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44 Cards in this Set
- Front
- Back
- 3rd side (hint)
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What bacteria requires acid fast staining?
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Mycobacterium sps and Nocardia (page 110)
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What is the mordant used in acid fast staining?
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Phenol and steam/heat
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Cord Factor
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Cord factor is a protein that helps the mycobacterium, tuberculosis, stick together giving it it's long and slender formation. It's part of its virulent factor and critical to its survival in the host.
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Examples of mycobacteria
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M. tuberculosis, M. leprae, M. smegmatis
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Is Methylene Blue positively or negatively charged?
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Methylene blue chloride is composed of a cationic chromophore (the part of the dye that is colored), therefore it is positively charged (page 109)
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What does Methylene blue bind to?
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Because Methylene blue is positively charged it binds to DNA (which is negatively charged), many proteins, and other negatively charged molecules. This is what gives the nucleus it's dark blue color (tightly packed DNA) and the cytoplasm it's lighter blue shade (less dense negatively charged particles). This is also why it's called a basic dye because they combine and stain acidic structures.
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What's an example of an anionic dye?
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Eosin
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Disadvantages of Gram staining
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1. Often disease causing bacteria don't have distinct stain characteristics
2. The stain cannot diagnose the cause of most infection 3. Some bacterial cells stain poorly or not at all. For example mycobacterium like tuberculosis. Gram staining also requires fresh bacteria. |
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Describe the process for acid fast staining and how it works?
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The primary stain, red carbolfuchsin stains all of the cells. The phenol and steam/heat then drives the stain through the waxy wall of the mycobacterium where it remains trapped. You then decolorize using hydrochloric acid. The acid only removes the stain from the non-acid-fast cells and the background. It cannot penetrate the waxy wall of the acid-fast cells so they remain pink. The counterstain, methylene blue then only stains the non-acid-fast cells making them blue.
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What color are acid-fast cells stained during the staining process?
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Pink.
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What color are gram positive cells at the end of the Gram's staining
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Purple
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What color are gram negative cells at the end of the gram staining process?
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Pink
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What bacteria create endospores?
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Clostridium sps and Bacillus sps.
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What are three diseases are caused by Bacillus and Clostridium sps ?
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B. Anthrax = anthrax, C. perfringens = gangrene, C. Tetani = tetanus
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Describe the endospores staining and how it works?
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Endospores are difficult to stain b/c their walls are practically impermeable to chemicals. Heat has to be used to drive the malachite green, the primary stain, into the endospore. Once the slide is cooled the slide is decolorized with water and counterstained with sanfranin. This leaves the vegetative cells red and the endospores green.
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What color are the endospores at the end of the spore staining?
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Green
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What color is the vegetative cell at the end of a spore staining?
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Pink
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What does an anionic dye bind to?
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Binds to positively charged molecules such as amino acids. Anionic dyes are also known as acidic dyes because they stain alkaline structures.
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Describe capsule staining
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Capsule staining is a negative staining. You use an acidic dye like eosin or nigrosin to stain the background which makes the encapsulated cells look like they have a halo around them.
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What type of dye is used in negative staining?
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Acidic dyes because most cells are negatively charged.
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What are three examples of basic dyes?
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Crystal violet, sanfranin, and methylene blue.
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Name the three types of differential staining we used.
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Gram's staining, acid-fast staining, and spore staining.
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Name the three special stainings listed in our lab manual
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1.Negative Stain (ex. Capsule staining)
2. Flagellar staining 3. Fluorescent stain |
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Three advantages to negative staining?
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1. Detect presence of capsule
2. Detect the cells size and shape 3. Cells arrangement |
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What is the primary stain in the Gram staining process?
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Crystal violet.
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Describe the Gram staining process and how it works
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First the primary stain, crystal violet is applied. The iodine then acts as the mordant and makes the primary stain less soluble. The Gram positive cells with their thicker cell wall (peptidoglycan layer) retain the primary stain,crystal violet. When the ethanol and acetone (decolorizing agent) is applied it breaks down the thin Gram negative cell walls allowing the primary stain to be washed away leaving them colorless. You then apply the counterstain, red safranin which dies all the cells pink. Because the gram positive cells are still purple it hides the pink but the Gram negative cells are now pink.
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Advantages of Gram staining..
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-relatively quick and useful in diagnosis of gram negative, neisseria gonorrhea and streptococcus pneumonia
- if gram positive cells you can begin penicillin treatment |
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What is the primary stain, mordant, decolorizing agent and counterstain in a Gram's stain?
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Crystal violet = P
Iodine = M Acetone-alcohol=D Sanfranin = C C.I.A.'S |
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What is the primary stain, mordant, decolorizing agent, and counterstain in acid-fast staining?
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Carbolfuschin=P
Phenol & Steam/Heat = M Acid Alcohol = D Methylene blue = C |
C.P.A.M
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Name two aseptic transfer techniques
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Streak plate method and pour plate (sandwich method)
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What is the importance of aseptic transfer techniques?
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1. Keep specimen free of contaminants from the environment
2. Keep culture from contaminating you or others (safety) 3. To isolate pure microbes 4. Contaminants are kept out which enables us to study metabolism of a particular organism |
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Precautions during sterile transfer
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A. Carefully label all media, tubes, & culture
B. Use loop when cooled. C. Avoid aerosols D. Keep transfer instruments sterile. Don't wave or blow on loop. E. Disinfect area before and after. |
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Common streak plate method errors
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1. Getting fresh cells on loop
2. Failure to pass through previous section two times 3. Failure to go from edge to edge 4. Holding loop vertically and gauging agar 5. Forgetting to shake the tube when obtaining cells for the 1st quadrant |
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Why are colony sizes are smaller when cells are closer together?
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1. Less competition for nutrients and available space
2. Waste accumulation at toxic levels impairs growth. |
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EMB agar components and their role
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EMB agar contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue; it is commonly used as both a selective and a differential medium. EMB agar is selective for gram-negative bacteria. The dye methylene blue in the medium inhibits the growth of gram-positive bacteria; small amounts of this dye effectively inhibit the growth of most gram-positive bacteria (8). Eosin is a dye that responds to changes in pH, going from colorless to black under acidic conditions. EMB agar medium contains lactose and sucrose, but not glucose, as energy sources. The sugars found in the medium are fermentable substrates which encourage growth of some gram-negative bacteria, especially fecal and nonfecal coliforms. Differentiation of enteric bacteria is possible due to the presence of the sugars lactose and sucrose in the EMB agar and the ability of certain bacteria to ferment lactose in the medium. Lactose-fermenting gram-negative bacteria (generally enteric) acidify the medium, and under acidic conditions the dyes produce a dark purple complex which is usually associated with a green metallic sheen. This metallic green sheen is an indicator of vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms. A smaller amount of acid production, which is a result of slow fermentation (by slow lactose-fermenting organisms), gives a brown-pink coloration of growth. Colonies of nonlactose fermenters appear as translucent or pink (6, 9).
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What are two differences between the streak method and pour plate method
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1. In the pour plate method colonies form at and below the surface of the medium.
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When using EMB agar as a differential media why did the e.coli turn metallic green
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Because the ecoli fermented the lactose causing the pH to become acidic. The eosin dye turned the agar dark purple with an associated metallic green color. This indicates vigorous lactose or sucrose fermentation.
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What is the biochemical basis for EMB also being a selective media?
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The methylene blue in the EMB inhibits the growth of gram-positive bacteria making it selective for gram-negative bacteria
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Lysozyme affects gram positive cells, gram negative cells, or both?
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Lysozyme affects gram positive cells more. The reason being that gram negative cells have an extra outer membrane layer that protects the peptidoglycan layer.
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What does lysozyme attack
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Lysozyme hydrolyzes the glycosidic bond between NAG-NAM units of the backbone of the peptidoglycan layer
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What enzyme's presence is confirmed by the starch hydrolysis test?
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Amylase
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What's the importance of the starch hydrolysis test?
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The starch hydrolysis test tells us whether or not a bacteria has the enzyme that allows it to break down starch so individual glucose units can be transported inside the cell easily.
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What's the significance and importance of the enzyme catalase?
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Catalase allows for the conversion of the highly reactive oxidant, peroxide anion contained in hydrogen peroxide. This would be detrimental to a bacteria but the catalase converts hydrogen peroxide to water and molecular oxygen. This is important in allowing a bacteria to grow in oxygenated environments.
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What's the primary stain, mordant, decolorizing agent and counterstain in endospore staining
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Malachite green = P
Heat = M Water = D Safranin = counterstain |
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