1. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings PowerPoint ® Lecture Slide Presentation prepared by Christine L. Case M I C R O B I O L O G Y a n i n t r o d u c t i o n ninth edition TORTORA  FUNKE  CASE 7 The Control of Microbial Growth

2. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Control of Microbial Growth  Sepsis refers to microbial contamination.  Asepsis is the absence of significant contamination.  Aseptic surgery techniques prevent microbial contamination of wounds.  Aseptic techniques in the lab prevents contamination of cultures and us!

3. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Terminology  Sterilization: Removal of all microbial life  Commercial sterilization: Killing C. botulinum endospores  Disinfection: Removal of pathogens  Antisepsis: Removal of pathogens from living tissue  Sanitization: Lower microbial counts on eating utensils  Biocide/Germicide: Kills microbes  Bacteriostasis: Inhibiting reproduction of (not killing) microbes

4. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Mechanisms of Microbial Control Agents 1.Alteration of membrane permeability 2.Damage to proteins 3.Damage to nucleic acids 4.Damage to organelles

5. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Thermal Methods of Microbial Control  Autoclaving  Steam  Pasteurization  Dry Heat

6. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Autoclaving  Moist heat denatures proteins  Autoclave: Steam under pressure Figure 7.2

7. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Dry Hot-airAutoclave Equivalent treatments170˚C, 2 hr121˚C, 15 min Dry heat sterilization: Takes Longer!  Kills by oxidation  Flaming  Incineration  Hot-air sterilization

8. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 7.3 Steam Sterilization  Steam must contact item’s surface.

9. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Pasteurization  Reduces number of microbes  Equivalent treatments  63°C for 30 min  High temperature, short time: 72°C for 15 sec  Ultra high temperature: 140°C for <1 sec  Thermoduric organisms survive

10. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Physical Methods of Microbial Control 1.Filtration 2.Low temperature 3.High pressure 4.Desiccation 5.High Osmotic pressure or pH 6.Radiation

11. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Filtration  physically removes microbes b/c they are bigger than micro-holes in the filter.

12. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Low Temperature  Bacteriostatic: Inhibits microbial growth (does not generally kill)  Refrigeration  Deep freezing  Lyophilization (freeze drying)

13. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings High pressure  Denatures proteins

14. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Desiccation (Drying Out)  Prevents metabolism  We’re 60% water!

15. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings High Osmotic Pressure (Very Salty or Sugary)  High Osmotic Pressure causes plasmolysis i.e. it shrivels up and dies when the water leaves !

16. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Radiation damages DNA  Ionizing radiation (X rays, gamma rays, electron beams)  Nonionizing radiation (UV)  (Microwaves kill by heat; not especially antimicrobial)

17. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Ionizing (X-ray) vs. Non-Ionizing (UV) Radiation Allows new bonds to form. Not enough energy to break covalent bonds. Breaks covalent bonds. Causes loss of electrons.

18. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Ionizing (X-ray) Radiation Causes Double Stranded DNA breaks

19. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Non-Ionizing UV radiation causes thiamine dimers to form. Thiamine dimers are much more easily repaired that double stranded breaks!

20. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings

21. Factors affecting degree of disinfection 1.Concentration of disinfectant 2.Amount/type/arrangement of organic matter 3.pH 4.Time 5.Temperature 6.Moisture

22. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Evaluating Disinfectants: Disk Diffusion Method  The size of the zone of inhibition relates to the potency, concentration, and diffusability of the disinfectant. Figure 7.6

23. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Alcohols -kills both bacteria and fungi; used as skin antiseptics to degerm (?) skin areas -mode of action: protein denaturation agent; also disrupts and dissolves membrane lipids Advantage: acts and evaporates quickly with no residues, inexpensive and non toxic; degerms skin but does not sterilize area of application Disadvantage: poor choice for wound application (?); short contact time to be effective in microbe killing; damages rubber and some plastics TEthanol (70% ?) and isopropanol (rubbing alcohol).

24. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Peroxygens and other oxidizing agents -mode of action : releases toxic oxygen radicals (peroxide anion); oxidizes protein components (including enzymes) of treated microbes - useful for irrigating deep wounds (?) -Cells’ catalase breaks down peroxides, limits it’s action as antiseptic for open wounds -Benzoyl peroxide: main ingredient of acne medication

25. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Halogens: Iodine and chlorine Iodine: common preparations are Betadine and Isodine -used for skin infections and wound treatment; backpackers / campers? -mode of action : not precisely known - available as either tincture or iodophore Tincture is solution of iodine in alcohol Iodophore is complex of iodine and an organic carrier molecule; iodine is released upon dilution Advantage: slow, steady and sustained release of iodine from iodophore, thus longer lasting

26. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Chlorine: -widely used in it’s physical form (as gas) or in combination with other chemicals (as chloramines & chlorine dioxide) -very effective but has limited use due to it’s irritating nature. -when added to water, hypochlorous acid (strong oxidizing agent) is formed - sodium hypochlorite (in bleach) are routinely used as disinfectant;

27. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Phenol and phenolics - Phenolics (derivatives of phenol): are compounds containing chemically altered phenol - Bisphenolics: are derivatives of phenol that contains two phenolic groups connected by a bridge; Lysol component (?) and triclosan (?) - mode of action: affects plasma membrane lipids; also denatures cell and membrane proteins Advantage: remains stable for an extended time once applied - active in the presence of organic compounds and detergents Disadvantage: phenolics have strong odor and skin irritant for some Hexachlorophene: brain damage in infants; available by prescription only for staphylococcal treatment

28. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Aldehydes -Formalin: is 37 % aqueous solution of formaldehyde -mode of action: forms covalent cross-links with functional groups (-NH2, -SH, -OH, -COOH ) of proteins and nucleic acids; inactivate their function and alters their property -Glutaraldehyde: less irritating and more effective (than HCHO) but is toxic; used routinely to disinfect hospital instruments (at 2%); need to be rinsed thoroughly

29. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Surface Active Agents (Surfactants) - decreases surface tension - includes soaps and detergents - low toxicity for mammalian cells - mode of action: contains alkyl group that interacts with membrane lipids and disrupts membrane functions - consequence: alteration of membrane permeability and loss of essential cell component; bactericidal in action Disadvantage: May support growth of Pseudomonas rather than killing them )

30. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Gaseous Agents: are used in closed chamber with control humidity, temperature and regulated gas concentration - commonly used agents are ethylene oxide, propylene oxide and beta propiolactone mode of action; denature protein and DNA by cross linking organic functional groups Ethylene oxide: hazardous proerties i. flammable and explosive ii. poisonous (potential carcinogen

31. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Heavy metals: Ag, Hg, As, Cu and Zn Mode of action: denatures protein and causes loss of function Oligodynamic action: refers to the potency of heavy metals in minute quantity to exert antimicrobial activity. Silver : one of the few metals still used as a disinfectant used as a 1% silver nitrate solution (?) used in surgical dressings (silver impregnated), burn creams Copper : is often used to kill algae Copper compounds are used in paint formulation to control mildew Mercury: limited use due to corrosiveness and toxicity properties Thimerosal in vaccines (?) Used as a component in paint for mildew control and prevention

32. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Germicide High level germicide Low level germicide Intermediate level germicide kills all pathogens (including spores) Ex: catheters, implants, heart lung machines, needles, forceps, scalpels, pap smear biopsy kills fungal spores, protozoan cysts, viruses but not spores Ex: non invasive equipment and endoscopes kills vegetative bacteria, fungi protozoa and some viruses Ex: electrodes, stethoscopes

33. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 7.11