Microbiology Test 1: Chapter 12
Phenols disrupt membranes, denature proteins, and inactivate enzymes; not impaired by organic matter
Phenols: denature proteins, disrupt membranes, inactivate enzymes Disinfect surfaces. Not affected by organic material Lysol - residue for several days Halogenated phenols - increases effectiveness Hexachlorophene - inhibit Staphylococci and fungi - neurotoxicity Chlorhexidine gluconate - surgical scrubs Triclosan - in antimicrobial soaps - bacteria resistance
Other Agents
Plant oils - used as antimicrobials Thymol is used as a preservative and eugenol is used in dentistry to disinfect cavities Sulfates, nitrates - as food preservatives
Soaps and Detergents Lower surface tension, make microbes accessible to other agents Surfactants: Dissolve lipids, disrupt membranes, denature proteins, andinactivate enzymes in high concentrations; act as wetting agents in low concentrations
Surfactants - lower surface tension, dissolve lipids, disrupt membranes, denature proteins Soaps - contain alkali and sodium Hand washing - prevent spread of infections (microorganisms) Cationic detergents are quaternary ammonium compounds (quats) - react with membranes - non-toxic, sanitize food, utensils Zephiran (benzalkonium chloride) - skin antiseptic - no longer is recommended -Soaps and detergents remove microbes, oily substances, and dirt. -mechanical scrubbing enhances their action -soaps contain alkali and sodium and will kill many species of Streptococcus, Micrococcus, and Neisseria and will destroy influenza viruses. -detergents when used in weak concentrations in wash water, allow the water to penetrate into all crevices and cause dirt and microorganisms to be lifted and washed away. -Detergents are said to be cationic if they are positively charged, and anionic if they are negatively charged. -Cationic detergents are used to sanitize food utensils, do not kill endospores but can inactivate some viruses. -Anionin detergents are used for laundering clothes and household cleaning agents, they are less effective sanitizing agents because the negative charges on bacterial cell walls repel them. -many cationic detergents are quaternary ammonium compounds or whats, which have four organic groups attached to a nitrogen atom. -a variety of quats are available as disinfecting agents; their chemical structures vary according to their organic groups. there effectiveness is decreased in the presence of soap, calcium or magnesium ions, or porous substances such as gauze. -support the growth of some bacteria of the genus pseudomonas rather than killing them.
Sterilization
The killing or removal of all microorganisms in a material or on an object.
Disinfection
The reduction of the number of pathogenic microorganisms to the point where they pose no danger on the disease.
Radiation
control microorganisms, preserve food
Acids and Alkalis Acids: Lower pH and denature proteins Alkalis: Raise pH and denature proteins
denature proteins Soaps are alkali Acids - inhibit fermentation - food preservatives -soap is a mild alkali and its alkaline properties help destroy microbes. -a number of organic acids lower the pH of materials sufficiently to inhibit fermentation. -several are used as food preservatives -lactic and propionic acids retard mold growth in breads and other products -benzoic acid and several of its derivatives are used to prevent fungal growth in soft drinks, ketchup and margarine -sorbic acid and sorbets are used to prevent fungal growth in cheeses and a variety of other foods -boric acid, formerly used as an eyewash, is no longer recommended because of its toxicity.
Ultraviolet Light Denature proteins and nucleic acids
denatures proteins, nucleic acids Endospores are resistant to UV -UV light consists of light of wavelengths between 40 and 390 nm, but wavelengths in the 200 nm range are most effective in killing microorganisms by damaging DNA and proteins. -UV light is absorbed by the purine and pyramiding bases of nucleic acids -this absorption can permanetly destroy these important molecules -effective in inactivating viruses Surfaces in operating rooms. Sewage treatment
Sterilant
destroy all microorganisms, endospores and viruses, Treat scalpels, heat sensitive instruments
Sonic and Ultrasonic Waves Cause cavitation
disrupt cells, proteins. Not for sterilization --cavitation: the formation of a partial vacuum in a liquid-in this case, the fluid cytoplasm in the bacterial cell -enzymes used in detergents are obtained by cavitating the bacterium Bacillus subtillus -Sonication: the disruption of cells by sound waves -useful in fragmenting cells to study membranes, ribosomes, enzymes and other components
Low-level disinfectant
fungi, vegetative bacteria except Mycobacteria, enveloped viruses -general purpose disinfectants
Freezing Greatly slows the rate of most enzyme controlled reactions
greatly slows enzyme reactions Freezing at -20C is used to preserve foods in homes and in the food industry Keep food few months, does not sterilize
Osmotic Pressure Removes water from microbes
increase osmotic pressure of environment (salt, sugar )- remove water from cell (Plasmolysis) Prevent food spoilage, food preservation
Freeze-Drying (lyophilization) Dehydration inhibits enzymes
inhibit enzymes. Food Preserve cultures of microorganisms -the drying of a material from the frozen state -microbiologists use this for long term preservation rather than for destruction of cultures of microorganisms.
Drying Inhibits Enzymes
inhibits enzymes Preserve foods - fruits, etc -the absence of water inhibits the action of enzymes -endospores can survive but don't produce toxins Minimizes spread of infection (Treponema pallidum-causes syphilis)
Microwave Radiation absorbs water molecules, then releases microwave energy to surroundings as heat
not reliable
Halogens Oxidize cell components in absence of organic matter
oxidize proteins, cell components Hypochlorous acid (chlorine in water) - drinking water, swimming pools, also used to disinfect food utensils, and in household bleach. Chlorine is easily inactivated by the presence of organic materials. Iodine - skin antiseptics Iodophores: slow-release of iodine, molecules act as surfactants -Betadine, Isodine, used for surgical scrubs and on skin where an incision was made -Bromine is sometimes used in the form of gaseous methyl bromide to fumigate soil, also used in pools and hot tubs Chloramine (chlorine and ammonia) - wound cleansing root canal therapy, eliminates taste and odor problems -
Moist Heat Denatures Proteins
protein denaturation in presence of water (disrupt 3-dimensional structure), disrupt membrane lipids, inactivate viruses, and under pressure disrupt nucleic acids Penetrates better than dry heat Boiling water kills cells, not endospores Pressure increases boiling point of water Autoclave - pressure 15 lb/square inch, temperature 121C for 15-20 min.- kill all ( Fig. 12.10, 12,11) Sterilization by autoclaving important in Labs., hospitals: media, bandages, equipment, Food canning
Heavy Metals Denature proteins
selenium, mercury, copper, silver Attach to SH groups - denature proteins Silver nitrate -silver nitrate was once widely used to prevent gonococcal infection in newborn infants, a few drops of silver nitrate solution were placed in a baby's eyes at the time of delivery to protect against infection of gonococci entering the eyes during passage through the birth canal. --organic mercury compounds, such as merthiolate and mercurochrome, are used to disinfect surface skin wounds. -such agents kill most bacteria in the vegetative state but do not kill spores, they are not effective against mycobacterium -Merthiolate is generally prepared as a tincture, dissolved in alcohol -Selenium Sulfide kills fungi, including spores -copper sulfate is used to control algal growth
Cold
slows growth by slowing the rate of the enzyme-controlled reactions that control metabolism Does not achieve sterility, does not kill many organisms
Refrigeration Slows the rate of enzyme controlled reactions
slows rate of enzyme-controlled reactions. -many fresh foods can be prevented from spoiling by keeping them at 5C Prevent fresh food from spoiling for few days
intermediate level disinfectant
vegetative bacteria, mycobacteria, fungi, most but not all viruses. Non-critical instruments - stethoscopes
High-level disinfectants
viruses, vegetative cells. Treat gastrointestinal endoscopes
Ionizing Radiation Denatures proteins and nucleic acids
(x-rays, gamma rays) - denatures proteins, nucleic acids Kills microrganisms, viruses Sterilize plastic, equipment, pharmaceuticals. Prevent food from spoiling -X-Rays which have wavelengths of .1 to 40 nm, and gamma rays, which have even shorter wavelengths, are forms of ionizing radiation, because they can dislodge electrons from atoms, creating ions. -kill microorganisms and viruses -a rad is a unit of radiation energy absorbed per gram of tissue -humans usually do not become ill from radiation unless they are subjected to greater doses greater than 50 rads -ionizing radiation damages DNA and produces peroxides which act as powerful oxidizing agents in cells -this radiation can also kill or cause mutations in human cells if it reaches them
Principles and Applications of Heat Killing
-A preferred agent of sterilization for all materials not damaged by it, it rapidly penetrates thick materials not easily penetrated by chemical agents. -Thermal Death Point: the temperature that kills all bacteria in 24 hour old broth culture at neutral pH in 10 minutes. -Thermal Death Time: the time required to kill all the bacteria in a particular culture at a specified temperature. -Decimal Reduction Time (DRT) or (D-Value): the length of time needed to kill 90 percent of the organisms in a given population at a specified temperature.
Potency
-The potency (effectiveness) of a chemical antimicrobial is affected by time, temperature, pH, and concentration. -the death rate of organisms is affected by the length of time the organisms are exposed to the antimicrobial agent. -Thus adequate time should always be allowed for an agent to kill the maximum number of organisms -death rate is accelerated with heat -increasing the temp by 10 degrees roughly doubles the rate of chemical reactions and increases the potency of the chemical agent -acidic or alkaline pH can increase or decrease potency -if it increases the degree of ionization of a chemical agent it often increases its ability to penetrate a cell -increasing concentration can increase the effects of antimicrobial chemical agents -High concentrations=Bactericidal -Low concentrations=Bacteriostatic -Alcohol is the exception to this, they are more potent at 70 percent than at higher concentrations, water must be present for alcohols to disinfect because they act by coagulating (permanently denaturing) proteins, and water is needed for coagulation reactions. - Dirt, grease, organic compounds can interfere with action of agents
Phenol coefficient
-compare disinfectants to standard phenol -if its phenol coefficient is less than 1 it is less effective than phenol if its phenol coefficient is more than 1 it is more effective than phenol.
Reactions that Affect Viruses
-control of viruses requires that they be inactivated, that is, rendered permanently incapable of infecting or replicating in cells -inactivation can be effected by destroying either the viruses nucleic acid or their proteins -alkylaating agents, such as ethylene oxide, nitrous acid, and hydroxyl amine, act as chemical mutagens--they alter DNA or RNA -if the alteration prevents DNA or RNA from directing the synthesis of new viral particles, the alklylating agents are effective inactivators -detergents, alcohols, and other agents that denature proteins act on bacteria and viruses in the same way -dyes render viruses susceptible to inactivation when exposed to visible light, this process disrupts the structure of the viral nucleic acid -viruses sometimes remain ineffective even after their proteins are denatured, so methods used to rid materials of bacteria may not be as successful with infectious viruses -also use of an agent that does not inactivate viruses can lead to lab acquired infections
Reactions that Affect Proteins
-in denaturation, hydrogen and disulfide bonds are disrupted, and the functional shape of the protein molecule is destroyed. -any agent that denatures proteins prevents them from carrying out their normal functions -when treated with mild heat or some dilute acids, alkalis, or other agents proteins are temporarily denatured. -after the agent is removed some proteins can regain their normal structure -reactions that denature proteins include: hydrolysis, oxidation, and the attachment of atoms or chemical groups
Reactions that Affect Membranes
-membranes contain proteins and so can be altered by all the preceding reactions -membranes also contain lipids and thus can be disrupted by substances that dissolve lipids -Surfactants: are soluble compounds that reduce surface tension, just as soaps and detergents break up grease particles in dishwater -these include: alcohols, detergents, and quaternary ammonium compounds such as benzalkonium chloride (dissolves lipids), phenols which are alcohols dissolve lipids and also denature proteins -detergent solutions, also called wetting agents, are often used with other chemical agents to help the agent penetrate fatty substances -detergents do not kill organisms but get rid of lipids so that agents can reach the organism
Reactions that Affect Other Cell components
-nucleic acids and energy producing systems are also affected by chemical agents -alkylating agents can replace hydrogen or amino or alcohol groups in nucleic acids -certain dyes, such as crystal violet, interfere with cell wall formation -some substances such as lactic acid and propionic acid, inhibit fermentation and thus prevent energy production in certain bacteria, molds and other organisms
Dry Heat Denatures Proteins
-penetrates substances more slowly than most heat protein denaturation by oxidation Sterilize metal objects, glassware, oils, powders aerosols: droplets released into the air
Evaluation of Effectiveness of Chemical Agents
-phenol coefficient -filter paper method -the use-dilution test
Pasteurization Denatures Proteins
-process invented by pasteur to destroy organisms that caused wine to sour, does not achieve sterility. denatures proteins Kills pathogens in milk, dairy products, beer, Salmonella, Mycobacterium Flash method 71.6C for 15 sec. Holding method 62.9C for 30 min. -Ultrahigh temperature processing: raises the temperature from 74 to 140 then drops it back to 74 in less than 5 seconds, prevents milk from touching surface hotter than itself
The Filter Paper Method
-simpler than determining a phenol coefficient -uses small paper disks each soaked with different chemical agents -disks are placed on the surface of an agar plate that has been inoculated with a test organism -after incubation a chemical agent that inhibits growth of a test organism is identified by a clear area around the disk where the bacteria have been killed
Filtration Mechanically removes microbes
-the passage of material through a filer, or straining device -mechanically remove microbes by using different size pore filters (T. 12.4, Fig.12.19) Sterilize materials damaged by heat: media, nutrients, pharmaceuticals, drugs, vitamins
The Use-Dilution Test
-uses standard preparations of certain test bacteria -a broth culture of one of these bacteria is coated onto small stainless steel cylinders and allowed to dry -each cylinder is then dipped into one of several dilutions of the chemical agent for 10 minutes, removed, rinsed with water, and placed into a tube of broth -the tubes are incubated and then observed for the presence or absence of growth -agents that prevent growth at the greatest dilutions are considered the most effective
Disinfectant Selection
1. Be fast acting even in the presence of organic substances, such as those in body fluids 2. Be effective against all types of infectious agents without destroying tissues or acting as a poison if ingested 3. Easily penetrate material to be disinfected without damaging or discoloring the material 4. Be easy to prepare and stable even when exposed to light, heat, or other environmental factors. 5. Be inexpensive and easy to obtain and use 6. Not have an unpleasant odor.
Antiseptic
A chemical agent that can safely be used externally on living tissue to destroy microorganisms or to inhibit their growth.
Sanitizer
A chemical agent typically used on food handling equipment and eating utensils to reduce bacterial numbers so as to meet public health standards. Sanitization may simply refer to thorough washing with only soap or detergent.
Disinfectant
A chemical agent used on inanimate objects to destroy microorganisms. Most disinfectants do not kill spores.
Alcohols denature proteins when mixed with water
Alcohols: skin antiseptics - not sterilize Mixed with water - denature proteins Lipid solvents - dissolve membranes Ethyl and isopropyl - 70%
Alkylating Agents Disrupt structure of proteins and nucleic acids
Alkylating agents: disrupt the structure of both proteins, and nucleic acids (Can cause cancer) Formaldehyte - inactivate viruses, toxins without destroying their antigenic properties Glutaraldehyde - kills microorganisms and spores, sterilize equipment Ethylene oxide (gas) - great penetrating power. Sterilize objects, extremely toxic to skin eyes and mucous membranes and can cause cancer
Germicide
An agent capable of killing microbes rapidly, some such agents effectively kill certain microorganisms but only inhibit the growth of others.
Viricide
An agent that inactivates viruses
Bacteriostatic Agent
An agent that inhibits the growth of bacteria
Bactericide
An agent that kills bacteria. Most such agents do not kill spores
Sporocide
An agent that kills bacterial endospores or fungal spores.
Fungicide
An agent that kills fungi
Strong Visible Light Oxidation of light sensitive materials
Bactericidal, due primarily to the UV rays in the sunlight. -oxidizing light sensitive molecules such as riboflavin and porphyrins (components of oxidative enzymes) in bacteria. -combination of a dye and strong light can be used to rid materials of both bacteria and viruses -light causes fluorescent dyes to absorb energy and cause oxidation of proteins and nucleic acids
The Control of Microbial Growth
Consideration should be given to: -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.
Dyes May interfere with replication or block cell wall synthesis
Dyes: Acridine - interferes with cell replication by causing DNA mutations - clean wounds methylene blue inhibits growth of some bacteria in cultures crystal violet blocks cell wall synthesis
Physical Antimicrobial Agents
Important in prevention of infectious diseases, and preservation of food.
Oxidizing Agents Disrupt disulfide bonds, therefore denature proteins
Oxidizing agents: disrupt disulfide bonds in proteins - protein denaturation Hydrogen peroxide forms superoxide - kills obligate anaerobes - wound cleansing Potassium permanganate - disinfect instruments
