluorescence: ability of substance to absorb short wavelengths of light (UV) and to give off light of a longer wavelength (visible)
What does the specimen look like when used fluorescence microscopy ? •Specimen appears bright against a dark background
How does immunofluorescence work? Immunofluorescence: fluorochrome is combined with an antibody that recognizes the surface of a specific bacterial cell•Causes cells to fluoresce when hit with UV light
What is a fluorochrome? Some organisms fluoresce naturally under UV light, otherwise it can be stained with a fluorescent chemical compound called a fluorochrome (fluorophore )
Describe confocal microscopy and when it would be used. Specimens are stained with fluorochromes •One plane of the specimen is illuminated at a time •Scanned planes resemble a stack of images •Computer used for reconstruction of planes into a 3D image
Define resolution Resolution: ability of the lens to distinguish fine detail and structure•The resolution of a light microscope is limited to 2000x•Make use of other electron microscopy to help improve resolution
What is the difference between light microscopy and electron microscopy? Electron microscopy is used on objects smaller than 0.2 nanometers and must have a beam of electrons go through the specimen instead of light; uses electromagnetic lenses to focus beam of electrons.
Describe transmission electron microscopy and what you can see with it. Transmission Electron Microscopy (TEM) -Ultrathin pieces (sections) of the specimen are used -Electrons are passed through the specimen and are scattered -The scattered electrons are used to make the image -Used to observe fine cell structure details
Describe scanning electron microscopy and what you can see with it. Use the intact specimen •Beam of electrons is directed over the surface of the specimen and knock electrons from its surface •Electrons that are knocked off are used to make the image •Provides a 3D view •Allows for observation of surface details
Created by: Nicofosho15



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