pt 1 - micro.docx

Unit 3: Control of Microorganisms Section 1: Control of microbial growth Microbial control • External measures to keep pathogens away from body • Fomites- Inanimate object that serves to transmit disease Resistance: Highest - bacterial endospores Moderate - protozoan cysts - zygospores - naked viru...

Unit 3: Control of Microorganisms Section 1: Control of microbial growth Microbial control • External measures to keep pathogens away from body • Fomites- Inanimate object that serves to transmit disease Resistance: Highest - bacterial endospores Moderate - protozoan cysts - zygospores - naked viruses: hepatitis B; poliovirus - bacteria: Mycobacterium tuberculosis, Staphylococcus aureus and Pseudomonas Least - other bacteria - fungal spores and hyphae - enveloped viruses - yeast - protozoan trophozoites Methods of microbial control for use on fomites • Sterilization-complete destruction/removal of all living microbes, spores, and viruses on an object or in area. • Aseptic technique- steps to prevent contamination of sterile surfaces • Sanitization-remove microbes or reduce their populations to safe levels as determined by public health standards. • Disinfection- process of killing or inhibiting the growth of pathogens • Does not lead to sterilization • Ideal disinfectants should be fast acting, stable, easy to prepare, inexpensive, and easy to use. • ex: bleach • Antisepsis- Application of chemicals for removal of potential pathogens from living tissue • antiseptics must be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage. (ex: hydrogen peroxide) • Degerming- protocol that significantly reduces microbial numbers by using mild chemicals (e.g., soap) and gentle scrubbing of a small area of skin or tissue to avoid the transmission of pathogenic microbes. ( ex: handwashing) Measuring Microbial control -cide = to kill • Bactericide: destroys bacteria • Fungicide: destroys fungi • Viricide: destroys viruses • Sporocide: destroys spores • Inactivates major enzymes of an organism and interferes with its metabolism so that it dies. - static = stay still Bacteriostatic: inhibits/prevents the growth bacteria Fungistatic: inhibits/ prevents the growth fungi • Disrupts minor chemical reactions and slows the metabolism, which results in a longer time between cell divisions. Controlling Microorganisms. Cleaning products Sterile Surgical Instruments. • Microbial death- Permanent loss of reproductive capability; even under optimum growth conditions • Microbial death curve- graphical representation of the progress of a particular microbial control protocol • decimal reduction time (DRT) or Dvalue -amount of time it takes for a specific protocol to produce a one order of magnitude decrease in the number of organisms— death of 90% of the population Microbial death is logarithmic and easily observed using a semilog plot instead of an arithmetic one. The decimal reduction time (D-value) is the time it takes to kill 90% of the population (a 1-log decrease in the total population) when exposed to a specific microbial control protocol, as indicated by the purple bracket. • A definite proportion of the organisms die in a given time interval • The fewer organisms present, the shorter the time needed to achieve sterility • Microorganisms differ in their susceptibility to antimicrobial agents • The most susceptible phase for most organisms is the logarithmic growth phase Factors that affect effectiveness of microbial control protocol: • length of exposure • microbial load • nature of the microbe • action of the agent • temperature • pH • presence of solvents, interfering organic matter and inhibitors: Large amounts of saliva, blood and feces can inhibit the action of disinfectants and even heat Microbial death curve 13.2 Using Physical Methods to Control Microorganisms 1)Heat • Fast, reliable, inexpensive • Heat is applied above the maximal range for microbial growth, destroying cellular enzymes, which become irreversibly denatured • Important application in food industry • Thermal death time –minimal time necessary for killing population at a given temperature • Thermal death point- minimal temperature at which organism dies in a given time Dry heat • kills bacteria by oxidizing cellular components • Slower than moist heat Applications of dry heat • Incineration- uses direct flame, burns to ashes • Examples: • Flaming a loop- Placing loop containing bacteria directly in flame • Flaming mouth of tube gets rid of dust, and organisms that come into contact with mouth of tube • Bactericenerator • Incineration of carcasses of animals infected with deadly disease: anthrax (a) Sterilizing a loop, often referred to as “flaming a loop,” is a common component of aseptic technique in the microbiology laboratory and is used to incinerate any microorganisms on the loop. (b) Alternatively, a bactericinerator may be used to reduce aerosolization of microbes and remove the presence of an open flame in the laboratory. These are examples of dry-heat sterilization by the direct application of high heat capable of incineration. Applications of dry heat • Hot-air oven sterilization- • Uses radiating dry heat for sterilization • Does not penetrate materials easily • Long periods of exposure to high temperatures necessary • Temperatures include 160 C for 2 hours( ensures destruction spores) • 180C for 1 hour • Preferred over moist heat when sterilizing: • glassware to avoid any residual moisture that can contaminate the surface • Metallic medical instruments since dry heat is non-corrosive for metals • Powders, oils, and petroleum jelly since moist heat cannot penetrate grease. Moist heat • Kills bacteria by coagulating/denaturing proteins • Protein denaturation requires less energy than oxidation. Thus, less heat is required. • Faster than dry heat since water molecules conduct heat better than air. • Can be used at a lower temperature and shorter exposure time than dry heat. Applications of moist heat • Boiling- • Most microorganisms, as well as eukaryotic spores, can be killed within 10 minutes with viruses requiring 30 minutes and bacterial spores requiring 2 hours • Not reliable as sterilization procedure • Autoclaving-moist heat as pressurized steam • Heating for 15- 20 minutes at 121 C with 15 pounds of pressure per inch2. The pressure allows the temperature to rise above 100oC to 121 C. • prions are highly resistant (134 C for 18 min) • Used to control microorganisms in both hospitals and labs • Used for blankets, bedding, utensils, instruments • Important in making media Monitoring the Effectiveness of the Autoclave The white strips on autoclave tape (left tube) turn dark during a successful autoclave run (right tube). • Prevacuum autoclave- • draws air out of sterilizing chamber at the beginning of the cycle • A vacuum pump operates at the end of cycle to remove the steam and dry the load • Minimal exposure time for sterilization and reduced time to complete the cycle. • 132C to 134 C at pressure of 28-30lb/in2 for as little as 4 minutes. • Fractional sterilization/Tyndallization-moist heat without pressurized steam • free flowing steam at 100oC for 30 minutes on each of three consecutive days • Kills all vegetative cells on first day but not spores • Then viable cells resulting from the germination of spores are killed on the following days • Relies on having the proper conditions for germination of spores • Used for materials that cannot be sterilized by other methods • Pasteurization-heating food to a specific temperature and then cooling immediately. • Invented by Pasteur to destroy microbes that caused wine to sour • Does not achieve sterility: use of lower temperatures to destroy only pathogenic organisms. • Does not affect bacterial spores. • In fruit juices, targets enteric bacteria like Salmonella and E. coli. • In the case of milk, destroys yeast, Mycobacterium tuberculosis, Coxiella burnetii (hay fever). • Holding(batch) pasteurization method • Heating at 63oC for 30 minutes • High-Temperature Short-Time(HTST)/ Flash pasteurization • 71.6oC for 15 seconds • Most commonly used method • Ultra pasteurization method (UHT) 140oC for 3 seconds • Kills all bacteria • Used for organic milk since product has to travel longer • Two different methods of pasteurization, HTST and UHT, are commonly used to kill pathogens associated with milk spoilage. 2) Cold: Refrigeration and Freezing • Low temperatures slow spoilage by lowering metabolic rate of microorganisms • Refrigerator: ~4 C • Freezer: ~-18oC • Ultra low freezer: ~-70 C • Liquid Nitrogen ~-196 C • Limitations: psychrotrophs • Cultures and other medical specimens can be stored for long periods at ultra-low temperatures. (a) An ultra-low freezer maintains temperatures at or below −70 °C. (b) Even lower temperatures can be achieved through freezing and storage in liquid nitrogen. 3) Desiccation (Dehydration) • Used in preparation of various meats, fish, cereals, and other foods • Salting induces water to diffuse out of microorganisms via osmosis • Lyophilization- item is rapidly frozen (“snap-frozen”) and placed under vacuum so that water is lost by sublimation. • Limitations: fungi can tolerate low water (a) The addition of a solute creates a hypertonic environment, drawing water out of cells. (b) Some foods can be dried directly, like raisins and jerky. Other foods are dried with the addition of salt, as in the case of salted fish, or sugar, as in the case of jam. 4) Radiation A. Ionizing radiation: X, gamma rays • X and gamma rays both undergo ionizing radiation • consists of photons and/or moving particles (alpha, beta)that have sufficient energy to knock electrons out of the shells from either an atom or molecule, thus forming an ion. • Ions generated quickly combine with H2O, affecting metabolism and physiology • Used to • sterilize • heat-sensitive pharmaceuticals like vitamins, hormones, antibiotics • plastic items like Petri plates, intravenous tubing • latex items like gloves • Sutures • Tissues for transplantation • Preserve and extend the shelf life of food: irradiation of gamma rays produced during the natural decay of cobalt-60 or cesium-137. Pasteurizing dose used. • Limitations: Does not kill bacterial endospores, inactivate viruses, or neutralize toxins 4)Radiation B. Nonionizing radiation: UV rays • Non- ionizing radiation • Has a wavelength between 100 and 400nm, with 265nm(germicidal) being most destructive to cells • When DNA absorbs UV light of sufficient intensity and for enough time, thymine dimers are induced. • This disrupts DNA replication, as well as protein synthesis and the cell dies • Limits airborne, surface contamination (a) UV radiation causes the formation of thymine dimers in DNA, leading to lethal mutations in the exposed microbes. (b) Germicidal lamps that emit UV light are commonly used in the laboratory to sterilize equipment. 4) Radiation B. Nonionizing radiation: Microwaves • are basically extremely high frequency radio waves, and are made by various types of transmitters. • In a microwave oven, the food is cooked inside out. • Microwaves vibrate mainly water molecules, causing friction and thus heat to defrost and cook foods. • Fats and sugars also absorb microwaves. • They are not absorbed by plastics, glass, ceramics. • 2 minutes of microwaving on full power is enough to kill or inactivate 99% of all bacterial cells and endospores on a sponge. A sponge can be microwaved to kill microbial contaminants. 4) radiation • Deinococcus radiodurans: Able to survive 1000X the amount of radiation that would kill a human 5) filtration • Mechanical method that can be used to remove microorganisms from a solution or gas. • Important in separation of viruses from bacteria (manufacture of vaccines) • Can be used to sterilize substances that are destroyed by heat (drugs, serum, vitamins, sucrose) • To collect microorganisms from air and water samples (water quality testing) • Types of filters • High-efficiency particulate air(HEPA) filter • Consists of a mat of randomly arranged fibers that trap particles, microorganisms, and spores. • Can trap over 99% of all particles, including microorganisms and spores with a diameter larger than 0.3um • Incorporated into biological safety cabinets, surgical units, burn units, specialized treatment facilities (a) HEPA filters like this one remove microbes, endospores, and viruses as air flows through them. (b) A schematic of a HEPA filter. • Types of filters • Membrane filter- pad of cellulose acetate or polycarbonate mounted in a flask along with a vacuum pump • As fluid passes through the filter, organisms are trapped in the pores of the filter pad • Filter pad placed on a media plate◊ plate incubated overnight◊ colony count • Membrane filters come in a variety of sizes, depending on the volume of solution being filtered. (a) Larger volumes are filtered in units like these. The solution is drawn through the filter by connecting the unit to a vacuum. (b) Smaller volumes are often filtered using syringe filters, which are units that fit on the end of a syringe. In this case, the solution is pushed through by depressing the syringe’s plunger. FIGURE 13.17: FIGURE 13.18: 13.3 Using Chemicals to Control Microorganisms Principles of chemical control • Physical control of microorganisms generally serves the process of killing all microorganisms(sterilization) • Chemical control primarily targets pathogenic microorganisms • Disinfection- process of killing or inhibiting the growth of pathogens • Disinfectant-A chemical used to kill or inhibit pathogenic microorganisms on a lifeless object such as a table top.( phenolic compounds, hypochlorites, quaternary salts) • Antiseptic-a chemical used to reduce or kill pathogenic microorganisms on a living object, such as the surface of the human body. • (alcohols, iodine solution, silver nitrate) • Note: A chemical agent can be used as both as disinfectant and antiseptic but the concentration would vary Types of disinfectants and antiseptics • Phenolic compounds • Phenol (carbolic acid) Remember Joseph Lister? • Standard for comparison against other disinfectants/antiseptics • Active against Gram positive • Inactivate enzymes, denature proteins, especially in cell membrane • Limitations: not effective as antiseptic: expensive, pungent odor, caustic to skin • Phenol derivatives- Greater germicidal activity and lower toxicity than phenol • Hexylresorcinol: mouthwash, topical creams, throat lozenges • Reduces surface tension • Phenol derivatives- • Bisphenols- combination of two phenol compounds • Prominent in modern disinfection and antisepsis Orthophenylphenol-used in Lysol and Amphyl, control bacterial and fungal growth on harvested citrus fruits Hexachlorophene (bisphenol) - toothpastes, deodorants, bath soaps is the active ingredient in pHisoHex. Triclosan- disrupts cell membranes by blocking synthesis of lipids Antibacterial soaps, lotions, kitchen sponges, cutting boards Chlorhexidine-surgical scrub, hand wash, superficial skin wound cleanser • Heavy metals- donate electrons and large atomic weight • Combine with proteins and inactivate them: form disulfide bridges between proteins, disrupting metabolism, and thereby killing the organism • Skin antiseptics, swimming pools, water supply • Limitations: not sporicidal • Heavy metals denature proteins, impairing cell function and, thus, giving them strong antimicrobial properties. (a) Copper in fixtures like this door handle kills microbes that otherwise might accumulate on frequently touched surfaces. (b) Eating utensils contain small amounts of silver to inhibit microbial growth. (c) Copper commonly lines incubators to minimize contamination of cell cultures stored inside. (d) Antiseptic mouthwashes commonly contain zinc chloride. (e) This patient is suffering from argyria, an irreversible condition caused by bioaccumulation of silver in the body. Types of disinfectants and antiseptics Mercury- very toxic and activity reduced in presence organic matter Less toxic when combined other compounds Merbromin, thimerosal- combined with carrier compounds and less toxic to skin Thimerosal used previously as preservative in vaccine Silver – as AgNO3, useful as antiseptic and disinfectant Copper- active against chlorophyll-obtaining organisms, especially algae Used in swimming pools, fish tanks, and municipal water supplies as copper sulfate Halogens- iodine and chlorine commonly used as disinfectants Oxidize cell components: inactivate enzymes by causing the release of atomic oxygen Combine with ions in water Active against Gram positive and negative bacteria, algae, protozoa, viruses, and biofilms Killing within 30 minutes of application Used in: municipal pools, hospitals, factories, purification of water Limitations: Cannot destroy spores • Available as inorganic compounds gas form(ClO2 ) bleach paper, sterilize medical equipment solid form (NaClO(bleach) , Ca(ClO)2 ) Clump cellular proteins Organic compounds Chloramines- disinfection drinking water Iodophors- iodine linked to solubilizing agent Reduces surface tension and reacts with enzymes in ETC and proteins in cell wall Release iodine over a long time without staining tissues or fabrics Povidone- iodine(Betadine)- wounds, antisepsis before incision Alcohols- EtOH Active against vegetative cells Denatures proteins, dissolves lipids Strong dehydrating agent • Hand sanitizers, used to treat skin before venipuncture Limitations: skin infections Isopropyl alcohol Rubbing alcohol Surfactants Soaps-chemical compounds of fatty acids combined with potassium or sodium hydroxide. Ph 8 Not considered disinfectants or antiseptics Work primarily as degerming agents, and surfactants- emulsify and solubilize particles attached to surfaces by reducing the surface Detergents Strong surfactants Attracted to phosphate groups of cellular membrane Most useful are quaternary ammonium salts-cationic derivatives of Ammonium chloride These react with cell membranes and can destroy some bacterial species and enveloped viruses Bacteriostatic, especially Gram-positive, little odor Benzalkonium chloride, cetylpyridinium chloride Sanitizing agents for food prep surfaces, industrial equipment, food utensils, skin antiseptics, mouthwashes, contact lens cleaners Limitations: reduced activity when mixed with soaps, certain Gram negative bacteria can grow in them (a) Two common quats are benzalkonium chloride and cetylpyridinium chloride. Note the hydrophobic nonpolar carbon chain at one end and the nitrogen-containing cationic component at the other end. (b) Quats are able to infiltrate the phospholipid plasma membranes of bacterial cells and disrupt their integrity, leading to death of the cell. Types of sterilizing agents Alkylating agents Cross link nucleic acids and proteins by reacting with amino and hydroxyl groups. Replace hydrogen atoms with alkyl groups Aldehydes Formaldehyde: embalming fluid, inactivation of viruses in certain vaccines, and toxins(toxoids) Glutaraldehyde: destroys bacterial cells within 10 to 30 minutes and spores in 10 hours Sterilization delicate objects like optical equipment Limitations: gives off irritating fumes, instruments must be thoroughly rinsed Gases Ethylene oxide Microbicidal and sporicidal cold sterilization, making it useful for the sterilization of heat-sensitive items Used to sterilize paper, plastics, leather, wood, metal, and rubber, interplanetary space capsules Good for items within plastic bags (highly penetrating) In medicine: catheters, artificial heart valves, heart-lung machine components, optical equipment Limitations: carcinogenic, skin irritant, and highly explosive (a) Alkylating agents replace hydrogen atoms with alkyl groups. Here, guanine is alkylated, resulting in its hydrogen bonding with thymine, instead of cytosine. (b) The chemical structures of several alkylating agents. Types of disinfectants and antiseptics Peroxygens Peroxides- compounds containing oxygen single bonds Hydrogen peroxide- used as a rinse in wounds, scrapes, and abrasions Broken down by catalase in tissue Results in superoxide radical which is toxic to bacteria Loosens dirt, debris, and dead tissue Benzoyl peroxide- active ingredient in teeth whitening and to treat acne 13.4 Testing the Effectiveness of Antiseptics and Disinfectants Categories of effectiveness of chemical disinfectants High-level germicides have the ability to kill vegetative cells, fungi, viruses, and endospores, leading to sterilization, with extended use. Intermediate-level germicides are less effective against endospores and certain viruses low-level germicides kill only vegetative cells and certain enveloped viruses, and are ineffective against endospores. What makes a chemical agent a good disinfectant or antiseptic? Able to kill or slow the growth of microorganisms Nontoxic to animals or humans, especially if it is an antiseptic Readily available Inexpensive Soluble in water Effective in diluted form Not separate on standing Substantial shelf life Non-corrosive Able to perform its job in a relatively short time Factors to consider when selecting a disinfectant or antiseptic Temperature- keep in mind that reaction that occurs at body temp may not occur at room temp pH- some chemicals only effective at particular pH Stability- May prefer slower reaction when long –term disinfection is desired Type of microorganism targeted- Gram positive more susceptible Pseudomonas can grow in disinfectants and antiseptics M. tuberculosis resistant to many disinfectants Type of surface to be treated- wound or bench Evaluating effectiveness of disinfectants and antiseptics Phenol coefficient (PC) - number indicating disinfecting ability of an antiseptic or disinfectant in comparison to phenol under identical conditions. Agent is mixed with standardized bacterial species Test which dilutions kill the organisms after a 10 minute exposure Limitations: Does not take into account temperature variations, toxicity, or presence in organic matter Disk Diffusion Method involves applying different chemicals to separate, sterile filter paper disks. The disks are then placed on an agar plate that has been inoculated with the targeted bacterium and the chemicals diffuse out of the disks into the agar where the bacteria have been inoculated. • A disk-diffusion assay is used to determine the effectiveness of chemical agents against a particular microbe. (a) A plate is inoculated with various antimicrobial discs. The zone of inhibition around each disc indicates how effective that antimicrobial is against the particular species being tested. (b) On these plates, four antimicrobial agents are tested for efficacy in killing Pseudomonas aeruginosa (left) and Staphylococcus aureus (right). These antimicrobials are much more effective at killing S. aureus, as indicated by the size of the zones of inhibition. Use-dilution test- Technique for determining the effectiveness of a chemical disinfectant on a surface standardized cultures are dried on small stainless steel cylinders, then exposed to the chemical agent, then transferred to a growth medium In-use test- a technique for monitoring the correct use of disinfectants in a clinical setting involves placing used, diluted disinfectant onto an agar plate to see if microbial colonies will grow can determine whether disinfectant solutions are being used correctly in clinical settings Example: floor is swabbed before and after application • Used disinfectant solutions in a clinical setting can be checked with the in-use test for contamination with microbes.