Control of microbial growth in the Environment ▪ Public health standards ▪ Health care facilities ▪ Laboratories ▪ Home Terminology of Microbial Control ▪ Sterilization is the removal and destruction of all microbes including endospores and viruses ▪ Commercial sterilization of canned food ▪ Targets endospores of Clostridium botulinum ▪ Aseptic- an environment or procedure free of contamination and specifically, absence of pathogens ▪ Aseptic surgery techniques prevent microbial contamination of wounds Terminology of Microbial Control ▪ Disinfection is the use of chemicals or physical agents to kill or inhibit the growth of microbes especially pathogens ▪ Disinfectants are used to treat inanimate objects or surfaces ▪ Antisepsis is the use of chemicals on skin or other tissue ▪ Chemicals used are called antiseptics ▪ Degermingis the removal of microbes by scrubbing /swabbing, soaps or alcohol are used ▪ Washing your hands ▪ A nurse prepares an area of skin for an injection Terminology of Microbial Control ▪ Sanitization ▪ Disinfection of places or things used by the public to meet accepted public health standards ▪ Steam, high-pressure hot water to sanitize restaurant utensils/dishes ▪ Chemicals to sanitize public toilets ▪ Pasteurization ▪ Heat is applied to kill pathogens ▪ Reduce the number of microbes that can cause spoilage of food and beverages Activity of chemical and physical agents ▪ Chemical/physical agents have “-static” or “-cidal’ activity ▪-stasis/-static: inhibit microbial metabolism and growth but do not kill the target microbes ▪-cide/-cidal: kill the target microbes- stasis/static Bacteriostatic Fungistatic Virustatic- cide/-cidal Bactericide Fungicide Virucide Germicides: antimicrobial chemical agents that kill pathogens Microbial death rates ▪ Microbial death is the permanent loss of reproductive capability ▪ The effectiveness of an antimicrobial agent is determined by calculating the microbial death rate, which is constant under a particular set of conditions ▪ A microbicidal agent will not kill all cells simultaneously but a constant % of cells over time ▪ Example shown in the graph - 90% of cells die after each treatment (1min) A constant death rate is indicated by a straight line What is an ideal antimicrobial agent? An ideal antimicrobial agent!! ▪ Inexpensive ▪ Fast-acting ▪ Stable during storage (chemicals) ▪ Control growth/reproduction of every type of microbe ▪ Harmless to humans, animals, and objects ▪ However, every antimicrobial agent has limitations, advantages and disadvantages Factors to keep into considerations when an antimicrobial method is selected ▪ Nature of the site/object to be treated ▪ Sensitive to heat or certain chemicals ▪ Risk of infection associated with medical instruments ▪ Invasive versus non invasive instruments ▪ Number/susceptibility of microbes ▪ Environmental conditions ▪ Temperature, pH, presence of organic material (feces, vomit, or blood) Antimicrobial chemicals & medical instruments High-level germicides kill all microbes, including endospores ▪ Invasive instruments-catheters, implants Intermediate-level germicidesdonot kill endospores ▪ Non-invasive instruments that contact mucous membranes ▪ Endoscopes Low-level germicidesnot effective against endospores/not all viruses ▪ Disinfect instruments that contact skin ▪ Stethoscopes, electrodes ▪ The effectiveness of germicides can be classified as high, intermediate , or low ▪ Based on their ability to destroy microbes on instruments that cannot be sterilized with heat Biosafety levels CDC and guidelines for four levels of safety in microbiological laboratories ▪ Each level raises personnel/environmental safety through strict laboratory techniques, safety equipment, design of facility ▪ Classification of pathogens by risk groups (BSL1-4) based on factors including ▪ Pathogenicity ▪ Routes of transmission ▪ Availability of effective treatments, vaccines Examples BSL1 – Nonpathogenic E. coli strains BSL2 – Staphylococcus aureus – MRSA BSL3 – M. tuberculosis, B. anthracis BSL4 – Ebola virus, Smallpox Heat treatment for microbial control ▪ Microbes exhibit a different degree of susceptibility to heat, which can be used to disinfect or sterilize Thermal death point Thermal death time Decimal reduction time (D) Lowest temperature that kills all cells in a broth in 10 minutes Time it takes to sterilize a defined volume of liquid at a set temperature Time required to kill 90% of microbes in a sample ▪ The canning industry uses the D value to destroy 90% of Clostridium botulinum endospores/each treatment at 121C Commercial Canning Process ▪ Uses industrial-sized autoclave called a retort ▪ Designed to reduce 1012 Clostridium botulinum endospores to only 1 (a 12 D process – A D value at 121C is 0.204 min) ▪ Endospores could germinate in canned foods and vegetative cells would produce/release the botulinum toxin (neurotoxin) that could lead to botulism when ingested in food (food poisoning intoxication) Heat treatment for microbial control Reliable, safe, relatively fast, inexpensive, non-toxic ▪ Include moist heat, dry heat ▪ Boiling kills most microorganisms and viruses ▪ It does not sterilize because endospores can survive ▪ Pasteurization kill pathogens (i.e., Brucella melitensis or Mycobacterium bovis), decreases the number of heat sensitive microbes (reducing spoilage organisms) Autoclave ▪ Sterilization using pressurized steam (autoclave) ▪ Typically achieved at 121°C /15 psi in 15/20 minutes ▪ Destroys endospores ▪ Not prions ▪ Dry heat is less effective than moist heat, requires longer times and higher temperatures (ovens) ▪ Incineration to destroy medical waste and diseased animal carcasses ▪ Sterilization of inoculating loops in the laboratory Spores of G. stearothermophilus used as a biological indicator of sterility Refrigeration & freezing, desiccation & lyophilization, osmotic pressure Recall that several of these methods are also used to store microbial cultures!!!! ▪ Refrigeration is a short-term storage! – It inhibits the growth of many pathogens and spoilage microbes ▪ Some pathogens, Listeria and Yersinia can reproduce in refrigerated food and blood products, respectively ▪ Freezing stops microbial growth – water is not available ▪ It does not kill microbes ▪ Survivors will grow and spoil food once thawed Refrigeration & freezing, desiccation & lyophilization, osmotic pressure ▪ Removal of water by_ ▪ Desiccation often supplemented by addition of salt, sugars, preservatives ▪ Lyophilization (freeze-drying) ▪ Coffee, milk, meats, fruits, vegetables ▪ Salting or adding sugars ▪ Addition of salt, sugars increases environmental solute concentration (hypertonic environment) causing cellular plasmolysis Mechanical Methods For Controlling Microbial Growth ▪ Filtration can be used to count microbes or ▪ Sterilize heat-sensitive materials ▪ Culture media, drugs, vitamins, enzymes, antibiotic solutions or vaccines ▪ HEPA filters are placed in safety cabinets, in air ducts of operating rooms, or rooms of immunocompromised (AIDS) and highly contagious (TB) patients Radiation Electromagnetic spectrum Ionizing radiation ▪ Gamma rays and X rays ▪ Removes electrons from atoms and produces reactive oxygen species (ROS) ▪ Damaging cell structures including DNA ▪ Sterilize medical equipment and drugs ▪ Approved by FDA/WHO/UNFAO on food to eliminate pathogens and decrease numbers of spoilage organisms ▪ Spices, fruit, vegetables, poultry, beef, lamb, pork Radiation Electromagnetic spectrum Non-ionizing radiation (UV) ▪ Ultraviolet radiation causes formation of thymine dimers in DNA and/or mutations ▪ UVGI (Ultraviolet germicidal irradiation) ▪ Disinfection method that uses the UVC range for food preservation, air purification or disinfection of surfaces, wastewater treatment ▪ UV lamps used in hospital rooms including nurseries, operating rooms, biological safety cabinets ▪ Poor penetrating power (turbid liquids, solid, plastic, and glass can screen out UV radiation) Chemical methods to control the growth of microbes on living tissue and inanimate objects Factors that affect the effectiveness of chemical agents include ▪ Concentration of the chemical ▪ Temperature, pH, time of exposure ▪ Presence of contaminating organic material ▪ Numbers/types of microbes ▪ Object/site being treated Chemical methods to control the growth of microbes on living tissue and inanimate objects ▪ Phenol was first used by Lister to prevent infections of surgical wounds ▪ Triclosan is an example of bisphenolic, used in many products such as diapers, cutting boards, garbage bags ▪ Phenolic compounds are effective in presence of contaminating organic material - vomit, pus or feces - therefore commonly used in health care settings Chemical methods to control the growth of microbes on living tissue and inanimate objects ▪ Isopropanol, ethyl alcohol ▪ Used as disinfectants or antiseptics ▪ Pure alcohol is not an effective antimicrobial!! ▪ Solutions of 70% to 90% alcohol typically used ▪ Alcohols evaporate rapidly and this is a disadvantage – they may not contact microbes long enough to be effective Chemical methods to control the growth of microbes on living tissue and inanimate objects Halogens: Iodine, chlorine, bromine, fluorine Iodine - Antiseptic ▪ Tinctures: solution of iodine in alcohol ▪ Iodophors: organic compound containing iodine that slowly releases it ▪ Betadine (Iodophor) ▪ Antiseptic used in preparation for surgery on a hand Chemical methods to control the growth of microbes on living tissue and inanimate objects Chlorine ▪ Chlorine disinfects drinking water, swimming pools, waste-water from sewage treatment plants ▪ Sodium hypochlorite (household chlorine bleach) & calcium hypochlorite are effective disinfectants ▪ Chlorine dioxide (gas) - in 2001 was used to decontaminate federal buildings from anthrax endospores due to bioterrorism attack ▪ Chloramine– disinfectant and antiseptic in wound dressings Chemical methods to control the growth of microbes on living tissue and inanimate objects ▪ Oxidizing agents are high level disinfectants and antiseptics ▪ H2O2is used as a disinfectant, antiseptic (not on open wounds – catalase would inactivate it) or for sterilization ▪ Ozonecan be used to disinfect drinking water – an expensive though safer alternative to chlorine ▪ Peracetic acid is used for sterilization (sporicide) of equipment by food processors & medical personnel/Not affected by organic contaminants Chemical methods to control the growth of microbes on living tissue and inanimate objects ▪ Surfactants include soaps & detergents ▪ Soaps are good degerming agents/poor antimicrobial activity ▪ Detergents include the quaternary ammonium compounds (quats), antimicrobials found in several products ▪ Benzalkonium chloride (Zephiran)/Cetylpyridinium in Cepacol mouth wash ▪ Their action is retarded by organic contaminants ▪ Pseudomonas aeruginosa thrives in quats! Chemical methods to control the growth of microbes on living tissue and inanimate objects Heavy metals are bacteriostatic and fungistatic agents – Low level disinfectants ▪ Silver, mercury, copper, zinc, and arsenic ▪ Silver nitrate was used to prevent blindness caused by Neisseria gonorrhoeae in newborns ▪ Silver still used as an antimicrobial in some surgical dressings, burn creams, catheters ▪ Heavy metals denature proteins Chemical methods to control the growth of microbes on living tissue and inanimate objects 2% solution of glutaraldehyde used for disinfection or sterilization, depending on the time of exposure 37% solution of formaldehyde (formalin) used to disinfect hospital rooms or instruments ▪ Gaseous agents, used as microbicidal/sporicidal agents, include ethylene oxide , propylene oxide, beta-propiolactone. Sterilize medical instruments/equipment sensitive to heat Alternative methods to control microbes or infectious agents ▪ An enzymatic method to control microbes in food (cheese) or to prevent spoiled wine involves the use of lysozyme ▪ Lysozymeis naturally produced by the human body as an antimicrobial agent. It breaks the link between NAM/NAG in the peptidoglycan ▪ A second example involves the enzyme Prionzyme against infectious agents called prions to remove them from medical instruments Methods to assess effectiveness of antimicrobial chemical agents ▪ Several methods are used to assess the effectiveness of an antimicrobial chemical agent ▪ The figure shows the disk-diffusion method