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Biol 140L Final Exam.docx

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University of Waterloo
BIOL 240
Cheryl Duxbury

Biol 240L Final Exam Bacillus subtilis (rod) Safety E. coli (Rod) Types of Fires Rhodospirillium rubrum (spirillium) A – ordinary combustibles B – flammable liquids Experiment 4: Gram Stain Intro C – electrical fires D – combustible metals - gram stain used to differentiate between Pass, Aim, Squeeze, Sweep types of bacteria - gram-positive and gram-negative Experiment 1: Simple Staining - stain fix smear with primary stain (crystal Intro violet), then add Gram’s iodine which acts like a - Colour compound stain used to increase mordant (fixes primary stain to cells), then wash contrast between specimen and the with a decolourizing agent (95% ethyl alcohol), background then a counterstain (safranin) - gram-positive organisms will not be easily - simple staining procedure (using a single stain) - distinguish morphology (cocci, rods, spirals, decolourized and will be purple (crystal violet) vibrios) - gram-negative organisms will be decolourized - methylene blue (blue), basic fuchsin (red), and will appear red (safranin) crystal violet (purple) Results - positive and negative Staphylococcus epidermis (purple, round, - positive: stain is basic, positively charged positive) (cationic) and the slightly negative charge on E. coli (red, rod, negative) the bacteria attracts the stain Experiment 5: Acid-Fast Experiment 2: Microscope Intro Intro - used to stain lipid rich cell walls - objective lens (nearest the specimen) (Mycobacterium) - ocular lens (eyepiece) - smear is flooded with carbolfuchsin (dark red, - condenser (collects and concentrates light) 5% phenol, high affinity for lipid rich material) - oil improves resolution providing sharpness - heated so the stain can penetrate - resolving power is the ability to distinguish - decolourized, acid-fast organisms stay red between two images as separate (0.2 um is best - methylene blue used to counterstain - Kinyoun method: concentrations of phenol for this microscope) Results and carbolfuchsin increased don’t need heat Staphylococcus epidermis (round) - acid-fast organisms are gram positive but not E. coli (rod, bacillus) all gram positive are acid-fast Results Experiment 3: Basic Shapes Staphylococcus epidermis (round, non acid) Intro M. tuberculosis (rod, fast, red) - rod (bacillus), round (coccus) and spiral (spirillium) Experiment 6: Spore Stain Intro - stalked forms (caulobacter), club-shaped (corynebacterium) and comma shaped (vibrio) - bacillus and clostridium capable of making - See page 21 for chart endospores - magnification = size of specimen in drawing / - made when conditions are not the greatest, estimated or actual size store vital cellular components Results - must drive stain into spore (heat) Staphylococcus epidermis (round) - malachite green to stain spores and then - autoclave is used (like a pressure cooker, counterstain with safranin 121°C, 15-16 pounds per square inch Results Results Clostridium sporogenes (rod, spore at end) Sterile tube: cleaner, yellow Bacillus megaterium (rod, spore in centre) Non sterile: cloudy, precipitate at bottom, light yellow Experiment 7: Negative Stain Intro Experiment 10: Selective, Differential - negative stain used acid dye nigrosin (India Intro ink) which has negative charge - selective: contains specific chemicals which - repels the negative outer surface of cells, does not affect the growth specific organisms, deposit forms around bacteria but discourages growth of others - cells are unstained and background is grey - sodium azide isolates lactic acid bacteria, lack - doesn’t need to be heated cytochrome system which is what sodium binds - mix culture with drop of nigrosin, smear across to slide - differential: contains dyes/chemicals which Results allow observer to distinguish between types of Bacillus megaterium (clear, round) bacterial colonies - EMB used to detect coliform rods, may also be Experiment 8: Capsule Stain considered a selective medium Intro - enriched: allows basically everything to grow, - bacteria can be surrounded by a capsule, slime fastidious: stringent nutrient requirements layer or glycocalyx Results - consists of polysaccharides, polypeptide and Media EC SE EA SF (EF) carbohydrate material TSA (E) +++ +++ +++ +++ - alcian blue is a basic dye, water soluble, forms KF (S) - - - +++ linkages with acid groups of acidic EMB(D) ++ + ++ (dark + mucopolysaccharides, staining them blue (black centres, - can use nigrosin to do a negative stain as well centres, pink - negative stain (dry)  flood with crystals green mucoid) violet add water to slide to float stain off slide sheen)  repeat with gram’s safranin Results Experiment 11: Streak Plate Klebsiella pneumonia (pink around capsule with Intro rod like structure inside) - pure culture: only type of microorganism) - four way streak plate: streak along one edge, Experiment 9: Culture Media and Sterilization turn 90, streak again, turn 90 streak again, turn Intro 90 streak again, spread out inoculum so it is - culture medium is the material in which thin bacteria are grown Results - defined medium: know make up of medium Staphylococcus aureus (round, grape clusters, - complex medium: don’t know make up for purple) medium (tissue or blood) E. coli (rod, clustered, red) - nutrient broth basically grows everything - agar made from galactan from alga, melts at 100°C and solidifies at 43°C - sterilized means free of life, including viruses - lethal agent in sterilization is heat Experiment 12: Pour Plate - hanging drop: drop of water on cover, placed Intro over depressed slide, look under microscope - dilute specimen into a series of fluid agar and Results poured into petri plates Lactobacillus plantarum: No motility, Brownian - need to make several dilutions to make sure Pseudomonas fluorescens: Motility, True one plate is countable Motility Results Plate 1: a lot of growth, equally distributed Experiment 16: Flagella Plate 2: little growth, spread out Intro Plate 3: no growth - peritrichous (all over) Control: no growth - polar (one end) - flagella beyond limits of microscope. Thickness Experiment 13: Aseptic Technique increased using a mordant (tannic acid, Intro potassium alum) then staining with silver - squeeze bulb, press S to suck up solution, nitrate press E to empty - mordant incorporates stain and increases Results contrast No growth (experiment went well, or - precipitate of mordant will occur on debris as contaminants unable to grow) well, need clean slides Results Experiment 14: Plate Count Proteus vulgaris (rod, small rod sticking out  Intro flagella) - direct count reveals total number of bacteria including both dead and viable cells in a given Experiment 17: Nutrition volume Intro - Standard Plate Count reveals information on - basic nutrients: water, carbon, energy, living cells only nitrogen, minerals, and growth factors - assumes each viable cell will develop into a - need a source of energy (sugars, starches, colony amino acids) - colony forming units (CFU), expressed as - nitrogen to manufacture protein molecules number of CFU/mL (protein, amino acids, ammonium phosphate, Results potassium nitrate) Number of organisms per mL = number of - metallic elements (sodium, potassium, colonies x dilution factor calcium) - dilution factor is the inverse of dilution Results - remember per mL (0.1, need to move decimal Plate 1: Agar only point one more spot) Plate 2: Agar + Minerals CFU/mL =# of colonies/dilution x amount plated Plate 3: Agar + Minerals + Carbohydrates Plate 4: Agar + Minerals + Carb
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