1. Chapter 9 Control of Microbial Growth

2. Ignaz Semmelweis and Joseph Lister developed the first microbial control methods. – Mid 1800s Before aseptic surgical techniques ~10% of all deaths were due to nosocomial infections Delivering mothers as high as 25% death rate During the American Civil War, a surgeon may have cleaned his scalpel by wiping it on his boot sole between incisions.

3. Sepsis refers to microbial contamination Asepsis is the absence of significant contamination Aseptic surgery techniques prevent microbial contamination of wounds The Terminology of Microbial Control

4. Sterilization: removing all microbial life – Prions are highly resistant to all forms of sterilization – The definition of sterilization implies the absence of prions. Disinfection: removing pathogens – Surfaces or substances Antisepsis: removing pathogens from living tissue

5. Effectiveness of Microbial Treatment Depends on: – Number of microbes The more you start with the longer it takes to eliminate – Environment Organic matter (vomit or blood) inhibit certain chemicals Biofilms cells are protected by polysaccharide matrix Temperature disinfectants work better under warm conditions – Time of exposure Chemical often requires extended exposure to microbes – Spray, wipe, re-spray – Microbial characteristics Endospores and Gram negatives are more resistant

6. Microbial Characteristics and Microbial Control Gram negative more resistant than Gram positive – Pseudomonas highly resistant to biocides Outer membrane provides protection Porins are highly selective of molecules that they permit into the cell Mycobacterium tuberculosis – Mycolic acid – Waxy lipid component in cell wall resists effect of chemicals

8. Microbial Characteristics and Microbial Control Prions: infectious proteins known to be heat and protease resistant – Autoclave in sodium hydroxide to destroy – Or incineration

9. Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides.

10. Effectiveness of Microbial Treatment Bacterial populations when treated with heat or chemicals die at a constant rate.

11. Table 7.2 Microbial Exponential Death Rate: An Example

12. Actions of Microbial Control Agents Alteration of membrane permeability – Damage results in cell contents to leak from cell Damage to proteins – Little bags of enzymes- enzymes are folded proteins unfolding due to chemical treatments makes enzymes not function Damage to nucleic acids – Heat, radiation, or chemicals are lethal to cells

13. Physical Methods of Microbial Control

14. Temperature: Heat Sterilization Moist heat denatures proteins Heat preserved canned foods, most common method of food preservation Autoclave: steam under pressure – 15 pounds of pressure per square inch – 121 degrees C – Kills all organisms and endospores but not prions! – Use: glassware, media, intravenous equipment….

15. Examples of sterilization indicators. Why do we need to use sterility indicators?

16. Figure 9.7 Sterility indicators Cap that allows steam to penetrate Flexible plastic vial Crushable glass ampule Nutrient medium containing pH color indicator Endospore strip Incubation After autoclaving, flexible vial is squeezed to break ampule and release medium onto spore strip. Yellow medium means spores are viable; autoclaved objects are not sterile. Red medium means spores were killed; autoclaved objects are sterile. Bacillus stearothermophilus

17. Kills by oxidation – Dry heat – Flaming – Incineration – Hot-air sterilization Hot-AirAutoclave Equivalent Treatments170˚C, 2 hr121˚C, 15 min Temperature: Dry Heat Sterilization

18. Temperature: Pasteurization Reduces spoilage organisms and pathogens – High-temperature short-time: 72°C for 15 sec Milk – Ultra-high-temperature: 140°C for <1 sec Organic Milks are commonly pasteurized with this method Coffee creamers

19. Temperature: Low Low temperature inhibits microbial growth – Refrigeration – Deep-freezing Sub optimal growth temperatures

20. Filtration HEPA removes microbes >0.3 µm =300 nm Membrane filtration (liquids) removes microbes >0.22 µm Viruses 5-300nm Influenza virus: ~ 125-150 nm in diameter Rhinovirus common cold: 18-28 nm in diameter

21. Figure 7.4 Filter sterilization with a disposable, presterilized plastic unit. Flask of sample Cap Membrane filter Cotton plug in vacuum line ensures sterility Vacuum line Sterile filtrate

22. Radiation Ionizing radiation (X rays, gamma rays) – Damages DNA – Low level radiation approved for use in US on spices, meats, and vegetables – Disposable dental and medical supplies such as plastic syringes and surgical gloves – Postal service uses electron beams to sterilize certain classes of mail Nonionizing radiation (Ultra Violet) – Damages DNA – Does not penetrate only good for surface applications Microwaves kill by heat; not especially antimicrobial

23. Figure 9.11 Irradiated and non-irradiated food Non-irradiatedIrradiated

24. Chemical methods of microbial control

25. Chemical Disinfection No single disinfectant is appropriate for all circumstances! See Lab 26 for clinically important Disinfectants Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA The chemical compounds are not to be used to treat disease only for controlling growth and preventing disease

26. Chemical Methods of Microbial Control Antimicrobial Soap: To much of a good thing? Page 281 Development of Resistant Microbes – Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health – Use of such products promotes development of resistant microbes – Use of antimicrobial soaps should be limited to food handlers, caring for newborns, and caring for high-risk patients (immunocompromised) – CDC recommends using regular soap and washing with warm water for at least 10-15 seconds

27. Chemical Methods of Microbial Control Development of Resistant Microbes – Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health – Use of such products promotes development of resistant microbes Triclosan

28. – Disrupt plasma membranes – Active ingredient in antimicrobial soaps, toothpaste, deodorants – Incorporated into cutting boards and knife handles – Widespread use has resulted in reports of resistance Bisphenols