Microbial Control

Terminology for Microbial Control Sterilization- removal or destruction of all forms of microbial life Commercial sterilization- subjects canned food to only enough heat to destroy the endospores of Clostridium botulinum Disinfection- is the destruction of vegetative pathogens on a surface, usually with chemicals Spores and viruses are not necessarily destroyed Antisepsis- is the chemical disinfection of living tissue, such as skin or mucous membranes

Terminology for Microbial Control Asepsis- is the absence of pathogens on an object or area, as in antiseptic surgery Degerming (degermation)- is the removal of transient microbes from the skin by mechanical cleansing or by antiseptic Sanitation- is the reduction of microbial populations on objects to safe public health levels A biocide or germicide- kills microorganisms Fungicides kill fungi, virucides kill viruses Suffix – cide means the killer of a specified microorganism Suffix- stat used in this way indicates only that the substance inhibits – for example bacteriostasis Example for asepsis is handwashing Example for bacteriostatic is a refrigerator

Rate of Microbial Death Bacterial populations killed by heat or chemicals tend to die at constant rates—for example, 90% every 10 minutes. Plotted logarithmically, these figures form straight descending lines.

Factors that influence effectiveness of an antimicrobial treatment: 1. Number of microbes -more cells, more time needed to kill all 2. Environmental influences -organics often inhibit chemical agents (blood, feces, vomit) -temperature (disinfectants work better in warm temperatures) -pH (heat is more effective in an acid pH) Environmental- any medium containing fats and protein will tend to protect bacteria

Factors that influence effectiveness of an antimicrobial treatment: 3. Time of exposure -Chemical antimicrobials require a certain amount of exposure; same agent may need longer on resistant organisms or spores -with heat, lower temps require longer to kill 4. Microbial characteristics -resistance genes, protective structures (e.g.capsules) etc. can inhibit action -bioflims prevent penetration susceptibility to different agents varies among microbes

Type and Age of Microbe Bacteria- susceptible to protein denaturing BUT mycobacteria is not because of its hydrophobic coat Growth cycles- physiologically young bacteria(early in growth cycle) susceptible to heat Endospores more resistant to heat the older they get

Properties of Materials: Plastic and rubber- not heat tolerant Cutting edges of surgical instruments- no moisture or corrosive chemicals Edges can become pitted due to rust and corrosion, microbes can hide there Fabrics- no chemical disinfection; some too fragile

Actions of Microbial Control Agents 1. Alteration of membrane permeability The plasma membrane controls the passage of nutrients and wastes into and out of the cell. Damage to the plasma membrane causes: leakage of cellular contents interferes with cell growth. = Leak lysis, death Cell lysis is a process in which a cell is broken down or destroyed

Actions of Microbial Control Agents 2. Damage to proteins and Nucleic Acids Enzymes and other proteins are essential for cell function Denatures proteins Enzymes (no reactions) Proteins necessary for bacteria metabolism Hydrogen bonds are broken Covalent bonds are also broken 3. Damage to nucleic acids Prevent replication, transcription, or translation Hydrogen bonds hold proteins in the characteristic 3-dimensional shape required for their functions. Heat and certain chemicals break these bonds and the shape is lost. This is called denaturation. DNA and RNA carry the cell’s genetic information and function in protein synthesis. Damage to these by heat, radiation, or chemicals usually kills the cell.

Physical Methods of Microbial Control -to disinfect objects, food, and solutions common methods: -Temperature: kill or inhibit growth Heat Low Temps. -Filtration: physical removal -Desiccation: inhibit growth -Osmotic pressure -Radiation: kill

Methods involving Heat: Heat: Common food preservation Denatures protein (changes shape) Thermal Death Point (TDP) = lowest temp at which all microbes in liquid suspension will be killed in 10 min Thermal Death Time (TDT)= minimal length of time for all microbes in liquid suspension to be killed at given temp *Both are different for different species due to microbial variation in heat tolerance Decimal Reduction Time (DRT) = Time in minutes in which 90% of bacteria at a given temperature will be killed Concept of equivalent treatments: With any heat treatment, the higher the temperature used the shorter the exposure time needed to achieve the same effect

Methods involving Heat: 1. Moist heat will always kill faster than dry heat at the same temperature Kills microbes by coagulation of cell proteins A. Boiling (100°C) kills vegetative forms of bacterial pathogens, many viruses, and fungi within 10 minutes Some mo’s and viruses are resistant Endospores (up to 20 hrs.) and some viruses (30 minutes) survive boiling for longer times. This occurs faster in the presence of water, so moist heat requires lower temperatures and less time of exposure than dry heat. Edospores-dormant stage of some bacteria that allow them to survive harsh conditions-ex. Extreme heat

Methods involving Heat: B. Autoclaves Moist Heat (steam) and pressure for sterilization Achieves higher temps. than boiling Preferred method (sealed chamber, air is exhausted, and steam under pressure is injected) for all materials that can withstand it Kills all organisms and their endospores in about 15 - 20 minutes An autoclave is shown in Figure 1.1 and 1.2 Here we use an autoclave to sterilize culture media. In hospitals, doctors’ offices, dentists’ offices, etc. this methods is used to sterilize medical equipment. Materials being autoclaved are often wrapped in paper, so that after sterilization the outside of the package can be handled without contaminating the sterile item inside. The size of the container, the volume of a liquid, and the type of wrapping can influence the time and temperature required for sterilization.

Autoclave Figure 1.1 Figure 1.2 Autoclave tape Autoclave tape works by changing color after exposure to temperatures commonly used in sterilization processes, typically 121°C in a steam autoclave. It is important to note that the presence of autoclave tape that has changed color on an item does not ensure that the product is sterile, as the tape will change color upon exposure only. For steam sterilization to occur, the entire item must completely reach and maintain 121°C for 15-20 minutes with proper steam exposure to ensure sterilization. Diagonal shaped more leat, words Autoclaved lead free Figure 1.2 Autoclave tape

Methods involving Heat: C. Pasteurization Louis Pasteur Mild Heating Kills most pathogens Kills bacteria that cause spoilage Preserves taste in product Lowers bacterial numbers Pasteurization or pasteurisation is a process of heating a food, which is usually a liquid, to a specific temperature for a predefined length of time and then immediately cooling it after it is removed from the heat. This process slows spoilage caused by microbial growth in the food. Unlike sterilization, pasteurization is not intended to kill all micro-organisms in the food. Instead, it aims to reduce the number of viable pathogens so they are unlikely to cause disease (assuming the pasteurized product is stored as indicated and is consumed before its expiration date).

Methods involving Heat: 2. Dry Heat Kills by burning to ashes or by oxidation Flaming- we use this on loops in labs Incineration- burning of contaminated paper Hot air sterilization Hot ovens Mainly used for items not suitable for autoclaving Oily substances, powders Large amounts of glassware

Low Temperature Lower temp inhibits growth, rapid freezing limits moisture (bacteriostatic) Refrigerator temperatures (0° to 7°C) slow the metabolic rate of microbes; however Psychrotrophic species still grow slowly. Some organisms grow at temperatures slightly below freezing, but microbes at the usual temperatures of freezer compartments are completely dormant.

Filtration Liquids Heat- sensitive materials Small pores prevent passage of bacteria High-efficiency particulate air filters (HEPA) Operating rooms Special clean rooms Masks Used with solutions for: renal dialysis, heart bypass machines, I.V.’s Catch particles autoclaves miss

Desiccation Microbes require water for growth, and adequately dried (desiccated) foods will not support their growth, therefore inhibiting growth Absence of Water Lyophilization- rapid freeze drying Used for blood products , serum products, enzymes, cultures Avoids ice crystal formation; cells burst when water expands Species vary in their susceptibility. Dry surroundings kill some vegetative cells in one hour or less. Others can survive for months or even years. Bacteria that produce endospores are extremely resistant to drying. Many viruses are also resistant to drying.

Osmotic Pressure High salt or sugar concentrations cause water to leave the cell; this is an example of osmosis. Generally, molds and yeasts resist osmotic pressures better than bacteria.

Radiation Ionization Radiation includes X rays, gamma rays, and high-energy electron beams very short wavelengths and high levels of energy Penetrate deeply Ionizes water to form hydroxyl radicals These destroy cell components, especially DNA Kills: vegetative cells, viruses, most endospores with adequate exposure Applications: food preservation, sterilization of pharmaceuticals, medical supplies, mail Recently, approval has been granted for use of low level radiation of fruits and meats. The post office is now using this method to sterilize some mail. This process does involve the use of dangerous radiation and can only be used in a properly shielded room, so it is mostly used in factories where widescale use of the setup makes it economical.

Radiation Nonionizing radiation Have a longer wavelength and less energy Ultraviolet (UV) light is the common example Causes the formation of thymine dimers, which interferes with DNA replication and formation of mRNA. UV lamps are used in hospitals and in food service This method does not sterilize, but it does reduce bacterial growth Penetrating power is very low, so any type of covering protects microbes. Sunlight has some weak antimicrobial effects (biocidal), but the wavelengths of sunlight are too long to work well.

Types of Chemical Agents 1. Phenols and Phenolics A. Phenol (carbolic acid) Irritating to skin and mucous membranes Bad odor Rarely used today B. Phenolics Chemicals derived from phenol Chemically altered to make it less irritating and more effective Damages plama mbs., inactivates enzymes, denature proteins Often used as disinfectants as they remain active in the presence of organic matter Its main use now is in throat lozenges and sprays, but the concentration is so low that there is little antimicrobial effect, although there is some local anesthetic action. Some throat sprays may have a concentration above 1% and these may show antibacterial action.

Types of Chemical Agents C. Bisphenols contain 2 phenolic groups connected by a bridge Hexachlorophene pHisoHex is an example Prescription antibacterial lotion Used in nurseries to control gram + bacteria Skin bacteria: Staphylococcus and Streptococcus Triclosan Found in antibacterial soap Effective against G+ and G- bacteria

Types of Chemical Agents 2. Biguanides Chlorhexidine is an example Hibiclens soap Used on skin and mucous membranes Similar to phenolics but less toxic Disrupt plasma mb. Broad spectrum Effective against most vegetative bacteria and fungi, but not against endospores and many viruses. Damaging to eyes

Types of Chemical Agents 3. Halogens- Effective alone or in compounds A. Iodine (I2) One of the oldest and most effective Very effective on: all bacteria, many endospores, fungi and some viruses Combines with amino acids in proteins and denatures proteins Alters plasma mb. Negative Aspects Staining Sometimes irritating to the skin May trigger allergies Applications Skin disinfection, wound treatment, water treatment

Types of Chemical Agents B. Chlorine (Cl2) Action: forms hypochlorus acid with water → oxidizing agent, denatures proteins Broad spectrum: bacteria, fungi, some endospores, some viruses Positive aspects: -effective against all vegetative cells including Mycobacterium -cost effective Negative aspects: -action inhibited by organics -can form carcinogenic compounds Applications: water and sewage treatment, surface and instrument disinfection Sodium hypochlorite (NaClO): Is active ingredient of bleach The U.S. military uses tablets of sodium dichloroisocyanurate for fielddisinfection of water

Types of Chemical Agents C. Alcohols- frequently used for skin degerming Kill bacteria, fungi, but not endospores or naked viruses. Act by denaturing proteins and disrupting cell membranes. Used to mechanically wipe microbes off skin before injections or blood drawing, instrument disinfection Not good for open wounds, because cause proteins to coagulate and leave bacteria unharmed Ethanol (ethyl alcohol): Optimum concentration is 70%. Isopropanol: Rubbing alcohol Better disinfectant than ethanol Usual concentration is 90% Also cheaper and less volatile.

Types of Chemical Agents 5. Heavy metals and their compounds Oligodynamic action- Very tiny amounts are effective Includes- silver, mercury, copper, zinc Bind sulfur groups causing inactivation or denaturing of proteins A. Silver: 1% silver nitrate used to protect infants against gonorrheal eye infections until recently. B. Mercury Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds. C. Copper Copper sulfate is used to kill algae in pools and fish tanks (fungicidal, algicidal) D. Zinc Used in mouthwashes Superficial fungal and bacterial infections However, because these drops often cause the baby's eyes to be irritated, most hospitals now use erythromycin ointment instead

Types of Chemical Agents 6. Surface-Acting Agents Disrupt plasma membrane & denature proteins Decrease surface tension Include soaps and detergents Washing with soap breaks up the oily film that covers skin and allows microbes and dirt to be washed away 7. Quaternary Ammonium Compounds Widely used surface active agents Denature proteins & disrupt cell membranes Cationic (positively charge) detergents Effective against gram positive bacteria, less effective against gram-negative bacteria. Also destroy fungi, amoebas, and enveloped viruses.

Types of Chemical Agents 8. Aldehydes These can act very effectively against microbes Inactivate proteins Action: cross-link (thus inactivate) nucleic acids and proteins High activity (sterilization) biocidal including endospores Positive aspects achieves sterilization Negative aspects unstable -toxic -volatile with noxious fumes Applications: specimen preservation (embalming), vaccine sterilization

Types of Chemical Agents 9. Gaseous Sterilizers Chemicals that sterilize in a chamber similar to an autoclave. Denature proteins, by replacing functional groups with alkyl groups A. Ethylene Oxide Kills all microbes and endospores , but requires exposure of 4 to 18 hours. Toxic and explosive in pure form. Highly penetrating. Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment.

Types of Chemical Agents 10. Peroxygens Oxidize cellular components A. Hydrogen Peroxide Common household antiseptic Not good for open wounds because quickly broken down by catalase present in human cells used in deep wounds because it releases oxygen as it breaks down, which makes conditions unfavorable for anaerobic bacteria Effective in disinfection of inanimate objects (kills endospores) Sporicidal at higher temperatures Used by food industry and to disinfect contact lenses

Types of Chemical Agents B. Benzoyl Peroxide Main ingredient in many acne treatments May be used in treating wound infections caused by anaerobes C. Peracetic Acid One of the most effective liquid sporicides available Sterilant : Kills bacteria and fungi in less than 5 minutes Kills endosporesand viruses within 30 minutes Used widely in disinfection of food and medical instrument because it does not leave toxic residues