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Lab Microbiology Midterm Review Wednesday, October 08, 2008 10:41 PM Experiment 1- Media Preparation and Sterilization Solid medium contains agar (from red algae) Melts @ 100C, Solidifies @ 40-42C Types Plate - isolation Slant - maintenance Broth - liquid Deep anaerobic growth Complex medium composed of digests of chemically undefined substances such as yeast and meat extracts Defined medium medium whose precise chemical composition is known Sterilization: killing or removal of all living organisms Autoclave: 121C (for media, glass) Dry heat: flaming, hot air (for loops, glass) Filtration: for heat-sensitive liquids (antibiotics) Radiation: UV, gamma rays (for surfaces) Cold sterilization: ethylene oxide (for heat-sensitive materials) Experiment 2- Culturing Microorganisms from the environment Minority of microorganisms are pathogenic- disease causing agent Nosocomial means hospital-acquired Purpose: This experiment will demonstrate the ubiquitous nature of microorganisms. Materials needed: 1 Trypticase Soy (TS) broth 2 TS agar plates 2 sterile swabs Experiment 3- Hand Washing Resident (normal) microbiota/flora occur naturally on or in body Transient flora temporary on body, cant grow on skin because skin is too dry and acidic Nosocomial infection hospital-acquired infection Infectious dose number of microorganisms required to establish an infection Purpose: This experiment will evaluate effectiveness of different soaps & hand washing times. Experiment 4- Colony Morphology Organism Color Colony shape Round Rhizoid Round Round Margin shape Smooth Lobate/ filament Smooth Smooth Elevation Optical Surface propertie characts eristic Flat/ convex Flat Convex translucen Shiny t opaque opaque Dull Shiny Shiny colony - a group of genetically identical bacteria, arising from a single cell on an agar plate colony morphology - colony characteristics; see next slide contaminant - an unwanted organism which has been accidentally introduced into the culture Purpose: To observe the colony morphology of several organisms and become familiar with the terms used to describe colony morphology Escherichia coli Bacillus cereus Micrococcu s luteus Beige White Yellow Saccharomy White ces cerevisiae Streptomyc es coelicolor Serratia marcescens Purple/ white red Convex/ opaque umbonate Flat opaque Irregular Smooth/ / lobate round round smooth Dull convex opaque shiny Experiment 5- Asceptic Technique See p. 20-21 for proper technique Experiment 6- Pure Culture Pure culture - one that contains only one kind of microorganism; an isolated colony is one example of this Streak plate - technique for diluting bacteria on agar plates; can be used to isolate individual bacteria from a mixture Purpose: To isolate a pure culture (in the form of an isolated colony) from a mixed culture using the dilution technique known as streak plating Important to use proper aseptic technique in lab to avoid contaminants Common streak plate mistakes: Taking lid off completely or placing on bench top Not flaming between quadrants Going back into previous quadrant too many times Labeling the lid Not letting loop cool sufficiently Streaking bacteria onto lid Writing on agar Gouging agar Holding loop at incorrect angle Not using entire quadrant Possible Results for isolated colonies: Escherichia coli - off-white, translucent Staphylococcus epidermidis - white, opaque, small Micrococcus luteus yellow, opaque, small Experiment 7- Smear Preparation Staining makes cells more visible by adding contrast between cells and background. Smear - 1st step in staining cells, can be made from solid or liquid culture. Heat fixation kills microorganisms and sticks them to slide to allow for easier visualization & staining. Purpose: To prepare a smear from solid and liquid media. See p. 31 for smear preparation procedure Experiment 8- Simple Stain Contrast - a result of staining, allows for visualization of cells. Simple stain: aqueous or alcohol solution of a single basic dye; highlights entire cell, stains every cell the same. Purpose: To study bacterial cellular morphology and arrangement using a simple stain. Expected Cell Morphologies: Staphylococcus epidermidis staphylococci Escherichia coli small, random bacilli Spirosoma linguale spirals Bacillus megaterium large streptobacilli Streptococcus mutans streptococci Bacillus subtilis random rods Streptomyces coelicolor filamentous Deinococcus radiodurans diplococci or tetrads Experiment 9- Use of the Microscope Brightfield light microscopy Compound microscope multiple lens Binocular microscopes 2 oculars Objectives 10x initial focus using coarse adjustment knob 100x fine focus only Total magnification = magnification of the ocular lens (10x) multiplied by the objective lens (4x, 10x, 40x, 100x) Experiment 10- Is the broth culture a pure culture? Determine if culture given (A through H) is pure or mixed, by doing a simple stain and looking for different cell morphologies (pg. 34) and doing a streak plate and looking for different colony morphologies (pg. 14) Procedure: pg.44 select a culture; write letter on page 44 do a streak plate; incubate it at 37C do a simple stain view under the microscope answer questions on page 44 Experiment 11- Gram Stain Differential stain Difference between simple and differential stains: Simple stain stains all cell types and does not distinguish cell types. Differential stain distinguishes cell types. The Gram stain distinguishes Gram-positive and Gram-negative cells. Steps in a Gram Stain: ***Very important, must be able to perform: Thin layer of gram standard (loopful) add to slide Make a thin smear from a portion of one isolated colony on streak plate Allow smear to dry and then heat fix by waving slide over burner Allow slide to cool and place on stain rack Cover smear with crystal violet; leave for 1 min Rinse with water Cover with iodine, leave for 1 minute Rinse with water Briefly drop ethanol on the smear until the crystal violet no longer runs from it (should only be a few seconds) Rinse with water Add counterstain, Safarin for 1 min Rinse with water, blot Observe under microscope Staining differences between Gram + and Gram - are related to cell wall structural differences. Gram + have a thick peptidoglycan layer. Alcohol is unable to leach out the crystal violet-iodine complex. Gram bacteria: alcohol penetrates lipopolysaccharide layer and the thin peptidoglycan layer does not prevent the alcohol from removing the crystal violet-iodine complex. Gram reaction is most valid on young cultures (24 hours or less). Some Gram + organisms lose their ability to retain the primary stain and appear Gram - (pink). Spirosoma linguale Gram-negative spirals Staphylococcus aureus Gram-positive staphylococci Staphylococcus epidermidis Purple Gram-positive staphylococci Escherichia coli Red Gram-negative random rods Experiment 12- Endospore Stain Differential stain Endospore - dormant survival structure formed by some Gram positive rods when conditions are unfavorable Endospores are extremely resistant to heat, dehydration, harsh chemicals, and radiation. They can survive indefinitely Sporulation process of spore formation Vegetative cell a cell that can grow and divide under optimal conditions Germination cell becomes hydrated, emerges from the spore coat, and returns to a vegetative cell Endospore a survival structure in a cryptobiotic state; contains dipicolinic acid complexed with calcium Gram Positive Rods that form endospores Clostridium C. botulinum botulism C. tetani tetanus C. perfringens gas gangrene Bacillus B. anthracis anthrax Shaeffer Fulton Endospore Stain: Primary Stain - Malachite green Mordant heat from steam drives stain into cells Decolorizer - waterto remove stain from vegetative cells Counterstain - safranin Results Pink/Red vegetative cells Green endospores Endospore staining Procedure pg 55 1 smear/person of either Bacillus megaterium or Bacillus sphaericus. Attach clothespin to slide. Place slide on steaming can. Put piece of paper towel on top of prepared smear. Flood smear with Malachite green while on steam can for 6 minutes. Keep moist dont allow the stain to dry out. Remove slide from steam can, allow to cool. Remove paper towel and rinse with water until green no longer flows off the slide. Flood smear with Safranin for 1 minute. Rinse with water, blot dry with bibulous paper. Observe under microscope. Experiment 13- Acid Fast Stain Differential stain Used clinically to diagnose illnesses caused by Mycobacterium species Unique lipid component called mycolic acid. Mycobacterium cells resist rigorous decolorization, even with acid alcohol, hence Mycobacterium is called acid-fast bacteria. On solid media, colonies are wrinkled in appearance due to the cells hydrophobic nature. Acid Fast staining Procedure page 60 1. smear/person add small amout of egg albumin to slide, then add a loopful E. of coli from broth culture, lastly add a small scraping of Mycobacterium from plate. 2. Attach clothespin to slide. 3. Place slide on steaming can. 4. Put piece of paper towel on top of prepared smear. Flood smear with carbol-fuchsin while on steam can for 6 minutes. Keep moist dont allow the stain to dry out. Remove slide from steam can, allow to cool. Remove paper towel and rinse with water until red no longer flows off the slide. Flood smear with acid-alcohol for 30 seconds. Rinse with water. Flood smear with methylene blue for 2 minutes. Rinse with water, blot dry with bibulous paper. Observe under microscope. Experiment 14- Capsule Stain Negative stain: background is stained but the capsule is not. Capsule composition Layers of polysaccharide or polypeptide Azotobacter - polysaccharide Bacillus - polypeptide Primary component of capsule is water. Functions of a capsule: Prevents desiccation Reserve energy source Attachment to host cell Resists phagocytosis May enhance the ability to cause disease VIRULENCE FACTOR Procedure of Capsule stain: Add small drop of congo red Mix a small loopful of aztobbacter Allow it to dry (do not heat fix) Flood smear with maneval's stain Rinse w/ water Blot and view Experiment 15 Bacterial Population Counts Methods for counting bacteria Direct microscopic count counts all cells Hemocytometer Standard plate count (viable count) counts only living cells Spread plate Pour plate Turbidimetric assay can be correlated to viable cell count Spectrophotometer Know how to do dilution problems!!! Experiment 16- The Most Probable Number (MPN) Technique for Fecal Coliform Detection Potable water - safe to drink; MPN coliform index of 2.2 coliforms per 100ml of water Water contaminated with animal/human feces should not be consumed because it may contain pathogens Indicator organism - A bacterium whose presence in water indicates fecal contamination ex. Escherichia coli Presence of coliforms is indicative of fecal contamination. Coliforms: Aerobic or facultatively anaerobic Gram negative Do not form endospores Rod shaped Ferment lactose and produce gas Examples: E. coli, Enterobacter, Serratia, Citrobacter, Klebsiella, Hafnia SELECTIVE MEDIUM Favors the growth of certain microbes and inhibits competitors Example: Lauryl sulfate lactose broth - lauryl sulfate inhibits Gram-positive organisms, therefore selects for Gram-negative bacteria DIFFERENTIAL MEDIUM Visible indication of a physiological characteristic Example: Lauryl sulfate lactose broth - lactose fermenters cause gas production MPN TESTS: Series of tests used to determin if water is potable Tests for coliform contamination Consists of 3 Tests: Presumptive (today) Confirmed Completed Presumptive Test (day 1): The medium for this test is Lauryl sulfate lactose (LSL) broth Selective component: lauryl sulfate Selects for intestinal bacteria (e.g. Gram negatives) Differential component: lactose Differentiates lactose fermenters from non-lactose fermenters Explanation: intestinal bacteria that ferment lactose and produce gas Positive Result: Gas in the Durham tube and turbidity Indicates presence of fecal coliforms, but more must be done to confirm this suspicion Follow up: Count number of LSL tubes with growth and gas bubble, then use MPN table on page 86 to find MPN index for your sample Confirmed Test: Medium: Brilliant green lactose bile broth Selective components: brilliant green and bile Selects for: inhibit the growth of non-coliforms thereby selecting for coliforms Differential component: lactose Explanation intestinal bacteria that ferment lactose produce gas (and acid) as byproduct, gas will be visible in Durham tube + Result: Gas in the Durham tube & Turbidity Normally you wait until you see positive results from the Confirmed test before you do the Completed test. Completed Test: Medium: Eosin methylene blue (EMB) agar Selective component: eosin and methylene blue dyes Selects for: Gram negative bacteria Differential component: lactose Explanation: Lactose fermenters produce acid and gas as byproducts. Acid production drops pH around colonies which causes colonies to take up dyes and become dark. Results: Only Gram negative bacteria grow on the plates. If the bacteria are lactose-fermenters, the colonies will range in color from somewhat purple to green due to uptake of the dyes. Experiment 17- Effect of Environmental Conditions on Growth and Survival of Microorganisms Exp. 17 b: pH describes the acidity or alkalinity of a solution Optimum pH pH at which the organism grows best Acidophiles - grow best at pH <6 yeasts, molds, some bacteria Neutrophiles - grow best at pH close to 7 most bacteria Alkalinophiles - grow best at pH >7 some bacteria pH range describes the range of pH at which the organism can grow Note difference between can grow and grows best Purpose: to determine optimum pH and pH range for 3 organisms The effects o f pH on microbes (Human relevance): Sodium benzoate (pH 4.5) fruit products, jams, relishes, beverages, dressings, salads, pie and pastry fillings, icings, olives and sauerkraut against yeasts, some bacteria (foodborne pathogens but not spoilage bacteria) and some molds Potassium sorbate (pH 6.5) cheeses, dips, yogurt, sour cream, bread, cakes, pies and fillings, baking mixes, doughs, icings, fudges, toppings, beverages, margarine, salads, fermented and acidified vegetables, olives, fruit products, dressings, smoked and salted fish, confections and mayonnaise yeasts, molds, and select bacteria In wine processing, sorbates are used to prevent refermentation Exp. 17 D: A solution is composed of a solute dissolved in a solvent Osmosis the flow of water from areas of less solute to areas of more solute If a bacterial cell is placed in a hypertonic solution, water will leave the cell resulting in plasmolysis More solute in the solution than the cell If a bacterial cell is placed in a hypotonic solution, water will enter the cell until the cell can no longer take in water More solute in the cell than the solution If a bacterial cell is placed in an isotonic solution, there will be no net flow of water in either direction Solute concentrations in the cell and solution are the same Halophile- can grow well in hypertonic or high salt solutions Ex: Vibrio fischeri Halotolerant- can grow in relatively high salt concentrations, but grow best at low salt-concentrations Ex: staphyloccus aureus; staphylococcus epidermis Extreme Halophile- likes extremely salty environment (require 15-30%) Ex: halobacterium salinarium 17 A: effect of temperature Organism Optimum Temperature Hyperthermoph Above 80C ile Thermophile Mesophile 45-80C 20-45C Psycrophile Below 20C Temperature range for growth the range of temperatures at which an organism can grow; usually 30-40 degrees. Which would you expect to survive best and why? E. coli intestinal bacteria B. megaterium soil bacteria, forms endospores **** D. radiodurans soil bacteria, can repair DNA damage caused by radiation Exp. 17 C: Ultraviolet radiation 10-400nm visible light is 400-700nm Used to sterilize surfaces 260nm is most damaging to nucleic acids Causes thymine dimers resulting in mutations (death) of cell Deinococcus radiodurans has extraordinary ability to repair DNA damage caused by UV Endospores are resistant to UV Experiment 18- Disinfectants and Antiseptics Antimicrobial agent - Chemical that kills or inhibits the growth of microorganisms Cidal Agent Static Agent Lethal inhibits growth kills microbes does not kill microbes Virucidal virustatic Fungicidal fungistatic bacteriocidal bacteriostatic Disinfectant antimicrobial agent used only on inanimate objects ammonia, bleach, ethanol) Antiseptic antimicrobial agent that can be applied to living tissue iodine, ethanol) (Ex. (Ex. Factors affecting the effectiveness of a chemical agent: Contact time and concentration of agent Effectiveness of an antimicrobial agent is determined by the size of the zone of inhibition Zone of inhibition - Clear area around antimicrobic disc where there is no bacterial growth Size of the zone of inhibition depends on Diffusion rate of the chemical agent into the media Concentration of the bacteria Type of growth medium Experiment 19- Antimicrobic Sensitivity Testing Antibiotic - Chemical substance produced by a microorganism that kills or inhibits the growth of other microorganisms Ex. Penicillin Mode of Action mechanism through which antibiotics kill or prevent the growth of pathogens Ex. Penicillin prevents proper synthesis of the cell wall, subjecting it to lysis Ribosomes (translation; protein synthesis), the cell wall and cell membrane (structural), DNA replication, and transcription are all targets of antibiotics Minimum Inhibitory Concentration - Smallest amount of agent needed to inhibit the growth of a microorganism Kirby Bauer method standard procedure used to measure antimicrobial activity ... View Full Document

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