EXAM UNIT THREE “Microbial Physiology & Growth”

1. EXAM UNIT THREE“Microbial Physiology & Growth” Microbial Physiology Microbial Genetics Microbial Growth

2. MICROBIAL PHYSIOLOGY • Study of microbial metabolic processes. • Microbes require essential elements (carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur) as well as various trace elements. • Nutrients are chemical compounds needed to sustain life. Essential nutrients are nutrients that are critical for life but cannot be synthesized by an organism. • All microbes must have both an energy source and a carbon source.

3. Phototrophs (“light feeders”) use light as an energy source. They use photosynthesis to convert light energy to chemical energy. Chemotrophs (“chemical feeders”) break chemical bonds to obtain energy. MICROBIAL ENERGY SOURCES

4. MICROBIAL CARBON SOURCES • Autotrophs (“self feeders”) use carbon dioxide (CO2) as their sole source of carbon. • Heterotrophs (“chemical eaters”) Use organic compounds other than CO2 as their carbon source.

5. Photoautotrophs (“light self feeders”) Use light as an energy source and CO2 as a carbon source. Ex. Plants, Algae, Cyanobacteria. Photoheterotrophs (“light different feeders”) Use light as an energy source and organic compounds other than CO2 as a carbon source. Ex. Purple and green nonsulfur bacteria. Chemoautotrophs (“chemical self feeders”) Use chemicals as an energy source and CO2 as a carbon source. Ex. Nitrifying, iron, and sulfur bacteria. Chemoheterotrophs (“chemical different feeders”) Use chemicals as an energy source and chemicals other than CO2 as a carbon source. Ex. All animals, protozoa, fungi, and most bacteria. Putting it all Together

6. METABOLISM • Metabolism is the sum of all chemical reactions in the cell. • Metabolic processes can be placed into two basic categories: Catabolism and Anabolism. • Catabolic reactions involve the breakdown of complex molecules. The breaking of chemical bonds releases stored energy. Ex. Cellular Respiration (the breakdown of glucose). • Anabolic reactions involve the synthesis of complex molecules. Energy is stored as chemical bonds form. Ex. Photosynthesis.

7. Metabolic Enzymes • Metabolic reactions can only be carried out if specific enzymes are present. • The genome (genetic code) of the bacterium codes for the specific enzymes used in its metabolic pathways. • Enzymes only function within specific environmental conditions. • Enzymes can be altered (denatured) by extreme pH and temperature. • The enzymes a bacterium contains determines the conditions it can live under.

8. Study of heredity: The acquisition and expression of traits. Involves both the genotype and phenotype of the individual. The Genotype is the compliment of genes (The DNA coding for traits) carried by an individual. The Phenotype is the expression of the genotype, the traits that are actually displayed. Genes direct all functions of the cell. MICROBIAL GENETICS

9. The Bacterial Genome • Most bacteria have a single chromosome consisting of a ring of naked DNA. • Some bacteria also have very small rings of DNA known as plasmids or “R” factors. These may code for resistance to antibiotics.

10. Bacterial Genetic Variation • Bacteria reproduce through binary fission. During this process, a microbe copies its genome, splits in half, and produces a clone (an exact copy). • Bacteria obtain new traits (genetic variation) through mutations, lysogenic conversion, transduction, transformation, and conjugation.

11. Mutations • Permanent change in the structure of the DNA. • Passed on to offspring. • Mutations may be beneficial, harmful, lethal, or silent (no effect). • Mutations may be spontaneous or induced.

12. Spontaneous Mutations • Do not have a known cause. • They occur by chance approximately every million DNA replications.

13. Induced Mutations • Induced mutations have a known cause. • Mutation-causing agents are known as mutagenic. • Ultra-violet radiation and nicotine are examples of mutagenic agents. • Approximately 90% of mutagenic agents are also carcinogenic (cancer-causing).

14. Lysogenic Conversion • Prophages are viruses that insert their DNA into a host bacterium. This viral DNA is then incorporated into the host genome, but is not completely read to make viruses. • This condition is called lysogeny. • The host bacterium will code for some of the viral DNA and produce new products or develop new traits.

15. Transduction • In transduction, a defective bacteriophage containing bacterial DNA infects a host cell. • The infected bacterial cell obtains new bacterial DNA, and therefore new variation.

16. Transformation • In transformation, a bacterial cell picks up fragments of DNA from its environment and incorporates them into its genome. • This is very rare and generally occurs between closely related species.

17. Conjugation • In conjugation, a bacterial cell replicates its plasmids, attaches to another bacteria cell using pili (small, hairlike structures), forms a conjugation tube, or sex pilus, and transfers the DNA.

18. Genetic Engineering • The transfer of genes (DNA segments) to other cells to produce specific products. • Human genes are often transferred into other cells such as bacteria and yeasts. • These products are typically protein-based compounds such as human growth hormone, insulin, antibiotics, enzymes, etc. • Plasmids are frequently used as vectors to transfer the genes to the host cell.

19. Examples of Transgenic Biotechnology • This is gene splicing between species. • E. coli are used to produce many products such as human insulin. • Goats are used to produce milk that has a human protein to prevent blood clots. This protein is then isolated from the milk. The human gene is inserted into the goat embryo (zygote).

20. Gene Therapy • Involves the insertion of genes into cells to correct genetic abnormalities. • First conducted in the U.S. in the 1990s. • Viruses are commonly used as vectors (ex. Adenoviruses, herpesviruses). • Often genes are inserted into liver cells that will produce a beneficial compound and release it into the bloodstream. • To date, most gene therapy attempts have failed.

21. MICROBIAL GROWTH • Many factors affect microbial growth such as nutrient availability, moisture, temperature, pH, osmotic pressure, and oxygen levels. • By manipulating these factors, we can encourage or discourage microbial growth.

22. Nutrient Availability • All living things must continually uptake nutrients to survive. • Nutrients are vital to growth, repair, maintenance, and reproduction. • All microbes must have a carbon source, an energy source, and various vitamins and minerals. • By regulating the essential nutrients required by a microbe, we can regulate its growth. • If even a single essential nutrient is missing, the organism cannot sustain life.

23. Moisture • All living things must have water to survive. • There is a wide range of variation in the water requirements of organisms. • Some microbes are very resistant to desiccation (drying) out and can survive extreme conditions. • The most resistant microbial structures are bacterial spores and protozoan cysts. They can survive for extended periods in the complete absence of moisture.

24. Temperature • Every microorganism has an optimal growth temperature, a minimal growth temperature, and a maximum growth temperature. • The optimal temperature range of microorganisms varies widely. • Microorganisms are typically placed in one of three temperature ranges: Thermophiles, Mesophiles, & Psychrophiles.

25. Thermophiles • Optimal growth range of 50 - 60 degrees Celsius (122 – 140 F) • Max. temp. = 113 C (147 F) • Found in compost heaps, silage, hot springs, and hydrothermal vents.

26. Mesophiles • Optimal growth range of 20 - 40 degrees Celsius (68 – 104 F) • Max. temp. = 45 C (113 F) • Human pathogens are mesophiles and typically thrive at body temp (37 C) • Boiling at 100 C (212 F) will kill most pathogens but not bacterial spores.

27. Psychrophiles • Optimal growth range of 10 - 20 degrees Celsius (50 – 68 F) • Max. temp. = 30 C (86 F) • Refrigeration is typically 5 C (41 F) and some organisms of decay are psychrophiles. • Psychroduric microbes can survive being frozen.

28. pH: The potential of Hydrogen • pH is a measure of the hydrogen and hydroxide ion concentration of a solution. • The pH scale runs from o to 14. • A pH of 7 is neutral. • Pure water has a neutral pH • Solutions with a pH below 7 are acidic. • Solutions with a pH above 7 are basic (alkaline). • Human blood and body tissue has a pH range of 7.2 to 7.4. This is optimal pH for most pathogens. • Most bacteria can survive at a pH range of 6.5 to 7.5.

30. Acidophiles • Acidophiles are microbes capable of surviving in very acidic conditions. • The pathogen, Helicobacter pylori is an acidophile that can survive in the human stomach and is known to cause ulcers. • Many fungi prefer acidic environments.

31. Alkaliphiles • Alkaliphiles are microbes capable of surviving in very alkaline (basic) conditions. • The bacterium Vibrio cholerae is the only human pathogen that grows well at a pH above 8. It causes cholera, a very severe diarrheal disease.

32. Tonicity and Osmosis • A solution is composed of a solute dissolved within a solvent. For example salt water. Salt is the solute and water is the solvent. • Tonicity refers to fluid tension on the cell membrane resulting from differences in solute concentrations in and out of the cell. • Osmosis is the diffusion of water across a cell membrane resulting from solute differences on either side of a membrane. • There are three basic forms of solutions relative to tonicity: Isotonic solutions, Hypotonic solutions, and Hypertonic solutions.

33. Isotonic Solutions • “Equal solute” solution in which the solute concentration is equal on both sides of the cell membrane. • There is equal tension on both sides of the membrane and water does not flow in or out of the cell (no osmotic flow). • This is a balanced state in which the cell will neither shrink or swell.

34. Hypotonic Solutions • “Low solute” solution in which there is less solute outside the cell. • Water enters the cell in an effort to equalize the solute concentrations and the internal pressure of the cell increases. • In cells without a cell wall, the cell will typically burst. In bacteria, the cell typically will not burst due to the cell wall. However, the increased internal pressure is detrimental to the cell and inhibits cellular growth and reproduction.

35. Hypertonic solutions • “High solute” solution in which there is more solute outside the cell. • Water leaves the cell in an effort to dilute the solute outside the cell. The cell crenates (shrivels) as it loses water. • In bacteria, the cell shrinks away from the cell wall. This inhibits or kills bacterial cells as they become dessicated. • The sugar solutions in jams and jellies, and salty brines are hypertonic and inhibit most bacterial growth by dessicating the microbes.

37. Oxygen Requirements • The term “obligate” means “must have”, the term “facultative” means “can tolerate” • “Aerobic” means “with oxygen”, “anaerobic” means “without oxygen”. • Most bacteria are facultative anaerobes. They can live without oxygen, but prefer to have it. • Some bacteria are obligate anaerobes and must have an oxygen-free environment. Oxygen will kill them, an example is Clostridium perfringens. • A small number of bacteria are “microaerophilic” and prefer environments with low oxygen levels. An example is E. coli.

38. Definition of Terms Related to Inhibiting Microbial Growth • Sterilization: The destruction of all microbes and their products. • Disinfection: The removal of pathogens from nonliving objects. • Disinfectants: Chemicals used for disinfection. • Antiseptics: Chemicals used to eliminate pathogens from living tissues. • Sanitization: Reduction of microbial populations to a safe level. • Germicidal: “germ” “to kill”, chemicals used to kill microbes. A.K.A: bactericidal, fungicidal, etc.

39. Physical Methods of Inhibiting In Vitro Microorganism Growth • In vitro growth is growth outside an organism, for example in a petri dish or on a cutting board. • There are numerous physical methods used to kill microbes in settings such as hospitals, clinics, laboratories and restaurants. • These include heat, heat and pressure, dessication, radiation, sonic disruption, and filtration.

40. Using Heat to Inhibit Growth • Most common and cost-effective method for heat tolerant objects. • Two factors determine the effectiveness of heat sterilization: Temperature and time of exposure. • The thermal death point (TDP) and the thermal death time (TDT) must be reached to kill the specific microbes. • Any insulating material around the microbes must also be accounted for. Pus, fecal material, blood, etc. can act as a protective barrier and must be removed or accounted for. The presence of heat-resistant spores must also be considered. • Two types of heat sterilization are used: dry and moist.

41. Dry Heat Sterilization • When dry heat is used, a temp. of 160 to 165 C (320 – 329 F) must be reached for 2 hours, or a temp. of 170 – 180 C (338 – 356 F) for 1 hour. • The heat must penetrate the material completely to be effective.

42. Moist Heat Sterilization • Much more effective than dry heat. • Effective at a lower temp. than dry heat. • Moist heat denatures cellular proteins such as enzymes. • Boiling (100 C, 212 F) for 30 minutes will kill most vegetative cells. • Boiling will typically not kill endospores such as those associated with botulism, tetanus, anthrax, and gangrene. Hepatitis viruses also can survive boiling. • Moist heat sterilization under pressure is necessary to effectively kill all pathogens. Autoclaves or pressure cookers are designed for this. • Increased pressure allows increased temps. to be reached. Autoclaving at 15 psi at 121.5 C (250.7 F) for 20 minutes kills all unprotected pathogens including spores and viruses.

43. Effect of Cold Temperatures • Most microbes are not killed by cold temperatures or freezing. • Cold temperatures do inhibit microbial metabolism and slow freezing can kill some microbes due to crystal formation. • Rapid freezing (ex. Liquid nitrogen) places bacteria and some other cells in a state of suspended animation. • Foods should never be thawed and refrozen. The freezing can preserve the bacteria and spores that are produced during thawing. Re-thawing can then result in rapid deterioration or toxin production.

44. Dessication • Drying prevents bacteria from reproducing. However, many remain viable and can resume growth when moisture is present. • Lyophilization (freeze-drying) is used to preserve food, microbes, antibiotics, toxins, antisera, and antitoxins. This technique combines drying with freezing within a vacuum chamber. • Since dried microbes are dormant but still viable, great care should be used in cleaning areas contaminated with dried pathogen-containing materials such as feces, blood, pus, etc. • Dried pathogens that are inhaled or land on a wound or burn may colonize the area.

45. Filtration • Numerous types of filters are used to trap microbes and microscopic particles. These are commonly called micropore filters. • Materials such as diatomaceous earth, porcelain, cellulose, and various synthetics are used. • HEPA (high-efficiency particulate air filters) are common used in hospitals. • Early scientists frequently used cotton to prevent microbial contamination of biological materials.

46. Chemical Methods of Inhibiting In Vitro Microorganism Growth • Chemical Disinfectants and antiseptics are common used to kill microbes and inhibit microbial growth. • Their effectiveness is influenced by many factors such as organic material present, the type and load of microbes, the concentration of the agent, the contact time, surface characteristics (smooth or porous), temperature, and pH. • The most effective antiseptic or disinfectant should be selected for the specific purpose, environment, and pathogens present.

47. Characteristics of Ideal Antimicrobial Agents • Wide spectrum • Fast-acting • Nontoxic to humans and noncorrosive • Residual effect • Water soluble • Stable • Low odor • Some examples are alcohols, iodine, chlorine, and phenol (carbolic acid).

48. Controlling Microbial Growth in Vivo • Chemotherapeutic agents are any drugs used to treat any condition or disease. • Chemotherapeutic agents that are used to treat infectious diseases are called antimicrobial agents. • These are classified as antibacterial, antifungal, antiprotozoal, and antiviral agents.

49. Antibiotics • Produced primarily by soil-borne molds and bacteria. • Penicillin and cephalosporins are examples of antibiotics produced by molds. • Bacitracin and erythromycin are examples of antibiotics produced by bacteria. • Semisynthetic antibiotics are chemically modified antibiotics such as ampicillin (synthetic penicillin).

50. Ideal Qualities of Antimicrobial Agents • Kill or inhibit pathogens. • Harmless to host. • Will not cause allergic reactions. • Stable when stored. • Have residual effect in tissues of host. • Kill pathogens quickly before they can mutate and develop resistance. There are no perfect antimicrobial agents, most have some side effects, produce allergic reactions, or allow resistance to develop.