Foundations in Microbiology

1. PowerPoint to accompany Foundations in Microbiology Fifth Edition Talaro Chapter 11 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Physical and Chemical Control of Microbes Chapter 11

3. Control of microbes Physical and chemical methods to destroy or reduce microbes in a given area Physical – heat and radiation Chemical – disinfectants and antiseptics

4. Relative resistance of microbes • Highest resistance • Bacterial endospores • Moderate resistance • Bacteria with resistant vegetative cells: • Pseudomonas sp. • Mycobacterium tuberculosis • Staphylococcus aureus • Protozoan cysts • Some viruses (naked more resistant than enveloped) • Least resistance • most bacterial vegetative cells • Fungal spores • Enveloped viruses • Yeast • Protozoan trophozoites

5. Terms • Decontamination - The destruction, removal, or reduction in number of undesirable microbes • Sepsis - The growth of microorganisms in the tissues • Asepsis - Techniques that prevent the entry of microorganisms into sterile tissues • Antiseptic - Chemicals applied to body surfaces to destroy or inhibit vegetative pathogens

6. Terms • Sterilization – a process that destroys all viable microbes, including viruses & endospores (autoclave and radiation) • Disinfection – a physical or chemical agent that destroys vegetative pathogens, not endospores, on inanimate objects (bleach, boiling water) • Sanitization – any cleansing technique that mechanically removes microbes (soaps and detergents) • Degermation – reduces the number of microbes from living tissue (surgical hand scrub)

7. Fig. 11.1

8. Microbial death • Involves permanent loss of reproductive capability, even under optimum growth conditions

9. Factors that influence action of antimicrobial agents: • Number of microbes • Nature of microbes in the population (young cells die more quickly) • Temperature & pH of environment • Concentration or dosage of agent • Mode of action of the agent • Presence of solvents, organic matter, or inhibitors

10. Factors that influence the rate at which microbes are killed by antimicrobial agent

11. Cellular targets of control • Cell wall • Cell membrane • Cellular synthetic processes (DNA, RNA) • Proteins

12. Surfactants inserting into the lipid bilayer, alter permeability, cause leakage both in and out of the cell

13. a) The native state is maintained by bonds that create active sites to fit the substrate. Agents denature by breaking 2o and 3o bonds c) Random bonding and incorrect folding

14. Selecting a workable method of microbial control Practical concerns • Does the application require sterilization? Or is disinfection adequate? • Is the item to be reused? • Can the item withstand heat, pressure, radiation, or chemicals? • Is the method suitable? • Will the agent penetrate to the necessary extent? • Is the method cost- and labor-efficient & is it safe?

15. Methods of Physical Control • Heat • Cold temperatures • Desiccation • Radiation • Filtration

16. 1. Heat Moist heat – use of hot water or steam • Mode of action – denaturation of proteins, destruction of membranes & DNA • sterilization • autoclave 15 psi/121oC/10-40min (raise the temp of steam by increasing the pressure) • intermittent sterilization – unpressurized steam at 100oC 30-60 min for 3 days • disinfection • Pasteurization <100oC for seconds; kills Salmonella, Listeria & overall microbe count • Boiling at 100oC for 30 minutes to destroy non-spore-forming pathogens

17. Sterilization using steam under pressure The Autoclave

18. Dry heat using higher temperatures than moist heat, can also sterilize, less efficient that moist heat • incineration – 600-1200oC combusts & dehydrates cells (limited to metals, ex. loop) • dry ovens – 150-180oC- coagulate proteins, not suitable for plastics/paper

19. Dry heat- Incineration Shield prevents splattering of microbial samples during flaming

20. 2. Cold temperatures • Microbistatic – slows the growth of microbes • refrigeration 0-15oC & freezing <0oC • Pathogens can survive several months • used to preserve food, media and cultures • -70oC to -135oC can preserve cultures of bacteria and viruses for long periods of time

21. 3. Desiccation • gradual removal of water from cells, leads to metabolic inhibition • not effective microbial control – many cells retain ability to grow when water is reintroduced • A valuable way to preserve foods

22. 4. Radiation Defined as energy emitted from atomic activities and dispersed at high velocity through matter or space

23. 4. Radiation Ionizing radiation – ejects orbital electron from an atom causing ions to form • Deep penetrating power, breaks DNA • gamma rays, X-rays, cathode rays • used to sterilize medical supplies & food products

24. 4. Radiation Nonionizing radiation: Ultraviolet Rays – excites atoms by raising them to a higher energy state, leads to the formation of abnormal bonds • Little penetrating power to sterilize air, water and solid surfaces • UV light creates thymine pyrimidines dimers, which interfere with replication

25. Dislodges electron from molecules Excites electron-abnormal bonds form A solid barrier cannot be penetrated by nonionizing radiation

26. Formation of pyrimidine dimers by the action of UV radiation Pyrimidine bases= Thymine and cystosine Dimers prevent that segment from correctly replicating or transcribing

27. An Ultraviolet (UV) treatment system for disinfection of water

28. 5. Filtration • physical removal of microbes by passing a gas or liquid through filter • used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms

29. Filtration Scanning electron micrograph of filter showing relative size of pores and bacteria trapped on surface (8um to 0.2um)

30. Chemical control • Halogens • Phenolics • Chlorhexidine • Alcohols • Hydrogen peroxide • Detergents & soaps • Heavy metals • Aldehydes

31. Halogens(used for disinfection for 200 yrs) • Chlorine – Cl2, hypochlorites (chlorine bleach), chloramines • Denaturation of proteins by disrupting disulfide bonds • Can be sporicidal • Iodine - I2, iodophors (Betadine) • Denature proteins • Can be sporicidal • Milder medical & dental degerming agents, disinfectants, ointments • Less susceptible to inactivation by organic material

32. 2. Phenolics • Phenolics-one or more aromatic carbon rings with additional functional groups • Disrupt cell membranes & precipitating proteins; bactericidal, fungicidal, virucidal, not sporicidal • Lysol • triclosan- antibacterial additive to soaps

33. All contain basic aromatic ring, but differ in the types of additional compounds

34. 3. Chlorhexidine • Hibiclens, Hibitane • A surfactant & protein denaturant with broad microbicidal properties • Not sporicidal • Used as skin degerming agents for preoperative scrubs, skin cleaning & burns • Mildness, low toxicity and rapid action

35. 4. Alcohols • Colorless hydrocarbons with one or more -OH functional groups • Ethyl or isopropyl alcohol in solutions of 50-95%. At 50% or greater concentration dissolves cell membrane lipids • 70% proteins coagulate • 100% dehydrates • Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells, fungi, and enveloped viruses • Skin degerming agent. Not sporicidal

36. 5. Hydrogen peroxide (H2O2) • A colorless, caustic liquid that decomposes in the presence of light into water and oxygen gas • Weak (3%) to strong (25%) • Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O2 gas – toxic to anaerobes • Strong solutions are sporicidal

37. 6. Detergents & soaps • Quaternary ammonia cpds act as surfactants that alter membrane permeability of some bacteria & fungi • Positively charged detergent • Not sporicidal • Soaps- mechanically remove soil and grease containing microbes • Alkaline cmpds made by combining the fatty acids in oils with sodium or potassium salts • Weak microbicides

38. Detergents are polar molecules with charged head and uncharged tail A common quarternary ammonium detergent

39. Graph showing effects of hand scrubbing

40. 7. Heavy metals-(relatively high atomic weight: mercury, silver, and gold) • Solutions of silver & mercury kill vegetative cells in low concentrations by inactivating proteins • Oligodynamic action-the property of having antimicrobial effects in exceedingly sm. amts • Not sporicidal • Bind to functional groups of proteins and inactivate them

41. A pour plate inoculated with saliva Contains silver and mercury

42. 8. Aldehydes • Organic substances bearing a reducing functional group on terminal carbon • Glutaraldehyde & formaldehyde kill by alkylating protein & DNA • glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments, high-level disinfectant • formaldehyde - disinfectant, preservative, toxicity limits use

43. Gases & aerosols • Ethylene oxide, propylene oxide, betapropiolactone & chlorine dioxide • Strong alkylating agents, sporicidal • Sterilizing inanimates • Ethylene oxide is the gas used in chemiclaves • Reactions with functional groups of DNA and proteins, therefore, blocks both DNA replication and enzymatic actions

44. Sterilization Using Gas An automatic ethylene oxide (ETO) sterilizer. Machine equipped with gas canisters containing ETO and carbon dioxide, chamber to hold items, and ways to evacuate and introduce air

46. Table 11.7

47. Fig. 11.1