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BIOL 140L lab study notes.docx

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University of Calgary
BIOL 241
Josh Neufeld

Experiment #1: Simple Staining Though all cells stain the same colour, a simple stain helps distinguish between various morphologies ∵ unstained bacterial cells are nearly transparent Two types of simple staining:  Positive - Basic stain, positively charged (cationic) chromophore o Bacteria outer surfaces are slightly negative (Eg Methylene blue) Inoculation - Placement of something that will grow or reproduce Inoculant/inoculum - Microorganism used in inoculation Flaming the test tube sterilizes to test t ube and prevents contaminants in cool air from entering the culture Heat-fixing helps the cell adhere to the slide so it can be stained Experiment #2: Operation of the Microscope System of two lenses:  Objective - Magnifies to produce a real image into the plane of the ocular o 4x = Scanning o 10x = Low power o 40x = High power o 100x = Oil immersion  Ocular - Magnifies it further (Magnifying power of 10x) Condenser - Collects and concentrates light to the stage Iris Diaphragm - Adjusts amount of light Experiment #3: Basic Bacterial Shapes Three general shapes of bacteria: rod (bacillus), round (coccus) or spiral (spirillium)  Bacillus and spirillium are the most motile while cocci is the most nonmotile Experiment #4: The Gram Stain Differential staining technique - A stain that differentiates bacteria depending on their abilities to retain a particular stain Age of culture and pH affect the reaction to Gram's stain Process:  Primary stain of crystal violet  Gram's iodine mordant (fixes primary stain into cell walls)  Smear is washed with a decolourizing agent (alcohol)  Counterstain of safranin is added Gram-Positive are not easily decolourized and retain the purple stain Gram-Negative become decolourized and turn red/pink Experiment #5: The Acid Fast Stain Mycobacterium have waxy cell walls due to large amounts of mycolic acids  Lipid-rich walls render the cell wall impermeable to most stains  Eg. Mycobacteria phlei Procedure:  Smear flooded with carbolfuchsin (has high affinity for lipid-rich material)  Heated to facilitate penetration of the stain  Alcohol applied and acid-fast organisms retain the carbolfuchsin  Counterstained with methylene blue Acid-fast organisms are red while non-acid-fast are blue All acid-fast organisms are Gram-positive. Experiment #6: The Spore Stain Some Bacillis and Clostridium are capable of condensing their vital cellular components into an endospore  Endospore highly resistant to environment influences (Eg Desiccation, temperature) Eg. Bacillus megaterium - central, Clostridium sporogenes - subterminal Process:  Malachite green is driven in with heat  Counterstained with safranin (light red) Terminal - At the end Subterminal - Not quite at the end Central - Middle Experiment #7: The Negative Stain Nigrosin (India ink) has negative charges on its chromophore and is repelled by the negatively charged outer surface and forms a deposit around the cell Advantages:  Doesn't require heat-fixing so the cells are not distorted Can be used on difficult to stain bacteria (Eg Mycobacterium)  Experiment #8: The Capsule Stain Cell wall can be surrounded by a capsule/slime layer called glycocalyx  Consists of polysaccharides, polypeptides or carbohydrate material Alcian blue is water soluble Polysaccharides are water soluble and uncharged, simple stains will not adhere to it  Thus bacteria is stained and capsule is left unstained (Basically a negative stain) Heavy capsule indicates highly virulent form of organism ∵ the capsule makes destruction of the microbe by phagocytic cells more difficult Experiment #9: Culture Media Preparation and Sterilization Defined Medium - Known composition General Purpose Medium - Supports the growth of a large number of organisms Nutrient broth consists of beef extract and peptone Agar melts when heated to nearly 100°C and remains liquid until cooled to about 43°C Sterile -Free of all life, including viruses Heat is a good way to sterilize ∵ enzymes and proteins are irreversibly destroyed Autoclave - Increases temperature by building up steam pressure and prevents boiling Experiment #10: Selective, Differential, and Enriched Media Selective Media - Media containing chemical substances that will prevent growth of one or more groups of bacteria without inhibiting the growth of the desired one  Eg Incorporation of NaCl or bile salts Differential Media - Possesses certain chemicals which permit differentiation between types of bacteria  Eg Eosin Methylene Blue (EMB) agar inhibits Gram-Positive bacteria o Considered selective as well as differential Enriched Media - Specific nutrients are included ∵ the microorganism's nutritional requirements are fastidious  Eg Blood or extracts of plant tissue Results:  All microbes grow in Tryptic Soy Agar  EMB inhibits Gram-Positive bacteria  KF Streptococcal Agar - Only Enterococcus faecalis grew Experiment #11: Streak-Plate Method - Isolation of Pure Cultures Three dilution methods for isolation of bacteria:  Streak plate - Dilution technique that spreads a loopful of culture over the surface of an agar plate  Spread plate - Spreading of diluted sample over an agar plate  Pour plate - Dilution of the specimen into a fluid-agar medium and pouring it into petri plates Process:  Four-way streak method performed on Stapylococcus aureus and E. coli Results:  E. Coli - Rods, large colonies S. aureus - Round, small colonies  Experiment # 12: The Pour-Plate Method Several dilutions required to ensure that at least one plate is countable Process:  Solution is continuously diluted to provide varying plates of growth Experiment #13: Aseptic Technique in Pipette Handling Pipette should be opened at the mouth end Never hold the pipette at the end with your hand Experiment #14: Plate Count Method Plate count is a direct count of the total number of dead and viable bacteria cells Aliquot - A portion Process:  Bacterium is diluted progressively and poured onto petri plates  CFU (Colony Forming Units) are counted rather than each individual cell Experiment #15: Demonstration of Motility Brownian motion - Organism stays in one place but vibrates/shakes  Due to water molecules bombarding the organism Process (Motility-Test Agar): Tubed agar is stabbed with bacterium   If there is growth spread out starting from the stab line then the organism is motile Preparation of Hanging Drop (Test for Brownian motion):  Bacterium placed on cover slip, attached to depression side and observed by microscope Results:  Lactobacillus plantarum shows Brownian motion  Pseudomonas fluorescens has flagella and shows turbidity in motility-test agar Experiment #16: Bacterial Flagella Thickness of the flagella can be increased by being coated with mordants and stained with silver nitrate  Eg. Mordants: Tannic acid and potassium Results:  Proteus vulgaris - Peritrichous, rod, dark red Experiment #17: Nutrition of Microorganisms Testing for the nutritional requirements of different microorganisms.  Such as minerals (Na, K), carbs or nitrogen Results:  None of the microorganisms can live on minerals and agar alone  Only Lactobacillus plantarum cannot grow on Agar + Minerals + Carbs  All microorganisms can live on Agar + Minerals + Carbs + Nitrogen ∴, L. plantarum requires nitrogen, others require carbs Experiment #18: Some Metabolic Activities of Bacteria Bacteria can be distinguished by their metabolic processes, such as: fermentation of carbohydrates, production of indole, breakdown of urease and the formation of hydrogen sulfide.  Fermentation occurs if the organism prefers the carb releases acidic gas as a product  Indole is created from the breakdown of tryptophan; Kovac's reagent is added to react with indole for a red colour  Urea can be broken down by urease releasing CO and 2H , and 4H is indicated by phenol red  Hydrogen sulfide is made when
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