Controlling Microbial Growth in Vitro

1. Controlling Microbial Growth in Vitro

2. Factors that affect microbial growth Temperature psychrophiles (cold loving microbes ) range 0 C - 20 C mesophiles (moderate temp. loving microbes) range 20 C - 40 C thermophiles (heat loving microbes) range 40 C - 100 C

3. Factors that affect microbial growth Availability of nutrients: Many nutrients are energy sources Nutrients serve as sources of C, O, N, P, & Sulfur Source for trace elements as Iron, Iodine, Zinc

4. Factors that affect microbial growth Moisture All living organisms need water for normal metabolic processes Moisture necessary for growing and reproduction

5. pH Most bacteria grow between pH 6.5 - pH 7.5 Very few can grow at below pH 4.0 Acidophilic microbes: Alkalophilic microbes:

6. Osmotic Pressure Microbes obtain almost all their nutrients in solution from surrounding water Tonicity isotonic hypertonic hypotonic

8. Barometric pressure Barophiles: prefer to live in a high atmospheric pressure like some archeans

9. Bacterial Growth - increase in the # of cells Binary Fission Generation Time (Doubling Time) time required for a cell to divide most about 1 Hr. To 3 Hrs. E. coli - 20 minutes Mycobacterium tuberculosis - 24 Hrs.

10. Limiting factors in the environment Lack of food, water or nutrients space accumulation of metabolic wastes lack of oxygen changes in pH temperature

11. Phases of Growth 4 Phases 1. Lag Phase 2. Log Phase 3. Stationary Phase 4. Death Phase

12. 1. Lag Phase Bacteria are first introduced into an environment or media Bacteria are “checking out” their surroundings cells are very active metabolically # of cells changes very little 1 hour to several days

13. 2. Log Phase Rapid cell growth (exponential growth) population doubles every generation microbes are sensitive to adverse conditions antibiotics anti-microbial agents

14. 3. Stationary Phase Death rate = rate of reproduction cells begin to encounter environmental stress lack of nutrients lack of water not enough space metabolic wastes oxygen pH

15. 4. Death Phase Death rate > rate of reproduction Due to limiting factors in the environment

16. Inhibiting the growth of microorganisms in Vitro

17. Methods to Control Microbial Growth 1. Physical 2. Chemical

18. Terms used: Sterilization vs. Disinfection Sterilization destroying all forms of life Disinfection destroying pathogens or unwanted organisms

19. Disinfectant vs. Antiseptic Disinfectant antimicrobial agent used on inanimate objects Antiseptic antimicrobial agent used on living tissue Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)

20. cidal vs. static Bactericidal - kills bacteria Bacteristatic - inhibits bacterial growth Fungicidal Fungistatic Algacidal Algastatic

21. Factors that effect Antimicrobial Activity 1. Temp 2. Time 3. Concentration of Antimicrobial agent 4. Type of Microbe 5. Activity of Microbe 6. Presence of organic matter

22. Targets of Antimicrobial Agents 1. Cell membrane 2. Enzymes & Proteins 3. DNA & RNA

23. Physical Methods of Microbial Control 1. Heat works by denaturing enzymes and proteins A. Thermal Death Point (TDP) lowest temp. at which all microorganism in a liquid culture are killed in 10 minutes B. Thermal Death Time (TDT) minimum length of time in which all microorganisms in a liquid culture are killed at a given temperature

24. Moist Heat 1. Boiling Water kills vegetative bacterial cells, Fungi and many viruses not effective for endospores and some viruses Hepititis (20 min) Some spores may survive boiling water for up to 20 hrs

25. Moist Heat 2. Autoclave (Steam under pressure) preferred method of sterilization Water boils at 100 C Increasing the pressure raises the Temp. 15 lbs./ per sq. inch (psi) ------> 121 C 121 C for 15 min.

26. Dry Heat 1. Direct Flaming Inoculating Loop and Needle 100% effective 2. Incineration disposable wastes (paper cups, bags, dressings) 3. Hot Air Sterilization Oven ( 170 C for 2 hours) used on substances that would be damaged by moist heat sterilization gauzes, dressings or powders

27. Filtration Removes microorganisms from solutions that might be damaged by heat culture media enzymes vaccines antibiotics

28. Radiation 1. Ionizing Radiation gamma rays & x-rays penetrates most substances Used on substances that could be damaged by heat plastic petri dishes plastic syringes catheters surgical gloves

29. Radiation 2. Non-Ionizing Radiation UV Light does not penetrate plastic, glass or proteinaceous matter Used to reduce microbial populations hospital rooms nurseries operating rooms

30. Pasteurization Disinfection - not sterilization (removes unwanted organisms) Mycobacterium tuberculosis 63 C for 30 minutes 72 C for 15 seconds Thermodurics able to survive high temps.

31. Methods used to control Microbial Growth 1. Heat Moist Heat Boiling Water Steam Heat (Autoclave) Dry Heat Direct Flaming Incineration Hot Air Sterilization (Oven) 2. Filtration 3. Radiation Ionizing Radiation Non-Ionizing Radiation 4. Pasteurization (Heat)