Chapter8 microfarid


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Chapter8 microfarid

  1. 1. MICROBIOLOGY for the Health Sciences ‫اعداد : أ. فريد ابو العمرين‬ 1 Chapter 8,9. Controlling Microbial Growth
  2. 2. Chapter 8. Controlling Microbial Growth in Vitro
  3. 3. Chapter 8 Outline 1. Introduction 2. Factors that Affect Microbial Growth 3. Encouraging the Growth of Microbes in Vitro 4. Inhibiting the Growth of Microbes in Vitro 3
  4. 4. Factors That Affect Microbial Growth • Availability of Nutrients – – – All living organisms require nutrients to sustain life. Nutrients are energy sources. Organisms obtain energy by breaking chemical bonds. • Moisture – – – Water is essential for life. It is needed to carry out normal metabolic processes. Certain microbial stages (e.g., bacterial endospores and protozoal cysts) can survive a drying process. 4
  5. 5. Temperature Every organism has an optimum growth temperature.  Thermophiles are microorganisms that grow best at high temperatures.  Mesophiles are microbes that grow best at moderate temperatures (e.g., 37o C).  Psychrophiles prefer cold temperatures (like deep ocean water). 5
  6. 6. pH • “pH” refers to the acidity or alkalinity of a solution. • Most microorganisms prefer a neutral or slightly alkaline growth medium (pH 7.0 - 7.4) • Acidophiles prefer a pH of 2 to 5 • Alkaliphiles prefer a pH > 8.5  pH range 1 6 Acid 7 Neutral 14 Alkaline
  7. 7. Osmotic Pressure and Salinity   7 Osmotic pressure is the pressure that is exerted on a cell membrane by solutions both inside and outside the cell. Osmosis is the movement of a solvent, through a permeable membrane, from a lower concentration of solutes (dissolved substances) to a higher concentration of solutes.
  8. 8. Osmotic Pressure and Salinity  When the concentration of solutes in the external environment of a cell is greater than that of solutes inside the cell, the solution in which the cell is suspended is said to be hypertonic.  When the concentration of solutes outside a cell is less than that of solutes inside a cell, the solution in which the cell is suspended is said to be hypotonic.  A solution is said to be isotonic when the concentration of solutes outside a cell equals the concentration of solutes inside the cell. 8
  9. 9. Factors That Affect Microbial Growth, cont.   In hypertonic solution a cell shrink If a bacterial cell is placed into a hypotonic solution, Cells swell up, and sometimes burst.   9 In isotonic soln. the cell has normal turgor. In the case of erythrocytes, this bursting is called hemolysis
  10. 10. Changes in Osmotic Pressure 10
  11. 11. Factors That Affect Microbial Growth, cont. – – 11 Organisms that prefer to live in salty environments are called halophilic organisms. Microbes that can survive in high atmospheric pressure are know as piezophiles.
  12. 12. Gaseous Atmosphere  Microorganisms vary with respect to the type of gaseous atmosphere that they require.  Obligate aerobes prefer the same atmosphere that humans do (~20-21% O2 and 78-79% N2, other gases < 1%).  Microaerophiles require reduced concentrations of oxygen (~5% O2).    Obligate anaerobes are killed by the presence of oxygen. Facultative grow in presence or absence of oxygen Capnophiles require increased concentrations of CO2 (5-10% CO2). 12
  13. 13. Encouraging the Growth of Microbes in Vitro 13
  14. 14. Bacterial Growth • Bacterial growth as an increase in the number of organisms rather than an increase in their size. • Bacteria divide by binary fission (one cell divides to become two cells) when they reach their optimum size. • Binary fission continues through many generations until a colony is produced on solid culture medium. • Binary fission continues for as long as there is a sufficient supply of nutrients, water, and space. • The time it takes for one cell to become two cells is called the generation time (e.g., E. coli = 20 minutes). 14
  15. 15. Binary fission of staphylococci. 15
  16. 16. Culture Media • Media are used in microbiology labs to culture (i.e., grow) bacteria. • Culture media can be liquid or solid. • An enriched medium is a broth or solid containing a rich supply of special nutrients that promote the growth of fastidious organisms. • A selective medium has added inhibitors that discourage growth of certain organisms while allowing the growth of a desired organism. 16
  17. 17. Culture Media • A differential medium permits the differentiation of organisms that grow on the medium. • The various categories of media are not mutually exclusive; e.g., blood agar is enriched and differential. • Thioglycollate broth (THIO) is a popular liquid medium in bacteriology labs; it supports the growth of all categories of bacteria from obligate aerobes to obligate anaerobes. – There is a concentration gradient of dissolved oxygen in the tube; organisms grow only in that part of the broth where the oxygen concentration meets their needs. 17
  18. 18. A Thioglycollate (THIO) Broth Tube 18
  19. 19. Bacterial colonies on MacConkey agar (a selective & differential medium) 19 S. aureus on mannitolsalt agar (a selective & differential medium)
  20. 20. Colonies of a β-hemolytic Streptococcus species on a blood agar plate (in this case, the blood agar is both enriched and differential) Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 20
  21. 21. Inoculation of Culture Media • Culture media are inoculated with clinical specimens (i.e., specimens collected from patients with a suspected infectious disease). • Inoculation involves adding a portion of a specimen to the medium. • Inoculation is accomplished using a sterile inoculating 21 loop.
  22. 22. Importance of Using “Aseptic Technique”  Aseptic technique is practiced to prevent • (a) microbiology professionals from becoming infected, • (b) contamination of their work environment. • (c) contamination of clinical specimens, cultures, and subcultures. 22
  23. 23. Incubation • After media are inoculated, they must be placed into an incubator which will maintain the appropriate atmosphere, temperature, and moisture level; the process is known as incubation. • 3 types of incubators are used in clinical microbiology laboratories: – – – 23 A CO2 incubator (contains 5-10% CO2) A non-CO2 incubator (contains room air) An anaerobic incubator (the atmosphere is devoid of oxygen)
  24. 24. Bacterial Population Growth Curve 24
  25. 25. A population growth curve of living organisms.  lag phase during which the bacteria absorb nutrients, synthesize enzymes, and prepare for cell division. • The bacteria do not increase in number during  Logarithmic growth phase (log phase) • the bacteria multiply so rapidly that the number of organisms doubles with each generation time. • Growth rate is the greatest during the log phase. 25
  26. 26. A population growth curve of living organisms.   Stationary phase   Death phase or decline phase  The microorganisms then die at a rapid rate. 26 As the nutrients are used up and the concentration of toxic waste products build up, the rate of division slows, such that the number of bacteria that are dividing equals the number that are dying. As overcrowding occurs, the concentration of toxic waste products continues to increase and the nutrient supply decreases.
  27. 27. Inhibiting the Growth of Microbes in Vitro 27
  28. 28. Definition of Terms  In Vitro: In an artificial environment, as in a laboratory setting; used in reference to what occurs outside an organism.  In Vivo: Used in reference to what occurs within a living organism. • Sterilization is the complete destruction of all microbes, including cells, spores, and viruses. • Disinfection is the destruction or removal of pathogens from nonliving objects. 28
  29. 29. Definition of Terms, cont. • The suffix –cide or –cidal refers to “killing.”  Germicidal agents, biocidal agents, and microbicidal agents are chemicals that kill microbes. • Bactericidal agents are chemicals that specifically kill bacteria. • Sporicidal agents kill bacterial endospores. – – – 29 Fungicidal agents kill fungi, including fungal spores. Algicidal agents kill algae. Viricidal agents destroy viruses.
  30. 30. Definition of Terms (cont.) • A static agent is a drug or chemical that inhibits growth and reproduction of microbes. • A bacteriostatic agent is one that specifically inhibits the metabolism and reproduction of bacteria. • Lyophilization is a process that combines dehydration (drying) and freezing. • This process is widely used in industry to preserve foods, antibiotics, microorganisms, and other biologic materials. • Sepsis refers to the presence of pathogens in blood or tissues, whereas asepsis means the absence of pathogens. 30 Antisepsis is the prevention of infection.
  31. 31. Physical Methods to Inhibit Microbial Growth • Heat  Heat is the most practical, efficient, and inexpensive method of sterilization of those inanimate objects and materials that can withstand high temperatures.  Because of these advantages, it is the means most frequently used. • Types of Heat – Dry heat – e.g., oven, electrical incinerator, or flame – Moist heat – boiling or use of an autoclave 31
  32. 32. Dry Heat Sterilization  Dry Heat. Dry-heat oven provides effective sterilization of metals, glassware, some powders, oils, and waxes.  These items must be at: 1. 160°C to 165°C for 2 hours 2. 170°C to 180°C for 1 hour.  The effectiveness of dry-heat sterilization depends on how deeply the heat penetrates throughout the material, and the items to be baked must be positioned so that the hot air circulates freely among them.  Incineration (burning) is an effective means of destroying contaminated disposable materials. 32
  33. 33. The autoclave   Increased pressure raises the temperature above the temperature of boiling water (above 100oC) and forces steam into materials being sterilized.  Autoclaving at: 1. a pressure of 15 psi 2. 121 oC 3. for 20 minutes.  33 A large metal pressure machine that uses steam under pressure to completely destroy all microbial life. To destroys vegetative microorganisms, bacterial endospores, and viruses.
  34. 34. The autoclave quality-control • Pressure-sensitive autoclave tape and commercially available strips or solutions containing bacterial spores can be used as quality-control measures to ensure that autoclaves are functioning properly. • After autoclaving, the spores are tested to see whether they were killed. 34
  35. 35. Physical Methods to Inhibit Microbial Growth • Cold; most microorganisms are not killed, but their metabolic activities are slowed. • Desiccation; For many centuries, foods have been preserved by drying. Many dried microorganisms remain viable, but they cannot reproduce. • Radiation; an ultra-violet (UV) lamp is useful for reducing the number of microbes in the air. • Ultrasonic waves; used in hospitals and medical and dental clinics to clean equipment. • Filters; used to separate cells/microbes from liquids or gases. 35 Gaseous atmosphere; can be altered to inhibit growth.
  36. 36. Radiation  UV lamp is useful for reducing the number of microorganisms in the air.  Sterility may also be maintained by having a UV lamp placed in a hood or cabinet containing instruments, paper and cloth equipment, liquid, and other inanimate articles.  Many biologic materials, such as sera, antisera, toxins, and vaccines, are sterilized with UV rays. 36
  37. 37. X-rays and gamma and beta rays  Radiation may be lethal or cause mutations in microorganisms and tissue cells because they damage DNA and proteins within those cells.  Studies performed in radiation research laboratories have demonstrated that these radiations can be used for the prevention of food spoilage, sterilization of heat-sensitive surgical equipment, preparation of vaccines, and treatment of some chronic diseases such as cancer, all of which are very practical applications for laboratory research. 37
  38. 38. Ultrasonic Waves  In hospitals, medical clinics, and dental clinics, ultrasonic waves are a frequently used means of cleaning delicate equipment.  Ultrasonic cleaners consist of tanks filled with liquid solvent (usually water); the short sound waves are then passed through the liquid.  The sound waves mechanically dislodge organic debris on instruments and glassware. 38
  39. 39. Filtration  Filters of various pore sizes are used to filter or separate cells, larger viruses, bacteria, and certain other microorganisms from the liquids or gases in which they are suspended.  Filters with tiny pore sizes (called micropore filters) are used in laboratories to filter bacteria and viruses out of liquids.  The variety of filters is large and includes sintered glass (in which uniform particles of glass are fused), plastic films, unglazed porcelain, asbestos, diatomaceous earth, and cellulose membrane filters.  Small quantities of liquid can be filtered through a filter-containing syringe, but large quantities require larger apparatuses.  Microbes, even those as small as viruses, can be removed from liquids using filters having appropriate pore sizes. 39
  40. 40. Gaseous Atmosphere  In limited situations, it is possible to inhibit growth of microorganisms by altering the atmosphere in which they are located. Because aerobes and microaerophiles require oxygen, they can be killed by placing them into an atmosphere devoid of oxygen or by removing oxygen from the environment in which they are living.  Conversely, obligate anaerobes can be killed by placing them into an atmosphere containing oxygen or by adding oxygen to the environment in which they are living.  For instance, wounds likely to contain anaerobes are lanced (opened) to expose them to oxygen. Another example is gas gangrene, a deep wound infection that causes rapid destruction of tissues.  Gas gangrene is caused by various anaerobes in the genus Clostridium. 40
  41. 41. Disinfection • Chemical disinfection refers to the use of chemical agents to inhibit the growth of pathogens, either temporarily or permanently. • Disinfectants are affected by: 41 – – – – – – – Prior cleaning of the object or surface The organic load (e.g., feces, blood, pus) The bioburden; types and numbers of microbes Concentration of the disinfectant Contact time Physical nature of the object being disinfected Temperature and pH
  42. 42. 42
  43. 43. 43
  44. 44. Using Chemical Agents to Inhibit Microbial Growth, cont. Characteristics of an ideal chemical antimicrobial agent: • Should have a broad antimicrobial spectrum • Fast acting • Not affected by the presence of organic matter • Nontoxic to human tissues and noncorrosive • Should leave a residual 44 antimicrobial film on surface • Soluble in water and easy to apply • Inexpensive and easy to prepare • Stable as both a concentrate and a working solution • Odorless
  45. 45. Using Chemical Agents to Inhibit Microbial Growth (cont.) • Antiseptics – – May safely be used on human tissues. Reduce the number of organisms on the surface of the skin; do not penetrate pores and hair follicles. • Antiseptic soaps and scrubbing are used by healthcare personnel to remove organisms lodged in pores or folds of the skin. • Antimicrobial chemical agents that can safely be applied to skin are called antiseptics. 45
  46. 46. Chapter 9. Controlling Microbial Growth in Vivo Using Antimicrobial Agents