Exam 1 Study Guide 1. Know the difference between prokaryotic and eukaryotic cells. a.

Prokaryote: A microorganism in the domain Bacteria or Archaea composed of single cells having a single chromosome but no cell nucleus or other membranebound compartments. Have nucleoid area. b. Eukaryote: An organism whose cells contain a cell nucleus with multiple chromosomes, a nuclear envelope, and membrane-bound compartments. 2. Know the different organelles. Such as nucleoid, protoplast, cytoplasm, flagella, pilli, endospore, etc. nucleoid: chromosomal region of a prokaryotic cell protoplast: 1) the living material of a cell, including the protoplasm and plasma membrane after the cell wall has been removed, or 2) a cell that had its cell wall completely or partially removed using either mechanical or enzymatic means, or 3) in general refers to a cells nucleus and the surrounding protoplasmic materials cytoplasm: a complex of chemicals and structures within a cell; in plant and animal cells excluding the nucleus flagella: long, hair-like appendages composed of protein and responsible for motion in microorganisms; found in some bacterial, protozoal, algal, and fungal cells pilli: short, hair-like structures used by bacterial cells for attachment capsule: layer around the cell, but not found in all prokaryotic cells, has great capacity to retain water metachromatic granules: stored phosphate regions (energy storage sites) that occur in some bacteria; they alter the color of particular stainsmethylene blue looks pink endospore: an extremely resistant dormant cell produced by some grampositive bacterial species; specially adapted to resist adverse environmental conditions nucleus: where DNA/RNA formed cell wall: every organism has one; located outside the cell membrane; provides support; acts as a filtering mechanism and pressure vessel; tough, but flexible protoplasmic (cell) membrane: every organism has one; separates the interior of cells from

the outside environment; selectively-permeable; controls the the movement of substances in and out of cells; consists of Do Do Do Do all all all all bacteria bacteria bacteria bacteria have have have have phospholipid bilayer with embedded proteins. endospores? No capsules? No ribosomes? Yes nucleoids? Yes

Almost every part of the cell is made of protein. 3. Pasteurs experiments (pg. 12-13) a. In 1859, took part in contest sponsored by the French Academy of Sciences to prove or disprove spontaneous generation. b. Discredited spontaneous generation c. Responsible for scientific experiment rules. 4. Know what a theory, a controlled experiment and a hypothesis is. a. Steps in Experimentation/Scientific Inquiry 1) Observations ex: Pasteur observed that organisms did not appear from nonliving matter 2) Hypothesis-provisional, but testable explanation for an observed phenomenon. Scientists usually put their best hypothesis forward first. ex: Pasteur hypothesized that airborne microorganisms were responsible for purported spontaneous generation of life in meat, gravy, broths, specifically he thought that life only arises from other life. 3) Controlled experiment-has a variable (an adjustable condition) and controls (the comparative condition ex: Pasteurs variable in exp. 1: open or closed flask neck; in exp. 2: heated or unheated neck. All other factors kept the same. Pasteurs control in exp. 1: flask was left open; in exp. 2: swan-neck flask was unheated. 4) Theory-a hypothesis that has been tested and shown to be correct every time by many separate investigations, or a general set of principles supported by large amounts of experimental evidence. At some point, sufficient evidence exists to say a hypothesis is now a theory. However, theories are not written in stone. They are open to further testing and may eventually be refuted. 5. Know the concepts of evolution and biogenesis. Evolution: the process in which the genetic composition of a population changes during

successive generations as a result of natural selection acting on the genetic variation among individuals, and resulting in the development of new species Biogenesis: the process in which life forms arise from similar life forms 6. Know the difference between passive and active diffusion. Passive diffusion: the movement of a substance from where it is in a higher concentration to where it is in a lower concentration; will continue until the concentration gradient has been eliminated Active transport: the movement of a substance from area of low concentration to area of high concentration by proteins or pumps in the cell membrane; referred to as moving the material against the concentration gradient. Requires energy. One type of energy is chemical; example is adenosine triphosphate (ATP). 7. Know the steps in metabolism for microorganisms. Metabolism-all biochemical reactions that take place in a cell 1) Digestion-taking large molecules and breaking them down into smaller molecules using hydrolysis (NEEDS WATER). Most of the time this happens outside of the cell. To do that the microorganism has to secrete enzymes called exo-enzymes. Exo-enzymes contribute to the virulence (killing power) of some bacteria. 2) Once large molecules are broken down they need to be taken inside the cell. This can be done in two ways: 1. active transport (requires energy-ATP) -most nutrients enter the cell this way 2: passive transport 3) Involves taking small molecules and reducing them down into more oxidized molecules. (ie. glucose into pyruvate and acetate). Whenever this happens, electrons and protons are exchanged and co-enzymes must be secreted to calm them down. The co-enzymes are: NAD and FAD. At this point there is some energy, but not much. Glycolysis nets only 2 ATP. 4) Complete oxidation of the nutrients for energy (ATP) for our cells which involves the Krebs cycle and the cytochrome system (electron transport system). Anabolism: takes energy and builds/assembles nutrients Catabolism: breakdown of nutrients for energy; when the metabolic process nets energy for the cells; provides the energy for anabolism 7. Two types of metabolism: 1) photosynthesis 2) chemoautosynthesis (one we are concerned with) making chemicals to get ATP (energy). Chemotropic metabolism involves complete oxidation of glucose.

Steps: GlycolysisKrebs cycleCytochrome system (aerobic) (anaerobic) 2 ATPs 2 ATPs 34 ATPs Fermentation: when glycolysis doesnt go on to the Krebs cycle. Not a bad thing. (fermenting bacteria dont go past glycolysis) Anaerobes stop just prior to the Krebs cycle. Organisms that go through the Krebs cycle are aerobes and into the cytochrome system which means they have more energy, grow faster, etc. Cytochrome system gives off water and oxygen (6 O2s and 12 H2Os) Metabolic pathway: Intracellular enzymes use/act upon substrates in a coordinated sequence -every substrate does a job. If metabolic pathway shuts down, microbe dies. Increasing the concentration of the substrate, increases the activity of the enzyme. 8. Enzymes: Are all proteins. Responsible for CALMING DOWN the electrons. Some are always inside the cell- constitutive enzymes. Some are adaptive (inducible) enzymes-only there when you need them. Co-enzyme: increase the rate at which a reaction proceeds; have a protein part and an organic part Prosthetic enzymes: have a protein part and an inorganic part (ie. calcium, manganese, magnesium). The inorganic parts are good. Chemical inhibition: Bad things (inorganics) can also attach to enzymes, like arsenic or lead which can slow down or stop the metabolic pathway by rendering the enzyme nonfunctional. Denatured enzyme: inactive enzyme that has reached its maximum activity. A denatured enzyme will stop the metabolic pathway. Denatured enzyme can occur if temperature rises above the optimum level. Vitamins-some enzymes need these as activators Factors that affect enzymes: 1) temperature. Higher temperature=increased enzymatic activity 2) pH Microorganisms store energy in nucleotides. The nucleotide is ATP (adenosine triphosphate). 8. ATP and ADP. a. ATP (Adenosine triphosphate)-A molecule in cells that provides most of the energy for metabolism. Is a nucleotide (not an enzyme). A major share of prokaryotic functions depends on a continual supply of ATP. Very unstable. b. ADP (Adenosine disphosphate)-A molecule in cells that is the product of ATP hydrolysis. Produce by the breaking of the last phosphate group of the ATP. 9. Know the electron acceptors.

Electron acceptor: a molecule that receives or accepts electrons from another molecule during a redox reaction. An electron acceptor is an oxidizing agent and is itself reduced during the process of redox reaction. Examples: oxygen, nitrate, iron (III) manganese (IV), sulfate, carbon dioxide In aerobic respiration (uses electron transport system), oxygen is the final electron acceptor. In anaerobic respiration (uses electron transport system), inorganic substances other than oxygen, such as nitrate ions or sulfate ions serve as the final electron acceptors. In fermentation, organic compounds serve as the final electron acceptor. 10. Microscopes and care of microscopes. 11. Dyeschromophores and auxchromes. Chromophore: the part of a molecule responsible for its color Auxochrome: group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light. 11. Know nitrogens, heterotrophic, autotrophic. Heterotrophic bacteria: cant synthesize their own energy-must consume organic matter for energy from the environment; . Autotrophic bacteria: can synthesize their food for energy from light or inorganic chemical substances such as nitrogen or sulfur(chemosynthesis)); aerobic (requires oxygen) 12. Gram stain procedure: a differential staining procedure used to identify bacterial cells as gram-positive or gram-negative. Step 1: Apply media to the slide. Step 2: Fix media to slide with water and let dry. (All 3 coagulate the protein which is in the cell wall of the bacteria.) Step 3: Flood slide with crystal violet. Leave on for approx. one minute. Step 4: Rinse slide with water. Step 5: Flood slide with iodine (the mordant). Leave on for approx. one minute. Note: Mordant: something that will fix the material to the substance being stained. Step 6: Rinse slide with water. Step 7: Flood slide with acetone alcohol. (Most important step.) Leave on for 15-30 seconds. Alcohol is the decolorizer. If cell is gram-positive it will adhere to the crystal violet. If cell is gramnegative the alcohol will leach out the crystal violet so it will become colorless. If you dont leave the acetone alcohol on long enough you can force everything to be grampositive. If you leave it on too long you can force all of the crystal violet to be leached out and everything will be gramnegative. Step 8: Rinse slide with water. Step 9: Flood slide with safranin (counterstain).

Step 10: Rinse with water. Gram-positive: a bacterial cell that stains purple/blue after Gram-staining (retains crystal violet) example: gram positive cocci Gram negative: a bacterial cell that stains red/pink after Gram-staining (release crystal violet, picks up safranin) example: gram negative rod How do we quality control our gram stains? With every batch you will use a KNOWN gram-positive and gram-negative. Can buy these. 12. Acid Fast Stain-really good stain because everything is divided into two categories: 1) Acid Fast Positive-retain the primary stain which is carbofusian (pink color) -only one genus of organism that is acid fast positive-mycobacterium. Mycobacterium is 1/2 fungus and 1/2 bacteria. One species in the genus mycobacterium, tuberculosis, is a pathogen. Tuberculosis takes 6-8 weeks to grow in the lab, but the acid fast stain can give a quicker presumptive positive. If test shows acid fast negative, IT IS NOT tuberculosis. 2) Acid Fast negative-lets go of the carbofusian (goes colorless), instead holds onto the secondary stain, methylene blue Procedure: Flood with carbofusian. Keep on for 3-5 minutes. Rinse with water. Flood with acid alcohol for ___ minutes. Rinse with water. Counterstain with methylene blue. 12. Other stains: 1) Capsular stains 2) Flagullar stains 13. What microorganisms require for growth. 1) Nutrients (food) 2) Temperature- in general, the higher the temp., the more easily microorganisms grow 3) pH most prefer a neutral pH 4) water- in general, more moisture means more microorganisms 5) elements- require certain elements in the air; nitrogen and oxygen are both necessary 14. Mesophile: an organism that grows in temperature ranges of 20C to 40C. Psychrophile: an organism that lives at cold temperature ranges of 0C to 20C. Thermophile: an organism that lives at high temperature ranges of 40C to 90C. 15. The growth curve. 1) Lag phase: no cell division; population numbers remain stable; preparation for division. 2) Logarithmic phase (log phase): active stage of exponential growth; numbers double with each generation; disease symptoms are usually seen in this phase 3) Stationary phase: reproductive and death rates are roughly equal

4) Decline phase (exponential death phase): cell death becomes substantial; happens if nutrients in the external environment; cell death can be avoided by endospore formation. 16. How to calculate a growth population that is going through binary fission. a. Binary fission: an asexual process in prokaryotic cells by which a cell divides to form two new cells while maintaining genetic constancy b. Example: Organism generation through binary fission 1, 2, 4, 8, 16, 32, 64 17. Know what constitutes sterility and different ways of achieving sterility. a. Sterility: complete freedom from living microorganisms, spores, and viruses. Freezing, boiling and pasteurization doesnt sterilize. b. How to achieve sterility: 1) autoclaves-use heated steam; need to have proper temp. time, and pressure. 2) incineration 3) flaming 4) dry heat oven-takes a long time 5) chemical sterilization (ie. ozone, bleach, formaldehyde) 6) Ethylene oxide gas 7) radiation 18. Disinfectant: a chemical used to kill or inhibit pathogenic microorgansims on a lifeless object such as a tabletop. Antiseptic: a chemical used to reduce or kill pathogenic microorganisms on a living object, such as the surface of the human body. 20. Phenol: A chemical compound that has one or more hydroxyl groups attached to a benzene ring and derivatives are used as an antiseptic or disinfectant; also called carbolic acid. 21. Phenol coefficient (PC): A number that indicates the effectiveness of an antiseptic or disinfectant compared to phenol. 21. Know the autoclave and spores that we use to monitor the autoclave. a. Autoclave tested with Geobacillus stearothermophilus one of the most heat-tolerant species of bacteria 22. Know how we sterilize plastics and other things that cant be exposed to high temperatures. a. Ethylene oxide 22. Selective media: growth media that contain ingredients to inhibit certain microorganisms while encouraging the growth of others. (ie. gram positive and gram negative bacteria) Differential media: growth media in which different species of microorganisms can be distinguished visually. (ie. blood agar-differential for hemolysis) One-purpose media (nonselective media): both selective and differential

23.

Terms: diplococci: a pair of spherical-shaped prokaryotic cells staphylococci: 1) an arrangement of bacterial cells characterized by spheres in a grapelike cluster 2) a genus of facultatively anaerobic, nonmotile, nonsporeforming, gram-pos. spheres in clusters hemolysis: the destruction of red blood cells; hemo means red blood cells, lysis means breaking apart. alpha hemolysis: when bacterial species plated on blood agar cause a partial destruction of red blood cells; seen by an olive green color in the agar around colonies SOME RED BLOOD CELLS AROUND THE STREP COLONY beta hemolysis: when bacterial species plated on blood agar COMPLETELY destroy the red blood cells; seen by a clearing in the agar around the colonies NO RED BLOOD CELLS AROUND THE STREP COLONY gamma hemolysis: when bacterial species dont have any effect on red blood cells; NORMAL RED BLOOD CELLS AROUND THE STREP COLONY aerobic respiration: process of transforming energy to ATP in which the final electron acceptor in the electron transport chain is oxygen gas (O2). anaerobic respiration: the production of ATP where the final electron acceptor is an inorganic molecule other than oxygen gas (O2); examples include nitrate and sulfate facultative: referring to an organism that grows in the presence or absence of oxygen gas (O2) microaerophilic: organisms that grow best in oxygen-reduced environments. Dr. Rebecca Lancefield: CLASSIFIED STREPTOCOCCI BASED UPON THEIR ANAGENIC DETERMINANTS. 1) knew it was hard to identify streps because there are so many types (cat, dog, etc...). Also normal streps (inside your mouth) 2) devised an antigen/antibody technique. Took known antibodies and devised a technique for identification by exposing the antibodies to the antigens 3) whenever you are invaded by an antigen, you will produce antibodies 4) developed/harvested antiseries (antibodies), A,B,C,D,E... by injecting animals/ humans with antigens so that they produced antibodies REMEMBER: streps are gram positive cocci