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Reasons for Microbial Control1. prevention of disease transmission and infection 2. prevention of decomposition and spoilage 3. prevention of contamination (communication of disease organisms through contact, dispersal, fomites) Microbial control is necessary to maintain a balance between man and microbes. The methods of control are: Physical and Chemical The results of control are: Stasality and Cidality Growth in most organisms is represented as an orderly increase in all cell components and activity within the cell as well as an increase in the overall number of cells. In microbiology, growth is usually defined more narrowly as an increase in the total number of cells or an increase in microbial mass (as in colony size). GROWTH RATE is defined as the amount of growth/unit of time. Multiplication usually takes place in a GEOMETRIC or EXPONENTIAL fashion rather than arithmetically. GENERATION TIME equals the length of time required for one organism to become two organisms. STASALITY (stasis; static) organisms do not reproduce; a permanent period of stasis is death, growth is inhibited; recovery is possible if cause of stasality is removed CIDALITY (cide; cidal) absolute death, no possibility of reproduction, all metabolic functions eradicated Mode of Action - How Control Agents Work
Damage usually occurs at the level of: a) the cell wall b) the cell membrane c) the internal proteins and cytoplasm
a) Damage to the cell wall - maintains the integrity of the cell cell and is the site of various enzymatic reactions; porous but protective; some Gram + bacteria are attacked by LYSOZYME (enzyme in tears, mucous secretions, white blood cells) which helps control skin staph; penicillins and cephalosporins inhibit cell wall synthesis in rapidly growing cultures b) Alteration of Cell Membrane Permeability - responsible for selective transport; "leakage" is term used when cell membrane is destroyed; phenolic compounds, detergents, quaternary ammonium compounds, all destroy selective permeability and cause leakage; alcohol (dissolve lipids and denature proteins) and heat have similar effects c) Alteration of Colloidal Nature of Cytoplasm - death is rapid when proteins are denatured; alcohol and high temperatures, organometallics; denature proteins and coagulate proteins d) Inhibition of Enzyme Action - cytochrome oxidase is inhibited by cyanide, trivalent arsenic interferes with the Krebs cycle' glycolysis is inhibited by halogens (chlorine, fluorine, etc.) mercury, hydrogen peroxide, sulfanilamide, some antibiotics, usually stop protein synthesis; oxidative phosphorylation is stopped by the nitrophenols e) Interference with Synthetic Processes - antimetabolites; blockage of folic acid by sulfonilamides needed for PABA; common TB treatment
Conditions Influencing Antimicrobial Action
1. Concentration & Kind of Agent usually the more concentrated the agent the more cidal the action; many products, if used in low concentrations, will act as a stimulus to microbes 2. Intensity & Nature of the Physical Agent the more intense the heat or radiation the more rapid the kill; consideration is needed as to what happens to other compounds present as result of action of agent 3. Time the longer the time in contact, the greater the kill 4. Temperature the higher the temperature the more effective the kill 5. Number of organisms greater numbers require longer time 6. Kinds of organisms Pseudomonas aeruginosa is one of the toughest to kill, Mycobacterium with its tough waxy walls and encapsulated forms are also difficult 7. Nature of the materials bearing the organism the microenvironment; organic matter inactivates certain chemicals; high protein concentrations may clog filtration systems
Standards are developed by the Food and Drug Acts; the first ones were developed by the Theodore Roosevelt administration. Over the years the standards became tougher as the attitudes of industry and laymen began to change and recognize the need for control. The biggest change occurred after 1969 with the adoption of the AOAC UseDilution Test (Association of Official Analytical Chemists). This test replaced the Phenol Coefficient Test because it provided a means of determining effectiveness of the agent under the conditions of its usage, rather than simply in vitro. Eventually it became the province of the Public Health Administration to determine what would be considered safe levels or standards. This is was purely administrative bureau which used the results of testing done by industry to determine the standards. The Public Health Laboratories are required to do the testing to determine if these standards are being met.
The 1st Food and Drug Act contained the following information: "the language used in the label is to be given the meaning ordinarily conveyed by it to those to whom it is addressed" This was the first attempt to require a label which would indicate the contents of the product, and use common language to explain it. This brought about a dearth of ambiguous terms. The following terms were finally agreed to by the FDA and Industry. Sterilization the act or process of destroying all forms of life on an object or in a material; this includes the destruction of endospores; sterilization is absolute, there are no degrees of sterilization HOWEVER, the literature is ambiguous about viruses; if viruses are considered life forms then they can be killed, if not then it is possible, at least semantically, for a solution to be sterile and contain viruses Disinfectant usually chemicals which kill growing forms (vegetative) of pathogens but not necessarily spore forms or encapsulated forms or viruses; they are commonly applied to inanimate objects and can be used as sprays or fogs; dependent on concentration they can be static or cidal; a minor consideration is the fact that our understanding of what constitutes a pathogen has changed since the original term was defined; the bottom line is simply to READ ALL LABELS CAREFULLY Antiseptic a chemical agent that arrests or prevents the growth or action of microbes; it controls by destruction or inhibition dependent upon concentration; works on cuts, scrapes, abrasions, wounds on or in the body; ERGO chemical disinfection of living tissue Sanitizers - chemical agents that reduce microbial populations to safe levels (as determined by the PHS); usually destroys 8090 percent; normally used on inanimate objects such as in dairy industry; more importantly to the public sanitizers imply cleanliness as well as disinfection PHYSICAL METHODS OF MICROBIAL CONTROL A. Temperature must take into account what is being subjected to high or low temperature Thermal Death Point (TDP) lowest temperature required to kill ALL microorganisms in a liquid suspension within 10 minutes (time is held constant, temperature varies) Thermal Death Time (TDT) minimal length of time in which all bacteria in a liquid culture will be killed at a given temperature (temperature is held constant, time varies) useful in situations such as pasteurization where certain products will be rendered unusable if temperature becomes too high 1. Flame or incineration used for inoculating loops and needles and for contaminated paper cups, bags, dressings. 2. Dry heat sterilization normally used for drying glassware requires minimum temperature of 1700 C for two hours to ensure sterilization 3. Boiling need a minimum of 10 continuous minutes of boiling to destroy spores of fungi, many viruses, and most bacterial pathogens; endospores and some viruses may require up to 25 HOURS for destruction 4. Autoclaving moist heat, greater penetration, steam under pressure; normally requires 1210 C at 1 atmosphere of pressure beyond normal atmospheric pressure; length of time varies with type of material being autoclaved 5. Low temperature normally used to restrict reproduction; may slow down enzymatic action but does not stop it; freezing or cold normally does not kill unless done slowly so that ice crystals may develop within the cells B. Filtration difficult to use in high protein content materials; will cause clogs in filter; cannot be trusted to remove all viruses; best used to determine water quality C. Dessication reproduction affected but viability of many organisms is unchanged; ineffective against endospores and some viruses; used in preservation certain foods D. Osmotic Pressure increase in sugar or salt concentration will lead to dessication and is common method of food preser- vation; may not be effective against all fungi, endospores, or some viruses E. Electricity poor method; hard to electrocute bacteria; really a combination of heat and dissociation of gases; ozone is formed; still used in milk and juice industries F. Sonic & Ultra Sonic - causes cavitation (thunder following lightning); molecules are moved out leaving a cone; as the vacuum becomes filled the molecules smash together; causes cracking or bursting more effective against rods than cocci; Gram + more resistant, acid fast and spores very resistant; no assurance of total sterility; used most frequently for disruption of eukaryotic cells G. Radiation ionizing radiation such as Xrays or gamma rays are most effective; Xrays are very penetrating but expensive; Gamma radiation are extremely penetrating Nonionizing radiation such as ultraviolet radiation causes changes in DNA molecules by causing thymine dimers to form and makes separation at the time of replication difficult if not impossible; UV rays at 260 nm or 2650 A is considered germi- cidal; UV converts oxygen into ozone which is an effective oxidant; poor penetration, usually only on the surface effects may be reversible; UV is adsorbed to protein and destroys enzyme systems;potentially damaging to eyes and skin CHEMICAL METHODS OF MICROBIAL CONTROL Important considerations for chemical killing agents include: 1. spectrum of kill (types of organisms) 2. effective dilution Ideal Disinfectant - Characteristics Looked for by FDA 1. Toxicity to microbes broad spectrum, effective against spores, waxy coats; lowest concentration still effective 2. Solubility in water, in tissue fluids; aesthetic qualities clear or cloudy 3. Stability shelf life water soluble products tend to last long; alcohol based products have reduced shelf life 4. Toxicity to man, other animals and plants skin tests, kidneys will usually show first; conjunctiva test and respiratory tests; if substance goes in or on the body it is under the control of FDA and the National Institutes of Health studies the protocol aspects 5. Homogeneity of mixture will it separate on standing 6. Combining power with organic substances want low affinity or will cause dilution test with organisms coated with mucin; does it combine with lipids in the body, toxic to man 7. Temperature toxicity at room temperature or body temperature; materials must not change at very high or very low temperature on the shelf 8. Penetrating power - few compounds penetrate skin (ex. DMSO); usually refers to cut, wounds, abrasions 9. Noncorroding and nonstaining 10. Deodorizing quality organometallics are especially good; usually the result of washing out of the atmosphere 11. Detergent action removal of dead tissue from wounded area 12. Availability in large quantities 13. Affordable