Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology

1. Environmental Microbiology-Laboratory Manual-prepared for Environmental Microbiology III Microorganism Growth Factors Controlling Growth Environmental Management technology Faculty of civil and environmental engineering Itb, 2010 anindrya.pahlawan@ymail.com

2. MICROBIAL GROWTH The capacity to grow, and ultimately to multiply, is one of the most fundamental characteristics of living cells (Posten & Coney).

3. GROWTH • In biophysics cells are open systems far from a thermodynamic equilibrium, exchange material and energy with their environment, and especially exhibit a large outflow of entropy. • In chemical engineering  growth is referred to as an increasing amount of biocatalyst • Mathematical growth are restricted to a couple of equations employing hyperbolic and exponential terms. • Growth is usually considered as an increase of cell material expressed in terms of mass or cell number. Ref: Posten & Coney

4. Microbial Nutrition • Why is nutrition important? • The hundreds of chemical compounds present inside a living cell are formed from nutrients. • Macronutrients : elements required in fairly large amounts • Micronutrients : metals and organic compounds needed in very small amounts This slide is taken from : MICR 300 : Microbiology, California State of University

5. Main Macronutrients • Carbon (C, 50% of dry weight) and nitrogen (N, 12% of dry weight) • Autotrophs are able to build all of their cellular organic molecules from carbon dioxide • Nitrogen mainly incorporated in proteins, nucleic acids • Most Bacteria can use NH3 and many can also use NO3- • Nitrogen fixers can utilize atmospheric nitrogen (N2) This slide is taken from : MICR 300 : Microbiology, California State of University

6. Other Macronutrients • Phosphate (P), sulfur (S), potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe) • Iron plays a major role in cellular respiration, being a key component of cytochromes and iron-sulfur proteins involved in electron transport. • Siderophores : Iron-binding agents that cells produce to obtain iron from various insoluble minerals. This slide is taken from : MICR 300 : Microbiology, California State of University

7. This slide is taken from : MICR 300 : Microbiology, California State of University

8. Need very little amount but critical to cell function.Often used as enzyme cofactors Micronutrients This slide is taken from : MICR 300 : Microbiology, California State of University

9. Growth factors This slide is taken from : MICR 300 : Microbiology, California State of University

10. Culture Media: Composition • Culture media supply the nutritional needs of microorganisms • defined medium : precise amounts of highly purified chemicals • complex medium(or undefined) : highly nutritious substances. • Inclinicalmicrobilogy, • Selective : contains compunds that selectively inhibit • Differential: contains indicator • terms that describe media used for the isolation of particular species or for comparative studies of microorganisms. This slide is taken from : MICR 300 : Microbiology, California State of University

11. Media

12. Culture Media: Physical Properties • Liquid • Bouillon or broth • Solid • Addition of a gelling agent (typically 1% agar) to liquid media • Immobilize cells, allowing them to grow and form visible, isolated masses called colonies (Figure 5.2). • Semisolid • Reduced amount of agar added • Allows motile microorganism to spread This slide is taken from : MICR 300 : Microbiology, California State of University

13. Bacterial Colonies on Solid Media P. aeruginosa (TSA) S. marcescens(Mac) S. flexneri(Mac) This slide is taken from : MICR 300 : Microbiology, California State of University

14. Laboratory Culture of Microorganisms • Microorganisms can be grown in the laboratory in culture media containing the nutrients they require. • Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique is practiced to prevent contamination by other microorganisms. This slide is taken from : MICR 300 : Microbiology, California State of University

15. Microbial growth involves an increase in the number of cells. • Growth of most microorganisms occurs by the process of binary fission Cell Growth and Binary Fission This slide is taken from : MICR 300 : Microbiology, California State of University

16. Microbial Growth Peptidoglycan layer This slide is taken from : MICR 300 : Microbiology, California State of University

17. Microbial Growth Pattern • Microbial populations show a characteristic type of growth pattern called exponential growth, which is best seen by plotting the number of cells over time on a semi- logarithmic graph. This slide is taken from : MICR 300 : Microbiology, California State of University

18. Growth Curve • Microorganisms show a characteristic growth pattern (Figure 6.8) when inoculated into a fresh culture medium. This slide is taken from : MICR 300 : Microbiology, California State of University

19. Measuring Microbial Growth • Growth is measured by the change in the number of cells over time. • Cell counts done microscopically measure the total number of cells in a population • whereas viable cell counts (plate counts) measure only the living, reproducing population. This slide is taken from : MICR 300 : Microbiology, California State of University

20. Total Cell Count

21. Viable Cell Count : Determination of Colony Forming Units

22. Environmental Effects on Bacterial Growth • Temperature • pH • Osmotic pressure • Oxygen classes This slide is taken from : MICR 300 : Microbiology, California State of University

23. Temperature and Microbial Growth • Cardinal temperatures • minimum • optimum • maximum • Temperature is a major environmental factor controlling microbial growth. This slide is taken from : MICR 300 : Microbiology, California State of University

24. Classification of Microorganisms by Temperature Requirements This slide is taken from : MICR 300 : Microbiology, California State of University

25. This slide is taken from : MICR 300 : Microbiology, California State of University

26. pH and Microbial Growth • The acidity or alkalinity of an environment can greatly affect microbial growth. • Most organisms grow best between pH 6 and 8, but some organisms have evolved to grow best at low or high pH. The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic. • Acidophiles : organisms that grow best at low pH • Alkaliphiles : organismsa that grow best at high pH This slide is taken from : MICR 300 : Microbiology, California State of University

27. This slide is taken from : MICR 300 : Microbiology, California State of University

28. Osmotic Effects on Microbial Growth • Osmotic pressure depends on the surrounding solute concentration and water availability • Water availability is generally expressed in physical terms such as water activity • Water activity is the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water. This slide is taken from : MICR 300 : Microbiology, California State of University

29. This slide is taken from : MICR 300 : Microbiology, California State of University

30. Halophiles and Related Organisms • In nature, osmotic effects are of interest mainly in habitats with high salt environments that have reduced water availability • Halophiles : have evolved to grow best at reduced water potential, and some (extreme halophiles) even require high levels of salts for growth. • Halotolerant : can tolerate some reduction in the water activity of their environment but generally grow best in the absence of the added solute • Xerophiles : are able to grow in very dry environments This slide is taken from : MICR 300 : Microbiology, California State of University

32. Oxygen and Microbial Growth • Aerobes : • Obligate : require oxygen to grow • Facultative : can live with or without oxygen but grow better with oxygen • Microaerphiles : require reduced level of oxygen • Anaerobes : • Aerotolerant anaerobes : can tolerate oxygen but grow better without oxygen. • Obligate : do not require oxygen. Obligate anaerobes are killed by oxygen

33. This slide is taken from : MICR 300 : Microbiology, California State of University

34. Factors Controlling Microbial Growth

36. Physical Methods

37. Heat Heat: Is the most practical, efficient, and inexpensive method of sterilization of those inanimate objects and materials that can withstand high temperatures. Two factors, temperature and time, determine the effectiveness of heat for sterilization. Sterilization is the process whereby all viable microbes including spores are removed or killed. NabeelAl-Mawajdeh RN.MCS

38. Heat (2) • Dry: At 160 to 165° C for 2 hours or at 170 to 180°C for 1 hour. - Red heat and incineration (burning): Direct exposure of material to flame till it becomes red. E.g., culture loops. - Direct flaming: Passing material over flame many times without reaching redness. E.g., flaming mouths of bottles, slides, flasks and culture tubes. - Hot air oven: Supplied with a fan from inside to distribute hot air in all chamber. It has temperature thermostat. It is used widely in hospitals, clinics, and laboratories. E.g., test tubes, glass pipettes, scissors, blades. • Moist: Heat applied in the presence of moisture, as in boiling or steaming, is safer and more effective than dry heat, and can be accomplished at a lower temperature for 30 min.; thus, it is less destructive to many materials. Moist heat causes proteins to coagulate (as occurs when eggs are hard boiled) - Boiling and steaming: At 100° C. clean articles made of metal and glass, such as syringes, needles may be disinfected by boiling for 30 minutes. Boiling is not always effective because heat- resistance bacterial endospores, mycobacteria and viruses may be present. - Autoclaving (steam under pressure): An autoclave is like a large metal pressure cooker that uses steam under pressure to completely destroy all microbial life. Nabeel Al-Mawajdeh RN.MCS

39. Cold • Cold: Most microbes are not killed by cold temperatures and freezing, but their metabolic activities are slowed, greatly inhibiting their growth. Nabeel Al-Mawajdeh RN.MCS

40. Radiation Radiation: a. Nonionizing radiation: - ultraviolet(UV) rays (has low degree ofpenetration): A ultraviolet lamp (germicidal lamp) is useful for reducing the number of microorganisms in the air and on surfaces. They do, however, penetrate cell and, thus, can causedamage to DNA. When this occurs, genes may be soseverely damaged that the cell dies. Many biologic materials, such as toxins, and vaccines, are sterilized with UV rays. b. Ionizing radiation: - X-rays and gamma rays (has high degree ofpenetration): Are used in industry for sterilization of plastic catheters, syringes, surgical equipments, preparation of vaccines…etc. Nabeel Al-Mawajdeh RN.MCS

41. Filtration • Filtration: Is the passage of a liquid or gas through a filter with pores small enough to allow microbes to pass. This method used for sterilization of serum, hormones, antibiotic solution. Nabeel Al-Mawajdeh RN.MCS

42. Gases • Gases (ethylene oxide, propylene oxide…etc): Suitable for plastics, hormones, surgical dressing, all antibiotics and thermo labile powder. Nabeel Al-Mawajdeh RN.MCS

43. Chemical Agents

44. Antiseptics • Antiseptics: chemicals inhibit the growth or kill microbes on living tissues like human skin and mucus membranes. NabeelAl-Mawajdeh RN.MCS

45. Disinfectants • Disinfectants: chemicals inhibit the growth or kill microbes. - Factors that determine the effectiveness of any disinfectant: a. Time. b. Temperature. c. Concentration. d. Type and number of microbes. e. Presence of spores. f. Presence of proteins in feces, blood, vomitus, pus. Nabeel Al-Mawajdeh RN.MCS

46. Disinfectants (2) Characteristics of good disinfectant: • Rapid action, easy to use. • Wide range of action. • Good penetration. • Capability of mixing with water. • Activity in organic matter (like blood, feces, vomit) • Resistance to decomposition. • Nonstainingand noncorrosive. • Odorless. • Stable in various temperature and light. • Cheep. Nabeel Al-Mawajdeh RN.MCS

47. Disinfectants (3) - Kinds of Disinfectants: a. Alcohol: 70% to disinfect skin and thermometer. b. Phenolics: 5% phenol is useful for disinfection of stool, sputum. 0.25% - 0.5% phenol for preservation of sera and vaccines. c. Chlorine: for sterilization of water supplies (1% in million) after the treatment of water by precipitation or filtration for removal of organic matters since they can not act efficiency in the presence of organic matter. d. Iodine (povidone): for skin disinfection (available as a tincture ,2% iodine with 70% alcohol). e. H2O2 (hydrogene peroxide) 3 – 6 % for wounds, ulcers, and mouth wash. f. Formaldehyde: for rubber, leather, shoes, books, and blankets. Nabeel Al-Mawajdeh RN.MCS

48. CHEMOTHEURAPEUTICS USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO Nabeel Al-Mawajdeh RN.MCS

49. Using Chemotherapeutic agents - Chemotherapeutic agent is any chemical (drug) used to treat an infectious disease, either by inhibiting or killing pathogens in vivo. a. Antifungal agents are used to treat fungal diseases. b. Antiprotozoal agents are used to treat protozoal diseases. c. Antiviral agents are used to treat viral diseases. d. Antibiotics Nabeel Al-Mawajdeh RN.MCS

50. Using Chemotherapeutic agents (Cont’d) d. Antibiotics are substances produced by microorganisms (usually a soil organism) that effective in killing or inhibiting the growth of other microorganisms. Some antibiotics (e.g., penicillin and cephalosporin) are produced by molds, whereas others (e.g., tetracycline, erythromycin, and chloramphenicol) are produced by bacteria. Many antibiotics have been chemically modified to kill a wider variety of pathogens or reduce side effects; these modified antibiotics are called semisynthetic antibiotics. Nabeel Al-Mawajdeh RN.MCS