Laboratory 4: The Microbes Around Us Nester et. al. Aspects of Diversity Recall that bacteria only come in a limited number of shapes and sizes Despite this apparent lack of physical diversity, bacteria are among the most adaptable and biochemically diverse of all living organisms-MAKE UP THE LARGEST BIOMASS ON EARTH! Easily disseminated Ability to tolerate harsh environments Display remarkable diversity in metabolism (both usage and byproducts) Allows for superior adaptability Important to biogeochemical cycling Without this diversity, life could not exist on earth Ecophysiology Diversity in physiological adaptations to sustain bacteria in specific environments Bacteria live in virtually all types of environmental niches (hydrothermal vents to frozen glaciers) Obviously, for some bacteria the need for specialized adaptations is absolutely essential to survival. Species diversity vs. individual species numbers-available nutrients and selection Terrestrial environments, aquatic environments, animal environments, extreme environments Biogeochemical Cycling Microbes are extremely important for sustaining life on earth because the replenish key elements/compounds necessary for biological processes: Act as saprophytes-organisms that break down organic materials. Act as elemental fixers (nitrogen and carbon fixation). See below for nitrogen fixation. Regardless of mechanism, microbes contribute to carbon, nitrogen, phosphorus, sulfur and oxygen cycling. www.uwsp.edu/.../biogeochemical_cycles.html Endosymbiotic nodulation Nitrogen fixation and root nodules Nester et. al. Techniques Featured in Today?s Lab Obtaining pure cultures from complex samples: What is a colony? Colony morphology (See Figures 10.2-10.4 in lab text) Purpose of selective, differential, and enrichment media Methods of colony isolation: The streak plate?Exercise 11 The spread plate?Exercise 13 The pour plate?Exercises 12 and 14 Nester et. al. Nester et. al. Methods of isolation: Pour plate vs. spread plate Nester et. al. Methods of Bacterial Enumeration Viable plate counting The use of serial dilutions Spread plate technique Pour plate technique What is a CFU? How are CFUs/ml calculated? Pg. 73. #colonies/dilutionxvolume plated Soil Microbiology Soil Physiology Soil varies greatly in physical features: Oxygen availability pH Temperature Salt/mineral content Soil varies greatly in nutrient availability: Availability of complex organic nutrients Most prevalent in the rhizosphere and other organic-rich soils These parameters (physical and chemical/nutrient) have a direct effect on the types and relative numbers of microorganisms found in a specific soil niche. Common Types of Soil Organisms Fungi: Role as primary decomposers and providers of usable nutrients. Actinomycetes: What are they? Metabolism of complex organic substances Smells like dirt! Production of antibiotics Bacteria: Specialized functions-root nodules and nitrogen fixation Growth and Enumeration of Soil Microbes GYE + Cyclohexamide Glycerol Yeast Extract Bacteria e.g. Actinomycetes SDA Sabouraud's Dextrose Agar Fungi TSA Tryptic Soy Agar Variety Water Microbiology Nester et. al. Water Microbes and Contamination Problems in evaluating water samples for contamination. The use of indicator organisms: What are they? Examples? Measuring fecal contaminants in water-the use of fecal coliforms: What are fecal coliforms? What is coliform bacteria? Exists in intestines of animals and humans Aerobic or facultatively anaerobic Gram (-), non-sporing rods Ferment lactose ? acid & gas Standard set for drinking water = 1 per 100ml Contamination Cont. How do we test for fecal contamination of water? Membrane filter method Water is filtered through 0.45µm pore size Bacteria trapped, count colonies, CFU/ml Benefits: Quick results Process large volumes Reproducible Microbes are Everywhere: Fresh Water Direct Plating M-Endo Agar with Coliforms Nester et. al. Membrane Filtration Nester et. al. Food Microbiology Fermentations What is fermentation? Microbial fermentations are extremely important in creating common foods/beverages: Beer, bread, cheese, yogurt, wine Microbial fermentations are also important in generating other useful industrial products: Acids, alcohols, etc. Fermentation Process used by obligate anaerobes, facultative anaerobes and aerotolerant microbes; aerobes for short durations only. Involves the reduction of pyruvate to something else Uses an organic molecule (like pyruvate) as the final electron acceptor Fermentation Cont. Generate very little ATP(2 ATP) (from glycolysis) Does not utilize Krebs cycle or electron transport chains Does not require oxygen or other inorganic electron acceptors Various microbes can ferment various substrates; can have numerous end products that depend on: microbe substrate enzymes involved Great identification/classification tool! Fermentation Pathways Nester et. al. Generated through glycolysis Pasteurization Pasteurization (not a form of sterilization): Kills pathogenic microbes in heat-sensitive solutions (Brucella spp., Listeria monocytogenes, Coxiella burnetii, Campylobacter spp.) High temp.-short-time (HTST) method: 72-82C for 15-20sec. Ultra-high-temp (UHT) method: 140-150C for ~ 5 seconds/rapid cool Used for dairy products and fruit juices Foodborne Illnesses Foodborne infections: What are they? Examples? Microbial intoxications: What are they? Examples? Kitchen hygiene and prevention of foodborne illnesses The Heterotrophic Plate Count Not practical to try to isolate and type pathogens from food preparations (unless screening for a specific organism). Can use a heterotrophic plate count: Total CFU/gram of food: CFU counted x 1/dilution factor x 1/weight of sample Testing for coliforms: Uses violet red bile glucose agar (w/neutral red) Using a Serological Pipette For a 1 ml serological pipette Total volume = 0.1 ml if draw up liquid to the line marked ?0.9? Dilutions Exercise 12, 13, 14 Undiluted sample 10-2 sample 1 ml 9 ml 9 ml 10 ?1 or .1 Sample is 1/10 of total volume 1 ml 10-2 or .01 .1 sample is 1/10 of total; .1 divided by 10 1 ml 99 ml 10-2 diluted by 100 10-4 1 ml 99 ml 10-4 diluted by 100 10-6 Dilutions Exercise 12, 13, 14 Undiluted sample 1 ml 9 ml 9 ml 10 ?1 Sample is 1/10 of total volume 1 ml 10-2 .1 sample is 1/10 of total; .1 divided by 10 1ml 100 same 0.1ml 10-1 0.1 ml 10-2 0.1 ml 10-3 Collecting a Biofilm Take 1 sterile swab Take 1 tube of saline Take these items with you and swab a biofilm of your choice. Put the swab with a biofilm on it into the saline Either leave the swab in the saline tube or mix the swab with the saline and throw the swab away Bring this to lab next Wednesday (Jan. 27th) Along with a paragraph summary of what the lab entails.
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