Control of Microbial Growth PDF

**Control of Microbial Growth** - Control of microbes in health care facilities, laboratories, and at home is a critical aspect of microbiology - Prevention of disease is more important than treatment of disease because it provides an economic savings and prevents morbi...

**Control of Microbial Growth** - Control of microbes in health care facilities, laboratories, and at home is a critical aspect of microbiology - Prevention of disease is more important than treatment of disease because it provides an economic savings and prevents morbidity and mortality associated with disease. **[Basic Principles of Microbial Control]** **Terminology** - It is important that scientists, healthcare workers, and microbiologists use correct terminology when describing microbial control to ensure that there is no miscommunication - **Sterilization** - The removal or destruction of **ALL** microbes, including viruses and bacterial endospores in or on an object - This term does **NOT** apply to prions (infectious proteins) because standard sterilization techniques do not eradicate them - **Aseptic** - An environment or procedure that is free of contamination by pathogens - E.g. surgeons use aseptic techniques in operating room to prevent spreading microbes to patients - **Disinfection** - The use of physical or chemical agents, known as disinfectants, (e.g. bleach, Lysol, UV light, heat, alcohol, etc\...) to destroy microorganisms - Unlike sterilization does not kill **all** pathogens. Disinfectants alone cannot kill endospores or certain viruses - The term disinfection is used **ONLY** when referring to the treatment of inanimate objects - When chemical is used on skin or other biological tissue it is called **antisepsis** - The chemical is called an **antiseptic** - **Degerming** - The removal of microbes from a surface through the **process of scrubbing** - Scrubbing is often used in concert with a chemical agent, but the term refers specifically to the act of scrubbing - **Sanitization** - The process of disinfecting places and utensils **used by the public** to reduce the number of pathogenic microbes to meet accepted public health standards - **Pasteurization** - The process of heating a food or beverage just enough to reduce the number of microorganisms but not affect the quality of the product - Does **NOT** kill all microorganisms. Just reduces number of microbial population enough to make illness or disease less likely - There are 2 primary means of controlling pathogen populations - **sterilization** and **disinfection** - Aseptic techniques are a type of sterilization - Degerming, sanitization, and pasteurization are types of disinfection - Additionally certain suffixes are used to denote how an agent acts to control microorganisms - -static - Agent inhibits microbial metabolism and growth - Does **NOT** kill microbes - e.g. bacteriostatic, virostatic, fungistatic, germistatic - **-cide/cidal** - Agent that destroys or permanently inactivates a microbe - e.g. bacteriocide, virocide, fungicide, gemicide **Microbial Death** - Microbial death: permanent loss of reproductive ability under ideal environmental conditions - To calculate the efficacy of an antimicrobial agent we must calculate the **microbial death rate**, which under normal conditions is constant over time - Microbicidal agents do not kill all cells at the same time. Rather they **kill a constant percentage of cells over time** (e.g. an agent may kill 70% of all cells per minute) **Action of Antimicrobial Agents** - There are many types of antimicrobial agents but they all fall into **2 main categories** - **Agents Which Interrupt Cell Walls or Cell Membranes** - The cell wall controls the structural integrity of the cell and if it is destroyed the cell dies - Protects the cell from the effects of osmosis - The cell membrane keeps the contents of cytoplasm within the cell and contains proteins which transport materials into and out of the cell which are necessary for cell survival - Without a cytoplasmic membrane a cell cannot survive - **Agents Which Interrupt Cell Metabolism** - \`Destruction of proteins - Proteins regulate cellular metabolism, function as enzymes, and form structural components of cells - As discussed in the lectures on cell structure and microbial growth proteins can become **denatured** by various chemical or physical agents - Denaturation: the process in which proteins or nucleic acids lose their quaternary, tertiary, or secondary structure (i.e. they lose their physical shape) - When a protein becomes denatured it **loses all function** and is no longer able to participate in metabolic or structural processes - Destruction of nucleic acids - Denaturation can also affect nucleic acids which are the components of the genetic material of cells and viruses - Ribosomes are proteins but they also have a nucleic acid portion to them - Destruction of this leads to **cessation of protein synthesis** **Selection of Microbial Control Methods** - In a perfect world an antimicrobial agent would kill every type of pathogen while being safe to humans, plants, and animals. Unfortunately these \"perfect\" agents do not exist - 3 Factors must be considered when choosing an antimicrobial agent - **Site to be treated** - Care must be taken to ensure that we do not cause harm to that which we are treating - e.g. Harsh chemicals and extreme heat are excellent at killing microorganisms but we do not want to use them to clean the human body, or delicate medical devices like a heart valve - e.g. a medical instrument which penetrates into the human body, like scalpels or needles, has a greater risk of causing an infection so a sterilizing agent needs to be used on these devices, while a device that only makes contact with the outside of the skin like a thermometer or the head of a stethoscope can be cleaned with a disinfectant - **Susceptibility of Microorganism** - Some microorganisms are harder to kill than others, so when selecting a treatment, you must choose an agent that is able to eliminate the toughest microbe present - If you can eliminate the most resistant than it should also eliminate any other more susceptible organisms that may be present - - The effectiveness of a germicide can be classified in the following ways based on their effectiveness at killing microrganisms - High Level Germicide - **Sterilizes** all pathogens including endospores - These are used to treat objects such as pacemakers, catheters, central lines, endotracheal tubes, etc.. - Intermediate Level Germicide - Kills up to the level of protoazoan cysts - Used to **disinfect** noninvasive (i.e does not penetrate into the body)medical instruments like respiratory masks, endoscopes, etc\... - Low Level Germicide - Eliminate vegetative bacteria, fungi, and some viruses - Used to **disinfect** items that only contact the skin like electrodes, stethoscope, furniture, etc\... - **Environmental conditions** - Temperature and pH affect microbial death rates and the efficacy of antimicrobial methods - e.g. warm disinfectants work better than cool ones because chemical reactions occur faster at higher temperatures - e.g. acidic conditions enhance the antimicrobial effect of heat **[Physical Methods of Control]** **Heat Related** - High temperatures denature proteins, interfere with the integrity of cytoplasmic membranes and cell walls, disrupts the function and structure of nucleic acids - **Moist Heat** - Used to disinfect, sanitize, sterilize, and pasteurize - Better than dry heat because water is better conductor of heat than air - **Boiling** - Kills vegetative cells of bacteria and fungi, protozoa, and most viruses in 10 minutes - Does **NOT** kill endospores, some viruses (e.g. Hepatitis) or protozoan cysts - NOT used for sterilization - **Autoclaving** - True sterilization requiring heat cannot be achieved via boiling because temperature does not get hot enough (T of boiling water is 100C or 212F) - To achieve higher temperature, you must add pressure - An autoclave is a machine that has a pressure tube and connecting pipes that allow water and air to be introduced or removed from the tube - A temperature of 121C (250F) with 15 PSI of pressure above normal air pressure can destroy all microbes in 10 minutes - **Pasteurization** - Heating a food or beverage product just enough to control the population of pathogenic microorganisms without damaging the product. - This is **NOT** sterilization - Dry Heat - Used for substances that cannot be sterilized with water (e.g. powders, oils, objects which might rust after repeated exposure to moisture) - Dry heat requires higher temperatures over longer periods of time to achieve sterilization - To achieve the same results as seen in an autoclave (sterilization in \< 15 minutes at 121C) an oven must be run at 171C (340F) for 1 hour - Complete **incineration is the ultimate means of sterilization** - e.g. heating an inoculation loop to until it glows red (1500C) to remove all microbial organisms. Incinerating the carcasses of diseased animals or contaminated clothing **Cold Related** - Refrigeration: maintaining a temperature between 0C and 7C (32F - 44F) - Halts metabolism and growth of most organisms except psychrophiles - A couple notable exceptions are the bacteria Listeria and Yersinia - Listeria can grow in refrigerated foods and cause food borne illness - Yersinia can multiply in refrigerated blood products and be passed on to transfusion recipients (Yesina pestis is the causative agent of bubonic plague) - Freezing: maintaining temperature below 0C - Slow freezing is better than quick freezing because this allows ice crystals to form which puncture plasma membranes of cells and destroying them - Many bacteria and viruses survive subfreezing temperatures so cold techniques do not achieve sterilization **Dessication & Lyophilization** - Dessication: to dry out/dehydrate - Inhibits microbial growth because metabolism requires water - Dried foods have been used for millennia to preserve foods - Lyophilication: a technique which combines drying out with freezing - This technique prevents the formation of ice crystals so bacteria can be stored but remain viable for future use **Filtration** - The passage of a liquid or gas through porous sieve designed to trap organisms - E.g. a HEPA filter in your vacuum cleaner or air conditioner. A water filter **Osmotic Pressure** - An ancient technique using high concentrations of salt or sugars to inhibit microbial growth via osmotic pressure - As previously discussed, osmosis is the net movement of water across a semipermeable membrane from an area of higher water concentration to an area of lower water concentration - Salt (or sugar) is used to draw water out of the cell and inhibit microbial growth - e.g. beef jerky, salted fish, honey, jam, jelly, pickles - Fungi are more resistant to osmotic forces than bacteria are **Radiation** - Particulate Radiation - High speed subatomic particles (e.g. protons) that have been freed from their atoms - Electromagnetic (**EM**)Radiation - Energy that is released from an atom after it has undergone some type of internal change. The wavelength of EM radiation determines how powerful it is - Shorter wavelengths are more powerful and have more penetrating power - **Ionic** Radiation - EM radiation with a wavelength \< 1nm - When it strikes molecules it has enough energy to dislodge electrons from atoms, creating ions which disrupt hydrogen bonding, oxidize double covalent bonds, and create highlt reactive hydroxyl (OH^-^) ions - **Nonionic** Radiation - EM radiation with a wavelength \> 1nm - **UV light** is the only type of EM radiation with a wavelength \> 1nm that has a antimicrobial function - UV light induces pyrimidine units in DNA to covalently bond which prevents them from forming Hydrogen bonds - This inhibits DNA transcription and replication **[Chemical Methods of Microbial Control]** **Phenolics** - Phenol, aka Carboxilic acid, is a weak acid that has mild antimicrobial properties - Phenolic compounds are phenol that have been modified by the addition of halogens or organic functional groups and have better antimicrobial properties and fewer side effects than phenols - E.g. chlorinated phenolics have one or more atoms of chlorine and increased antimicrobial action - Bisphenolic compounds are the antimicrobial agents found in Lysol - Act by denaturing proteins and disrupting cell membranes in a wide variety of cells - In our intro lecture we learned that British surgeon Joseph Lister was the first person to practice antispetic technique with the use of Phenol on incision sites **Alcohols** - Alcohols kill bacteria, fungi, and enveloped viruses - Does **NOT** kill bacterial endospores or fungal spores - Alcohols are intermediate level disinfectants and act by denaturing proteins and disrupting cell membranes - Surprisingly pure alcohols are ineffective antimicrobials because the denaturation of proteins require water - Examples of alcohols - Rubbing alcohol (isopropanol), wood alcohol (methanol), drinking alcohol (ethanol) **Halogen** - Halogens are highly reactive, nonmetallic chemical elements - So reactive because they are all one electron short of filling their valence shell - Halogens are Intermediate level disinfectants which act by denaturing proteins, including enzymes - Examples of halogens - Chlorine (used to sanitize pools), iodine (used as an antiseptic), fluorine (used to sanitize toothpaste and drinking water) - Fluorine in particular prevents tooth decay by interrupting the development of biofilms **Oxidizing Agents** - High level disinfectants and antiseptics that work by oxidizing enzymes which prevent metabolism - Healthcare workers use oxidizing agents to **kill anaerobes** in deep puncture wounds - Examples of oxidizing agents - Hydrogen peroxide, ozone, paracetic acid **Surfactants** - \"surface active\" chemicals - Reduce the surface tension of solvents by decreasing the attraction of water molecules which results in the solvent becoming more effective at dissolving solute molecules - Examples of surfactants - Detergents and soaps - Low level Disinfectant **Heavy Metals** - Heavy metals are metallic elements that have a high atomic weight and tend to have toxic properties - Heavy metal ions such as arsenic, zinc, mercury, lead, silver, etc\...combine with sulfur atoms in a molecule of cysteine (an amino acid) and causes proteins to denature - Low Level Antimicrobial and with a few exceptions are no longer used - Exceptions - At birth newborns eyes are treated with silver nitrate cream to prevent infections from maternal Neisseria gonorrhea - Copper solutions used to interfere with algae formation in fish tanks, storage tanks, and water reservoirs **Aldehydes** - Aldehydes are molecules which contain a terminal CHO group with a double bond between the carbon and oxygen - Denatures proteins by crosslinking (which interferes with)the functional groups of microbial organisms - E.g. formadehyde, glyceraldehyde **Gaseous Agents** - Many items such as heart lung machines components, sutures, mattresses, pillows, prosthetic heart valves, catheters, etc\... cannot be sterilized easily so highly reactive microbicidal and sporicidal gasses are used - e.g. ethylene oxide, propylene oxide, beta-propiolactone - These gasses penetrate paper and plastic wraps and penetrate every crack of an object **Enzymes** - Some enzymes target bacterial cells and kill them through various processes (e.g. osmosis, oxidation, destruction of cell wall and plasma membrane, etc\...) - Examples - Lysozymes - Secreted in tears and target the cell walls of bacteria - Prionozyme - Recently approved in Europe to target prions on surgical equipment  table\_09\_05\_labeled.jpg