Controlling Microbial Growth Vocabulary • Bactericidal: Bacteria-killing • Bacteriostasis: Bacterial growth inhibiting • Disinfection: Destruction of vegetative pathogens • Antisepsis: Destruction of vegetative pathogens on living tissue • Aseptic: Absence of a pathogen from an object or an area Vocabulary Cont. • Sanitation: Reduction of pathogens on eating utensils, etc. to public health standards • Antiseptic: chemical used for disinfection of skin or mucous membranes • Disinfectant: Chemical used on inanimate objects to kill or inhibit bacterial growth • Sterile: Without life, free of microorganisms Measuring the effectiveness of bacterial control methods • Not all genera of bacteria are equally susceptible to a given method of control. – Example: • E. coli bacteria are highly susceptible to heat and die quickly when they reach boiling temperatures. • Pathogens that create endospores on the other hand, can survive long periods of boiling. • How do we determine how hot and how long we need to keep a microbe at to kill it? Three acceptable ways to measure microbial tolerance to heat 1. Decimal reduction time (DRT): time in which 90% of the population of bacteria at a given temperature will die. 2. Thermal death time (TDT): Time in which all cells in a suspension are killed at a given temperature. 3. Thermal death point (TDP): The lowest temperature in which all microorganisms will be killed in 10 minutes. Death Curve Microbes die at a constant, logarithmic rate when treated. Death Curve What is the DRT for the above graph? What is the TDT? Physical Methods of Controlling Microbial Growth Moist Heat Methods • More effective than dry heat • Kills by coagulation of proteins • Three methods: boiling, autoclaving, pasteurization Boiling • 100 C for 10 min: kills vegetative bacteria, most viruses, and fungal spores. – Endospores and some viruses (hepatitis) can survive for long boiling periods. • Good for making drinking water and food safe for consumption. – Unreliable for sterilization of surgical equipment (endospores survive without stomach acid to kill them). Free flowing unpressurized steam is equivalent to boiling. Autoclave • Pressurizing of steam to make it hotter. 1 atm temp = 121 C kills all organisms and their endospores. Most effective and most preferred method of sterilization. Steam must contact all solid surfaces since solids don’t have convection currents like liquids do. Autoclave Pasteurization • Mildly heating food products to kill particular spoilage microorganisms or pathogens. 72 C for 15 seconds with refrigeration needed afterwards. Many heat resistant bacteria survive pasteurization but these are unlikely to cause disease. • Ultra high temperature pasteurization 140 C for 3 seconds sterilizes milk. Dry Heat • Kills and sterilizes by oxidation effect. • Direct flaming or incineration • Hot air sterilization (170 C for 2 hours) Filteration • Filters are made of cellulose or plastic polymers with pores of about .2 micrometers. Viruses can still get through. • There are filters with pore size as small as .01 micrometers for viruses but filtration is very slow. • Filters are important when you can’t heat something up because you’ll destroy what you’re trying to keep. Cold • Cold is bacteriostatic • Slow freezing is more harmful as crystals can set up better. • Deep freezing is quick freezing at temp between -50 and -95 degrees. This is usually done to preserve the specimen. Desiccation • Desiccation is the absence of water. Microbes need water in order to grow and reproduce. However, microbes in a desiccated state can be viable for years. When water is added, they can resume growth. • Desiccation is bacteriostatic, though the ability to survive varies by species. Lyophilization (freeze drying) • A suspension of microbes is quickly frozen at temp between -54 and -72 C and water is removed by high vacuum. Microbes can be stored for years in this state. Chemical Methods for Controlling Microbial Growth Test Guide to Chemotherapy • Different strains of microbes have different levels of susceptibility to different chemotherapeutic agents. • Susceptibility of a microorganism can change over the course of a treatment. • Physicians need to know the sensitivities of the pathogen before treatment can be started. Various tests are employed to give Physicians the knowledge they need. Methods for Testing Disinfectants • The American Official Analytical Chemist’s Use-dilution method (Use-Dilution Method) • The Disk-diffusion Method • Three bacteria, Salmonella, Staphylococcus, and Pseudomonas, are used as standards to examine the effectiveness of a given disinfectant. Use-Dilution • Small stainless steel cylinders are dipped and coated with these bacteria and than allowed to dry. • The cylinder is than dipped into one of several dilutions of the chemical agent to be tested for 10 minutes, removed, rinsed with water, and placed into a tube of nutrient broth. • Chemicals that prevent growth at the greatest dilutions are considered to be the most effective. Example Results Disinfecting Agents Dilution Ratio Agent 1 Agent 2 1:1 1:10 + 1:50 + 1:500 + + indicates growth - indicates no growth Agent 3 + + Disk-diffusion (Filter Paper) Method • Most common testing method. • Small disc of filter paper is soaked with chemical and placed on surface of agar plate that has been previously inoculated with the test organism. Disk-diffusion Cont. • The chemical is concentrated in one area, and as it diffuses out, it is less concentrated. The effectiveness is based on how big of a circle “kill-zone” or zone of inhibition is around where the chemical is introduced. • Multiple chemicals can be tested against one bacteria at a time with this method. Example Results Chemical Disinfectants • Can kill in three ways: – Damage of Plasma Membrane – Inactivate Enzymes – Denature Proteins Phenol and Phenolic • Phenol: First used by Joseph Lister in an operating room. It proved too toxic for skin and had a terrible odor. • Phenolic: Adapted version of phenol that is less potent and more useful. • Kills in all three ways. Iodine (a halogen) • Kills by inactivating enzymes by attaching to the amino acid tyrosine. • Effective against bacteria, endospore, fungi, and some viruses. • Iodine is available as tincture solution (in solution with alcohol). Tincture solutions often stain and patients may develop hypersensitivity to it. • Iodine can come in Iodophor (combined with an organic molecule for slower releasing). Chlorine (a halogen) • Chlorine is mostly just a disinfectant. Chlorine turns to hyochlorous acid, its active form, when it’s added to water. • Kills by deactivating enzymes. The acid oxidizes cellular molecules into inactive forms. Alcohol (ethanol and isopropyl) • Alcohols work in two ways: by denaturing proteins and dissolving lipids (plasma membrane). • Effective against bacteria and fungi but not endospores and non-enveloped viruses. Alcohol is most effective when it is diluted by water. (ethanol 70%, isopropyl 90%). Nonwatered alcohol doesn’t carry into cells very well. Don’t drink isopropyl alcohol. That is all. Heavy Metals • Gold, silver, copper and zinc are germicidal. Silver nitrate is used in infants’ eyes. • Selenium and zinc in shampoos control fungi that cause dandruff. • Heavy metals combing with –SH groups to denature proteins.