The Control of Microbial Growth

1. The Control of Microbial Growth Chapter 7 Dr. G. López-de-Victoria

2. History • Use of drying and salting to preserve foods • Ignaz Semmelweiss (1840)- aseptic techniques • Joseph Lister (1867)- use of phenolics, aseptic surgery GLV

3. The Terminology of Microbial Control • Sterilization is the process of destroying all microbial life on an object. • Commercial sterilization is heat treatment of canned foods to destroy C. botulinum endospores. • Disinfection is the process of reducing or inhibiting microbial growth on a non-living surface. • Antisepsis is the process of reducing or inhibiting microorganisms on living tissue. • Degerming removes microbes from a limited area. • Sanitization lowers microbial counts on utensils to safe public health levels. • The suffix –cide means to kill • The suffix – stat means to inhibit • Sepsis is bacterial contamination GLV

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5. The Rate of Microbial Death • Bacterial populations die at a constant rate when subjected to heat or antimicrobial agents. • The effectiveness of a treatment is affected by • The number of microbes- the higher the number the longer it takes to eliminate the population. • Environmental influences- presence of organic matter inhibits the action of the chemical. E.g. blood, vomit, feces. • Time of exposure • Microbial characteristics GLV

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7. Fig. 7.1 A microbial death curve. GLV

8. Conditions influencing Microbial Control • Temperature • Types of Microorganism (see Fig. 7.11) • Gram positive • Gram negative • Pseudomonads • Physiological state • actively growing • endospores • Environment GLV

9. Fig. 7.11 Decreasing order of resistance GLV

10. Actions of Microbial Control Agents • Alteration of membrane permeability • The susceptibility of the plasma membrane is due to its lipid and protein components. • Certain chemical control agents damage the plasma membrane by altering its permeability. GLV

11. Actions of Microbial Control Agents • Damage to proteins and nucleic acids • Some microbial control agents damage cellular proteins by breaking hydrogen bonds and covalent bonds. • Other agents interfere with DNA and RNA replication and protein synthesis. GLV

12. Physical Methods of Microbial Control • Heat • Heat is frequently used to eliminate microorganisms. • Moist heat kills microbes by denaturing proteins (enzymes). • Dry heat kill organisms by oxidation. • Thermal Death Point (TDP)- is the lowest temperature at which all the microbes in a liquid culture will be killed in 10 minutes. GLV

13. Heat (cont.) • Thermal Death Time(TDT) is the length of time required to kill all bacterial in a liquid culture at a given temperature. • Decimal Reduction Time (DRT) is the length of time required to kill 90% of a bacterial population at a given temperature; D value GLV

14. Moist Heat • Boiling- 100 °C kills many vegetative cells and viruses within 10 minutes. • Hepatitis virus can survive 30 min boiling; endospores can survive 20 hr. • Autoclaving (steam under pressure) is the most effective method of moist heat sterilization. Steam must be directly in contact with the material to be sterilized. • The higher the pressure the higher the temperature. • Steam at a pressure of about 15 psi (121 °C) will kill all organisms and their endospores in about 15 min. GLV

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16. Fig. 7.2 An Autoclave GLV

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18. Fig. 7.3 Examples of sterilization indicators GLV

19. Moist Heat (cont.) • Pasteurization • In HTST pasteurization, a high temperature is used for a short time (72 °C for 15 seconds) to destroy pathogens without altering the flavor of food. • Ultra-high-temperature (UHT) treatment is used to sterilized dairy products (140 °C for 3 seconds). GLV

20. Dry Heat • Methods of dry heat sterilization include • direct flaming- inoculating loops • incineration- to dispose of contaminated cups, dressings etc. • hot-air sterilization- oven; 170 °C for 2 hours • Dry heat kills by oxidation. GLV

21. Filtration • Filtration is the passage of liquid or gas through a filter with pores small enough to retain microbes. • Microbes can be removed from air by high-efficiency particulate air filters (HEPA). • Membrane filters composed of nitrocellulose or cellulose acetate are commonly used to filter out bacteria, viruses, and even large proteins. GLV

22. Fig. 7.4 Filter sterilization GLV

23. Low Temperatures • The effectiveness of low temperatures depends on the particular microorganism and the intensity of the application. • Most microorganisms do not reproduce at ordinary refrigerator temperatures (0 - 7 °C); bacteriostatic. • Many microbes survive (but do not grow) at the subzero temperatures used to store foods. GLV

24. Freeze-Drying • Microbes are placed in a suspending medium and frozen quickly at temperatures between -52 and 95 °C. • Water is removed by vacuum (sublimation)- lyophilization. • Powder-like product can be reconstituted to bring culture back to viable conditions. GLV

25. Desiccation • In the absence of water, microorganisms cannot grow but can remain viable. • Viruses and endospores can resist desiccation. • There is disruption of metabolism. • Method used in food preservation. GLV

26. Osmotic Pressure • Microorganisms in high concentrations of salts and sugars undergo plasmolysis. • Used as food preservation. • Mold and yeasts are more capable than bacteria of growing in materials with low moisture or high osmotic pressure. GLV

27. Radiation • The effects of radiation depend on its wavelength, intensity, and duration. • Ionizing radiation (gamma rays, X rays, and high-energy electron beams) has a high degree of penetration and exerts its effect primarily by ionizing water and forming highly reactive hydroxyl radicals. GLV

28. Radiation • Ultraviolet (UV) radiation, a form of non-ionizing radiation, has a low degree of penetration and causes cell damage by making thymine dimers in DNA that interfere with DNA replication. • The most effective germicidal wavelength is 260 nm. • Microwaves can kill microbes indirectly as materials get hot. GLV

29. Fig. 7.5 The radiant energy spectrum. GLV

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32. Chemical Methods of Microbial Control • Chemical agents are used on living tissue (as antiseptics) and inanimate objects (as disinfectants). • Few chemical agents achieve sterility. GLV

33. Principles of Effective Disinfection • Properties of disinfectant • Concentration of disinfectant • Presence of organic matter • Degree of contact • Temperature GLV

34. Evaluating a Disinfectant • In the use-dilution test, bacterial (S. choleraesuis, S. aureus, and P. aeruginosa) survival in the manufacturer’s recommended dilution of a disinfectant is determined. • Viruses, endospore-forming bacteria, mycobacteria, and fungi can be used in the use-dilution test. GLV

35. Evaluating a Disinfectant • In the disk-diffusion method, a disk of filter paper is soaked with a chemical and placed on an inoculated agar plate, a clear zone of inhibition indicated effectiveness. • Phenol coefficient- compares the effectiveness of a disinfectant against the effectiveness of phenol. GLV

36. Fig. 7.6 Evaluation of disinfectants by the disk-diffusion method. GLV

37. Types of Disinfectant • Phenol and Phenolics • Phenolics exert their action by injuring the lipid-containing plasma membrane which results in leakage of cellular contents. • Mycobacteria are susceptible to phenolics due to their rich lipid content. • Cresols- O-phenylphenol, main ingredient in Lysol. • Bisphenols • Hexachlorophene, used in pHisoHex; effective against G+ cocci • Triclosan- soap, toothpaste, plastics kitchenware; G+ and fungi. GLV

38. Fig. 7.7 The structure of phenolics and bisphenols. GLV

39. Types of Disinfectant • Biguanides • Chlorohexedine damages plasma membranes of vegetative cells. • Broad spectrum • Used for microbial control of skin and mucous membranes. • Used for surgical hand scrubs. • Effective against most vegetative bacteria and fungi. • Mycobateria, endospores, and protozoan cysts are not affected. GLV

40. Types of Disinfectant • Halogens • Some halogens (iodine and chlorine) are used alone or as components of inorganic or organic solutions. • Iodine may combine with certain amino acids to inactivate enzymes and other cellular proteins. • Iodine is available as a tincture (in solution with alcohol) or as an iodophor (combined with an organic molecule) like in Betadine. GLV

41. Types of Disinfectant • The germicidal action of chlorine is based on the formation of hypochlorous acid when chlorine is added to water. It is an excellent oxidizing agent. • Chlorine is used as a disinfectant in gaseous form (Cl2) or in the form of a compound, such as calcium hypochlorite, sodium hypochlorite, sodium dichloroisocyanurate, and chloramines. GLV

42. Types of Disinfectant • Alcohol exert their action by denaturing proteins and dissolving lipids. • In tinctures, they enhance the effectiveness of other antimicrobial agents. • Aqueous ethanol (60-90%) and isopropanol are used as disinfectants. • Not effective against spores or non-enveloped viruses. GLV

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44. Heavy Metals and their Compounds • Silver, mercury, copper, and zinc are used as germicidals. • They exert their antimicrobial action through oligodynamic action. When heavy metal ions combine with sulfhydryl (-SH) groups, proteins are denatured. • Ex. 1% Silver nitrate solution, mercuric chloride, copper sulfate (algicide) GLV

45. Fig. 7.8 Oligodynamic action of heavy metals. GLV

46. Surface-active agents • Soaps and acid anionic detergents • The agents decrease the surface tension among molecules of a liquid; soaps and detergents are examples. • Soaps have limited germicidal action but assist in the removal of microorganisms through scrubbing. • Acid-anionic detergents are used to clean dairy equipment. GLV

47. Quaternary Ammonium Compounds • Quats are cationic detergents attached to NH4+. • By disrupting the plasma membranes, they allow cytoplasmic constituents to leak out of the cell. • Quats are most effective against Gram-positive bacteria. • They do not kill endospores or mycobacteria. • Examples • Zephiran (benzalkonium chloride) • Cepacol (cetylpyridinium chloride) • Pseudomonads are highly resistant, can even live in quats. GLV

48. Fig. 7.9 The ammonium ion and a quaternary ammonium compound, benzalkonium chloride (Zephiran). GLV

49. Chemical Food Preservatives • Organic Acids and Derivatives • Inhibit metabolism • Effective mostly against mold • Interferes with mold metabolism or the integrity of the plasma membrane • Examples include sorbic acid and benzoic acid; calcium propionate used as a fungistat in bread • Widely used in foods and cosmetic industry. • Nitrate and nitrite salts prevent germination of Clostridium botulinum endospores in meats. GLV

50. Antibiotics • Nisin and Natamycin are antibiotics used to preserve foods, especially cheese. • Aldehydes • Aldehydes such as formaldehyde and glutaraldehyde (Gidex) exert their antimicrobial effect by inactivating proteins. • They are among the most effective chemical disinfectants. GLV