Control of Microbial Growth

How to minimize the microbial growth by different methods.

1. The Control of Microbial Growth

2. Sterilization Commercial sterilization - for canned food - Sterilization is the removal or destruction of all living microorganisms. Heating is the most common method used for killing microbes, including the most resistant forms, such as endospores. A sterilizing agent is called a sterilant. Liquids or gases can be sterilized by filtration. Complete sterilization is often not required in some settings. For example, the body’s normal defences can cope with a few microbes entering a surgical wound. A drinking glass or a fork in a restaurant requires only enough microbial control to prevent the transmission of possibly pathogenic microbes from one person to another endospores of Clostridium botulinum

3. Term Definition Comments Sterilization Destruction or removal of all forms of microbial life, including endospores Usually done by steam under pressure or a sterilizing gas, such as ethylene oxide. Disinfection Destruction of vegetative pathogens on inanimate objects. May make use of physical or chemical methods. Antisepsis Destruction of vegetative pathogens on living tissue. Treatment is almost always by chemical antimicrobials. Degerming Removal of microbes from a limited area, such as the skin around an injection site. Mostly a mechanical removal by an alcohol- soaked swab. Sanitization Treatment is intended to lower microbial counts on eating and drinking utensils to safe public health levels. May be done with high-temperature washing or by dipping into a chemical disinfectant. Terminology Sepsis, indicates bacterial contamination, as in septic tanks for sewage treatment or a disease condition. Aseptic means that an object or area is free of pathogens. Bacteriostatic: Inhibits bacterial reproduction. Bactericidal : Kills bacteria

4. Rate of Microbial Death Bacterial populations subjected to heat/antimicrobial chemicals die at a constant rate. Rate: 90% / min 1. Alteration of Membrane Permeability 2. Damage to Proteins and Nucleic Acids

5. Several factors influence the effectiveness of antimicrobial treatments: The number of microbes The more microbes there are to begin with, the longer it takes to eliminate the entire population. Environmental influences Warm conditions/solutions Presence of organic matter e.g, blood, vomitus, or feces Microbes in surface biofilms, are difficult for biocides to reach effectively Suspending medium, especially protective medium (fat, protein) protects microbes Heat is also measurably more effective under acidic conditions Time of exposure Chemical antimicrobials often require extended exposure to affect more-resistant microbes or endospores. Microbial characteristics Microbial characteristics affect the choice of chemical and physical control methods.

6. Physical Methods of Microbial Control Drying (desiccation) and salting (osmotic pressure) were probably among the earliest techniques Heat is very effective (fast and cheap). Heat appears to kill microorganisms by denaturing their enzymes; the resultant changes to the three-dimensional shapes of these proteins inactivate them Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min. Thermal death time (TDT): Time to kill all cells in a culture. Decimal Reduction Time (DRT): Minutes to kill 90% of a population at a given temperature. These are useful guidelines that indicate the severity of treatment required to kill a given population of bacteria.

7. Moist Heat Sterilization Moist heat kills microorganisms primarily by coagulating proteins (denaturation) – breakage of the hydrogen bonds that hold the proteins in their three-dimensional structure. Example? Boiling kills vegetative forms of bacteria, almost all viruses, fungi and their spores about 10 minutes. Free-flowing (unpressurized) steam is same temp as boiling water but some bacterial endospores can resist boiling for more than 20 hours. Not always a reliable sterilization procedure. However, brief boiling will kill most pathogens. Examples ? Steam under pressure in an autoclave - The higher the pressure in the autoclave, the higher the temp. Autoclaving is the preferred method of sterilization in health care environments, unless the material to be sterilized can be damaged by heat or moisture .

8. Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment, solutions, syringes, transfusion equipment etc. Sterilization in an autoclave is most effective when the organisms either are contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure of about 15 psi (121°C) will kill all organisms and their endospores in about 15 minutes. Sterilizing the surface of a solid requires that steam actually contact it.

9. Pasteurization Pasteur used mild heating, which was sufficient to kill the organisms that caused the particular spoilage problem without seriously damaging the taste of the product. Significant number reduction (esp. spoilage and pathogenic organisms)  does not sterilize! Classic holding method: 63  C for 30 min Flash pasteurization (HTST): 72  C for 15 sec. Thermo-tolerant organisms survive Ultra High Temperature (UHT): 140  C for 4 sec then rapidly cooled in vacuum chamber. Technically not pasteurization because it sterilizes. Products other than milk, such as ice cream, yogurt, and beer, all have their own pasteurization times and temperatures, which often differ considerably. The phosphatase test (phosphatase is an enzyme naturally present in milk). If the product has been pasteurized, phosphatase will have been inactivated

10. Dry Heat Sterilization It kills by oxidation effects. Flaming of loop Incineration of carcasses Anthrax Foot and mouth disease Bird flu Hot-air sterilization Hot-air oven 170˚C, 2 hr

11. Fil t r a tion Filtration is the passage of a liquid or gas through a screen- like material with pores small enough to retain microorganisms. A vacuum is created in the receiving flask; air pressure then forces the liquid through the filter. Filtration is used to sterilize heat-sensitive materials, such as some culture media, enzymes, vaccines, and antibiotic solutions. High-efficiency particulate air (HEPA) filters remove almost all microorganisms larger than about 0.3 μm in diameter. Membrane Filters Industrial Filters = 1 mm Filters for bacteria = 0.25 to 0.45 μm Filters for viruses = 0.01 μm

12. Low Temperatures Slows enzymatic reactions  inhibits microbial growth Freezing forms ice crystals that damage microbial cells Refrigeration, deep freezing, lyophilization Other Methods High pressure in liquids denatures bacterial proteins and preserves flavor Example ? Desiccation prevents metabolism – Example ? Osmotic pressure causes plasmolysis – Example ?

13. Radiation Ionizing Radiation: Shorter Wavelength < 1nm, so more energy X-rays, Gamma-rays , electron beams  dislodge e- from atoms  production of free radicals and other highly reactive molecules. eg, Cobalt - 60 radioisotope The principal effect of ionizing radiation is the ionization of water, which forms highly reactive hydroxyl radicals . These radicals kill organisms by reacting with organic cellular components, especially DNA, and damaging them. Non-ionizing Radiation: Wavelength > 1nm Ultraviolet (UV) light damages DNA of exposed cells by causing bond formation between adjacent pyrimidine bases, usually thymines. The thymines-dimers inhibits correct DNA replication. UV is used to control microbes in the air. A UV “germicidal,” lamp is commonly found in hospital rooms, nurseries, operating rooms, and cafeterias. UV light is also used to disinfect vaccines and other medical products. Disadvantage of UV, not very penetrating, so the organisms to be killed must be directly exposed. Also damage human eyes, and prolonged exposure can cause burns and skin cancer

14. Wavelength: 1 mm – 1m H 2 O quickly absorbs energy  release as heat to environment Do not have much direct effect on microorganisms Moisture-containing foods are heated by microwave action, and the heat will kill most vegetative pathogens. Solid foods heat unevenly because of the uneven distribution of moisture. Microwave

15. Chemical Methods of Microbial Control Few chemical agents achieve sterility. Consider presence of organic matter, degree of contact with microorganisms, and temperature Disinfectants regulated by EPA, Antiseptics regulated by FDA Use-dilution test Metal rings dipped in test bacteria are dried. Drie d cul t u r e s o f S . aur e u s and P . aer u gi n os a a r e p l ac e d in disinfectant for 10 min at 20  C. 3. Rings a r e t r an s f er r e d t o cul t u r e m e d i a t o d e t er m ine wh e t h er bacteria survived treatment.

16. Types of Disinfectants Phenol = carbolic acid (historic importance) Phenolics : Cresols (Lysol) - disinfectant Bisphenols Hexachlorophene (pHisoHex, prescription), hospitals, surgeries, nurseries Triclosan (toothpaste, antibacerial soaps, etc.) Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls Remain active in presence of organic compounds

17. Chlorine Oxidizing agent Widely used as disinfectant Forms bleach (hypochlorous acid) when added to water. Broad spectrum, not sporicidal (pools, drinking water) Iodine More reactive, more germicidal. Alters protein synthesis and membranes. Tincture of iodine (solution with alcohol)  wound antiseptic Iodophors combined with an organic molecule  iodine detergent complex (e.g. Betadine ® ). Halogens

18. Ethyl (60 – 80% solutions) and isopropyl alcohol Denature proteins, dissolve lipids No activity against spores and poorly effective against viruses and fungi Easily inactivated by organic debris also used in hand sanitizers and cosmetics Al c ohols

19. Soaps and Detergents Major purpose of soap: Mechanical removal and use as wetting agent Definition of detergents Acidic-Anionic detergents  Anion reacts with plasma membrane. Nontoxic, non-corrosive, and fast acting. Laundry soap, dairy industry. Cationic detergents  Quarternary ammonium compounds ( Quats ). Strongly bactericidal against wide range, denature proteins, disrupt plasma membrane esp. Gram+ bacteria Surface Acting Ingredients / Surfactants Soap Degerming Acid-anionic detergents Sanitizing Quarternary ammonium compounds (cationic detergents) Strongly bactericidal,

20. Aldehydes (alkylating agents) Inactivate proteins by cross-linking with functional groups: (–NH 2 , –OH, –COOH, –SH) Glutaraldehyde: Sterilant for delicate surgical instruments (Kills S. aureus in 5, M. tb in 10 min) Formaldehyde: Virus inactivation for vaccines Aldehydes and Chemical Sterilants

21. Plasma Luminous gas with free radicals that destroy microbes Use: Tubular instruments, hands, etc.

22. Hydrogen Peroxide: Oxidizing agent Inactivated by catalase  Not good for open wounds Good for inanimate objects; packaging for food industry (containers etc.) 3% solution (higher conc. available) Esp. effective against anaerobic bacteria: Effervescent action, may be useful for wound cleansing through removal of tissue debris