Chapter 9: Controlling Microbial Growth in the EnvironmentWhy is this Important? • Everyone should understand at least a little about how to control or eliminatemicrobial growth – Prevent contamination of food– Prevent human illness• Understand how and why different cleaners (bathroom and kitchen cleaners)work and how to make them work more effectivelyMicrobial Death Rates• Microbial death – the permanent loss of reproductive ability under ideal environmental conditions• Organisms can’t metabolize or go through replication, transcription, translation, etc. • Microbial death rate – usually found to be constant over time for any particular microbe under a particular set of conditionsTerminology of Microbial Control- Cide/cidal vs. stasis/static- Antisepsis vs. disinfection- Pasterization: not sterilization- Sanitization- Sterilization: used in canned food industry Action of Antimicrobial Agents• There are many types of chemical and physical microbial controls, but their modes of action (the way they work; how they affect microbial cells) fall into two basic categories– Those that disrupt the integrity of cells by adversely altering their cell walls or cytoplasmic membranes– Those that interrupt cellular metabolism and reproduction by interfering with the structures of proteins and nucleic acidsAlterations of Cell Walls and Membranes• Cell wall maintains cellular integrity by counteracting the effects of osmosis when the cell is in a hypotonic solution– If the wall is disrupted, it no longer prevents the cell from bursting as water moves into the cell by osmosis• Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell– When damaged, cellular contents leak out– Viral envelope is a membrane composed of proteins and phospholipids (usually acquired from the host cell) that is responsible for attachment of virus to target cell– Damage to envelope interrupts viral replication – can’t live without the envelope – Non-enveloped viruses have greater tolerance of harsh conditionsDamage to Proteins and Nucleic Acids• Proteins regulate cellular metabolism, function as enzymes in most metabolicreactions, and form structural components in membranes and cytoplasm• Protein function depends on 3-D shape that is maintained by hydrogen and disulfide bonds– When these bonds are broken by extreme heat or certain chemicals, the protein’s shape changes (denaturation) and the proteins cease to function– Not an active enzyme anymore – can’t do its job • Chemicals, radiation, and heat can alter or destroy nucleic acids– Can produce fatal mutations– Can halt protein synthesis through action on an enzymatic RNA moleculeThe Selection of Microbial Control Methods• Ideally, agents should be– Inexpensive– Fast-acting– Stable during storage: it will stay active – Capable of controlling growth and reproduction of every type of microbe while being harmless to humans, animals, and objects• Such ideal products do not exist and every agent has limitations and disadvantages• What affects the efficacy of antimicrobial control methods?– Site to be treated– Relative susceptibility of microorganisms– Environmental conditionsFactors Affecting the Efficacy of Antimicrobial Methods - Site To Be Treated• Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects• When performing medical procedures, the method and level of microbial control based on site of procedure because the site greatly affects the potential for subsequent infection- Relative Susceptibility of Microorganisms• Death rates do vary among microorganisms and viruses• Effectiveness of germicides classified as high, intermediate, or low– High-level kill all pathogens, including endospores– Intermediate-level kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria– Low-level kill vegetative bacteria, fungi, protozoa, and some virusesEnvironmental Conditions• Temperature and pH affect both death rates and the efficacy of antimicrobial methods– Warm disinfectants generally work better than cool ones: reflected in the graph; takes shorter amount of time when warmer – Acidic conditions enhance the antimicrobial effect of heat• Organic materials such as fat, feces, vomit, blood, and the intercellular matrix in biofilms interfere with penetration of heat, chemicals, and some forms of radiations, and in some cases these materials inactivate chemical disinfectants– Need to remove the organic things before disinfectionPhysical Methods of Microbial Control• Heat-Related Methods• Refrigeration and Freezing• Desiccation and Lyophilization• Filtration• Osmotic Pressure• RadiationHeat-Related Methods• For proteins to be active they need to be in tertiary structure– Heat takes the structure back to primary structure• Effects of high temperatures– Denaturation of proteins– Interference with integrity of cytoplasmic membrane and cell walls• Phospholipids start flying off– Disruption of structure and function of nucleic acids• Hydrogen bonds help the 2 strands stay together• Breaks the double helix• Thermal death point – lowest temperature that kills all cells in broth in 10 minutes– Temperature• Thermal death time – time to sterilize (killing all the organisms) volume of liquid at set temperature– Time• Decimal reduction time (D) – time required to destroy 90% of the microbes in a sample– Heat related – usually – Helps in canning foods Moist Heat• Used to disinfect, sanitize, sterilize, and pasteurize• Kills by denaturing proteins and destroying cytoplasmic membranes• More effective than dry heat; water (moisture) better conductor of heat than air– Boiling water on the stove and the water touching you vs. putting yourhand in the oven at a higher temperature– Water holds heat better• Methods of microbial control using moist heat– Boiling– Autoclaving– Pasteurization– Ultrahigh-temperature sterilization• Boiling– Kills vegetative cells (actively growing cells) of bacteria and fungi, protozoan trophozoites-life cycle in protozoan growth, and most viruses within 10 minutes at sea level– Boiling time is critical– Water boils at lower temperatures at higher elevations; requires longer boiling time– Endospores, protozoan cysts (not endospores but they are analogous to them), and some viruses can survive boiling–