Microbiological Control Methods Overview PDF

INTRODUCTION Control of microorganisms is essential to  prevent the transmission of diseases and infection,  stop decomposition and spoilage,  and prevent unwanted microbial contamination. Antimicrobial agents have traditionally tended to be divided into two groups: antibiotics chemical biocide...

INTRODUCTION Control of microorganisms is essential to  prevent the transmission of diseases and infection,  stop decomposition and spoilage,  and prevent unwanted microbial contamination. Antimicrobial agents have traditionally tended to be divided into two groups: antibiotics chemical biocides chemical biocides Antiseptic: Disinfectant: Preservatives: Principal target sites/mode of action Their principal mechanisms of action are disruption of cell wall and membrane structure and function, intracellular coagulation and chemical modification of cellular proteins and nucleic acids. For this reason, resistance to biocides tends to occur less readily than is found with antibiotics. Factors influencing the activity of biocidal agents Temperature Concentration pH Solubility Interaction with excipients and packaging materials Interaction with organic material/inorganic material Number and location of Microorganisms Innate resistance of microorganisms Biofilms  THE THREE LEVELS OF DISINFECTANTS  High-level Disinfectant kill endospores and, if properly used, are sterilant. Materials that necessitate high- level control are medical devices—for example, catheters, heart-lung equipment, and implants—that are not heat-sterilizable and are intended to enter body tissues during medical procedures.   Intermediate-level Disinfectant kill fungal (but not bacterial)spores, resistant pathogens such as the tubercle bacillus, and viruses. They are used to disinfect items (respiratory equipment, thermometers) that come into intimate contact with the mucous membranes but are noninvasive.   Low levels Disinfectant eliminate only vegetative bacteria, vegetative fungal cells, and some viruses. They are used to clean materials such as electrodes, straps, and furniture that touch the skin surfaces but not the mucous membranes.   The choice and appropriate use of antimicrobial chemical agents are of constant concern in medicine and dentistry. Although actual clinical practices of chemical decontamination vary widely, some desirable qualities in a germicide have been identified, including: Rapid action in low concentrations, CHOOSING A  Solubility in water or alcohol and long-term MICROBICIDAL stability, CHEMICAL  Broad-spectrum microbicidal action without being toxic to human and animal tissues,  Penetration of inanimate surfaces to sustain a cumulative or persistent action,  Resistance to becoming inactivated by organic matter,  Noncorrosive or non-staining properties, ANTISEPTICS, DISINFECTANTS AND PRESERVATIVE S CATEGORIES OF CHEMICAL AGENTS Phenolic Hydrogen Halogens Chlorhexidine Alcohols compounds peroxides Quatnary Aldehyde and Acid, alkali ammonium Heavy metals sterilant gases and dyes. compound Halogen Compounds The halogens are fluorine, bromine, chlorine, and iodine, a group of nonmetallic elements with similar chemical properties. Most halogens exert their antimicrobial effect primarily in the nonionic state, not the halide state (chloride, iodide, for example). Because fluorine and bromine are difficult and dangerous to handle and no more effective than chlorine and iodine, only the latter two are used routinely in germicidal preparations. These elements are highly effective components of disinfectants and antiseptics because they are microbicidal, and they are sporicidal with longer exposure. For these reasons, halogens are the active ingredients in nearly one-third of all antimicrobial chemicals currently marketed Mode of action: Hypochlorous acid oxidizes the sulfhydryl (S—H) groups on the amino acid breaks disulfide (S—S) bridges on numerous enzymes. The resulting denaturation of the enzymes is permanent and suspends metabolic reactions. It also damages the structure of DNA, RNA, and fatty acids. Death of almost all microorganisms usually occurs within 30 minutes, although endospores may require several hours. Chlorine compounds are less effective and relatively unstable if exposed to light, alkaline pH, and excess organic matter. Chlorine Chlorine Gas (Cl2) Large-scale disinfection of drinking water, sewage, and wastewater Chlorination to a concentration of 0.6 to 1.0 parts of chlorine per million parts of water destroys most vegetative pathogens.* Hypochlorites (HClO) Bleach : Used extensively in sanitization and disinfection of food equipment, treatment of swimming pools, spas, fresh foods; routine medical and household. Common household bleach is a 5% solution of sodium hypochlorite; dilutions of 1:10-1:1,000 are highly disinfection, deodorizing, and stain removal Chloramines (Dichloramine Halazone),An alternative to pure chlorine in treating water Because standard gas chlorination of water is now believed to for produce unsafe levels of trihalomethanes Also used as sanitizers and disinfectants;, treating wounds and skin surfaces.  Iodine Iodophors Most common iodine for skin and mucous membranes; antiseptic prep for surgery and injections surgical hand scrubs; to disinfect equipment surfaces; possibly for burns alternative and may be preventive for eye infections in newborns. A complex of iodine and a neutral protein polymer provides slow release and reduced toxicity or irritation of tissues; less prone to staining. Common products are povidone-iodine ; (PVI; Betadine), which contain 2% to 10% available iodine. Elemental Iodine Aqueous or Tinctures Topical antiseptic prior to surgery; : for burned or injured skin. Medium-level disinfection for plastic instruments, thermometers ,tablet form is also available for disinfection of contaminated water. Weak solutions of 1% to 3% in water or in alcohol tinctures. ; Aqueous solutions or tinctures of 5% to 10%; somewhat limited by their toxicity and tendency to stain. Phenol and Its Derivatives Phenol (carbolic acid) is an acrid, poisonous compound derived from the distillation of coal tar. First adopted by Joseph Lister in 1867 as a surgical germicide, phenol was the dominant antimicrobial chemical until other phenolics with fewer toxic and irritating effects were developed. it remains one standard against which other phenolic disinfectants are rated. The phenol coefficient quantitatively compares a chemical’s antimicrobic properties to those of phenol. Substances phenolics are alkylated phenols (cresols), chlorinated phenols (see triclosan, and bisphenols Mode of Action: In high concentrations, phenolics are cellular poisons, rapidly disrupting cell walls and membranes and precipitating proteins; In lower concentrations they inactivate certain critical enzyme systems. The phenolics will destroy vegetative bacteria (including the tuberculosis bacillus), fungi, and many viruses (not hepatitis B), but they are not reliably sporicidal. Their sustained activity in the presence of organic matter and their detergent actions contribute to their usefulness. Unfortunately, the toxicity of many of the phenolics makes them a questionable choice as antiseptics. Chlorhexidine The compound chlorhexidine (Hibiclens, Hibitane) is a complex organic base containing chlorine and two phenolic rings. Its mode of action targets cell membranes by lowering surface tension and causing denaturation of proteins. At moderate to high concentrations it is bactericidal for both gram-positive and gram-negative bacteria but inactive against spores. Alcohols are colorless hydrocarbons with one or more —OH functional groups. Of several alcohols available, only ethyl and isopropyl are suitable for microbial control. Methyl alcohol is not particularly microbicidal, and more complex alcohols are either poorly soluble in water or too toxic for routine use. Mode of Action Alcohol’s mechanism of action depends in part upon its concentration. Concentrations of 50% and higher dissolve membrane lipids, disrupt cell surface tension, and compromise membrane integrity. Alcohol that has entered the protoplasm denatures proteins through coagulation but only in alcohol-water solutions of 50% to 95%. Alcohol is the exception to the rule that higher concentrations of an antimicrobial chemical have greater microbicidal activity. Because water is needed for proteins to coagulate, alcohol shows a greater microbicidal activity at 70% concentration (that is, 30% water) than at 100% (0% water). Absolute alcohol (100%) dissolves cell membranes and inhibits cell growth but is generally not a protein coagulant Although useful in intermediate- to low-level germicidal applications, alcohol does not destroy bacterial spores at room temperature. Alcohol can, however, destroy resistant vegetative forms, including tuberculosis bacteria and fungal spores, provided the time of exposure is adequate. Alcohol tends to inactivate enveloped viruses more readily than nonenveloped viruses because of the surfactant effect on the envelope Hydrogen Peroxide and Related Germicides Hydrogen peroxide (H2O2) is a colorless, caustic liquid that decomposes in the presence of light, metals, or catalase into water and oxygen gas. The germicidal effects of hydrogen peroxide are due to the direct and indirect actions of oxygen. Oxygen forms hydroxyl free radicals (—OH), which, is bactericidal, virucidal, fungicidal, and, in higher concentrations, sporicidal. Hydrogen peroxide: 3 % hydrogen peroxide is most commonly used treating anerobic bacterial infection because of the lethal effect of oxygen release. Sterilizing hydrogen peroxide : 35% vaporized hydrogen peroxide is a major sterilant for industrial parts, medical items ,isolators, clean room Peracetic acid :used to sterilized rooms and medical devices Ozone: used to disinfect air, water industrial air conditions, however difficult to handle Aldehyde Sterilant and Disinfectants Organic substances bearing a —CHO functional group on the terminal carbon are called aldehydes. The aldehydes used most often in microbial control are glutaraldehyde and ortho-phthaldehyde (OPA), Formaldehyde The mechanism of activity involves cross-linking protein molecules on the cell surface. In this process, amino acids are alkylated, meaning that a hydrogen atom on an amino acid is replaced by the glutaraldehyde molecule itself. This irreversibly disrupts the activity of enzymes and other proteins within cells. Glutaraldehyde is rapid and broad-spectrum and is officially labeled as a sterilant and high-level disinfectant. It is one of the few chemicals that can be used to sterilize delicate reusable medical and dental instruments Formaldehyde : is a sharp, irritating gas that readily dissolves in water to form an aqueous solution called formalin. Pure formalin is a 37% solution of formaldehyde gas dissolved in water. Formalin is an intermediate- to high-level disinfectant, but its extreme toxicity (it is classified as a carcinogen) and irritating effects on the skin and mucous membranes greatly limit its clinical usefulness Gaseous Sterilant Ethylene oxide (ETO) and its relative propylene oxide (PO) are colorless gases at room temperature. ETO is very explosive in air, a feature that can be eliminated by combining it with a high percentage of carbon dioxide or fluorocarbon. Like the aldehydes, ETO is a very strong alkylating agent, and it reacts vigorously with functional groups of DNA and proteins. Ethylene oxide is one of a very few gases generally accepted for chemical sterilization because, when employed according to strict procedures, it is a sporicidal. A specially designed ETO sterilizer is equipped with a chamber, gas ports, and controls for temperature, pressure, and humidity ETO Sterilizer Chlorine dioxide: is another gas used for sterilization. Despite the name, chlorine dioxide works in a completely different way from the chlorine compounds It is a strong alkylating agent, which disrupts proteins and is effective against vegetative bacteria, fungi, viruses, and endospores. Chlorine dioxide has mainly been used for the treatment of drinking water, wastewater, food-processing equipment, and medical waste, but it also has numerous applications in decontamination of rooms, buildings, and large spaces or objects. Betapropiolactone (BPL) is a substance somewhat similar to ETO in applications. It is rapidly microbicidal when used as an aerosol or liquid to disinfect whole rooms and instruments, to sterilize bone and arterial grafts, and to inactivate viruses in vaccines. Chemicals with Surface Action: Detergents and Soaps Detergents are polar molecules that act as surfactants. Most anionic detergents have limited microbicidal power. This includes most soaps. Much more effective are positively charged (cationic) detergents, particularly the quaternary ammonium compounds (usually shortened to quats). They disrupt the disrupts the cell membranes of sensitive microbes After a few minutes of contact, the cells burst and die. Soaps are alkaline compounds made by combining the fatty acids in oils with sodium or potassium salts. In usual practice, soaps alone are weak microbicides, and they destroy only highly sensitive forms Soaps function primarily as cleansing agents and sanitizers in industry, the home, and the medical setting. Combining germicides such as chlorhexidine or iodophor with soaps produces highly active antiseptics, degermers, and disinfectants. Heavy Metal Compounds Various forms of the metallic elements such as mercury, silver, gold, copper, arsenic, and zinc have been applied in microbial control over several centuries. These are often referred to as heavy metals because of their relatively high atomic weights. However, from this list, only preparations containing mercury and silver still have any significance as germicides higher molecular weight metals (mercury, silver, gold) can be very toxic, even in minute quantities (parts per million). This property of having antimicrobial effects in exceedingly small amounts is called an oligodynamic*. Mercury, silver, and most other metals exert microbicidal effects by binding onto functional groups of proteins and inactivating Them. This mode of action can destroy many types of microbes, including vegetative bacteria, fungal cells and spores, algae, protozoa, and viruses (but not endospores). A newer use of metals is their incorporation into medical supplies and hospital facilities. Silver is now added to catheters, IV lines, and prostheses to prevent biofilm formations and infections. Organic mercury :Thimerosal (tincture very effective but toxic to mucous membrane) Silver Sulfadiazine Ointment Silver Nitrate (AgNO3) Metallic Silver Colloidal Silver  The main drawbacks to heavy metals are (1) metals (especially mercury) are very toxic to humans if ingested, inhaled, or absorbed through the skin, even in small quantities, for the same reasons that they are toxic to microbial cells; (2) they commonly cause allergic reactions; (3) large quantities of biological fluids and wastes neutralize their actions; (4) microbes can develop resistance to metals Dyes as Antimicrobial Agents Dyes are important in staining techniques and as selective and differential agents in media; they are also a primary source of certain drugs used in chemotherapy. Their antimicrobial effects are apparently due to the way they insert into nucleic acids and cause mutations. There is also some evidence they can interfere with cell wall synthesis. Because aniline dyes such as crystal violet and malachite green are most active against gram-positive species of bacteria and some fungi, they are incorporated into solutions and ointments to treat skin infections (ringworm, for example). The yellow acridine dyes, acriflavine and proflavine, are sometimes utilized for antisepsis and wound treatment in medical and veterinary clinics. For the most part, applications for dyes will continue to be limited because dyes stain and have a narrow spectrum of activity. Acids and Alkalis Conditions of very low or high pH can destroy or inhibit microbial cells, but they are limited in applications due to their corrosive, caustic, and hazardous nature. A Organic acids are widely used in preservation because they prevent spore germination and bacterial and fungal growth and because they are generally regarded as safe to eat. Acetic acid (in the form of vinegar) is a pickling agent that inhibits bacterial growth; propionic acid is commonly incorporated into breads and cakes to retard molds; lactic acid is added to prevent growth of anaerobic bacteria (especially the clostridia); and benzoic and sorbic acids are added to beverages, syrups, and margarine to inhibit yeasts and pharmaceuticals REFERENCE ◼ Alcamo's Fundamentals of Microbiology ◼ Talaro's Foundations in Microbiology Chapter 11 ◼ Prescott's Microbiology ◼ Alcamo Microbiology Testing efficacy of Disinfectants  All disinfectant tests have the same purpose i.e measuring the antimicrobial activity of a chemical substance or preparation, disinfectants, manufacturers must evaluate the new formulations as per the standard international guidelines for the microbicidal (bactericidal, fungicidal, sporicidal or virucidal) efficacy. To get approval from the regulatory bodies, application must be filed along with the test reports with the claimed efficacy data. There are several regulatory bodies such as FDA, EPA,, AOAC,, that help to demonstrate disinfectant products and their efficacy claims. FDA U.S. Food and Drug Administration EPA U.S. Environmental Protection Agency AOAC Association of Official Analytical Chemists Disinfectant Testing Products There is a wide range of disinfectant products that can be tested Household disinfectants Hygienic handrub and handwash Surgical handrub and handwash Instrument and surface disinfectants used in medical areas Germicidal spray products Germicidal wipes Veterinary disinfectants Many testing methods has been described for the testing/evaluation of Disinfectants. They are subdivided into 1) Suspension tests, 2) Capacity test 3) Surface disinfection tests Suspension tests This is the simplest test where the microorganisms are added to disinfectant diluted with water and the removed samples inoculated into broth containing a neutralizer. An example of this type of test is the Phenol coefficient type test and that include: Rideal-Walker test The chick Martin Test Phenol Coefficient Test Capacity tests Capacity test determine the ability of a disinfectant to retain activity in the presence of an increasing load of microbes. In a capacity test, the disinfectant is challenged repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted. Capacity tests simulate the practical situations of housekeeping and instrument disinfection. Best known capacity test is the Kelsey-Sykes test Surface tests These tests are designed to evaluate the effect of disinfectant on microorganisms dried onto surfaces. The specified microorganisms are dried onto either stainless steel or borosilicate glass cylinders. The cylinders are then placed into a solution of disinfectant normally in the presence of 5% serum (again to simulate organic matter) for a given period of time. The cylinders are then removed and placed into broth containing a neutralizer and incubated to determine if there are any survivors. Reference : Bentley, Arthur Owen. and Rawlins, Ernest Alexander. Bentley's textbook of pharmaceutics Bailliere Tindall London Physical and Physical and Chemical Chemical agents agents for for Microbial Microbial control control  Sterilization is defined as a process of complete elimination or destruction of all forms of microbial life (i.e., both vegetative and spore forms), which is carried out by various physical and chemical and most Sterilization recently biological methods. While most prevalent in the manufacture of sterile products it can be used in a variety of settings where microbes have potential impact on patients or products  The definition of sterility, ‘the absence of all viable microorganisms’, as essentially correct, the evidence that something is sterile can only be considered in terms of probability. This is because absolute sterility can only be proved by testing every single item produced (and with technology that will give an Sterilization undisputable result). However, the act of testing destroys the very item which is required for administration to the patient, so sterility cannot be proven empirically. Therefore, the concept of what constitutes ‘sterile’ is measured as a probability of sterility for each item to be sterilized. Probability can be considered in relation to components that are sterilized and to products that can be terminally sterilized in relation to the concept called the Sterility Assurance Level (SAL).  Number and location of Microorganisms Innate resistance of microorganisms Factors affecting Physical and chemical factors (temperature, pH, sterilization relative humidity) Organic and inorganic matter Duration of exposure Biofilm Ref:CDC  Adequate sterilization requires that both temperature and length of exposure be considered. As a general rule, higher temperatures allow shorter exposure times, and lower temperatures require longer exposure times. A combination of these two Practical Concerns in the variables constitutes Use of Heat: The thermal death time, or TDT, defined as the Thermal Death shortest length of time required to kill all test microbes at a specified temperature. The TDT has Measurements been experimentally determined for the microbial species that are common or important contaminants in various heat-treated materials. Another way to compare the susceptibility of microbes to heat is The thermal death point (TDP), defined as the lowest temperature required to kill all microbes in a sample in 10 minutes.  Terminal sterilization and Aseptic processing are two approaches to Terminal obtain a sterile drug Sterilization and product; regulatory bodies in the Aseptic United States (US) and European Processing of Union (EU). Pharmaceutical Products Terminal sterilization is preferred and should be considered first to minimize the risk of contamination and its consequences. Physical Agents Dry heat oven Dry Heat (Dehydration, Incineration combustion, oxidation) Red Hot Flaming Heat Temperature below 100 ºC Pasteurization Temperature at 100 ºC Moist Heat Non pressurized steam (coagulates protein) Boiling Water Temperature above 100º C Autoclave Red hot Flaming Exposure of wires and forceps to the Bunsen flame until it becomes red hot, then cool down and use. Used for loop, forceps, and metal rods. Dry heat oven Electric or gas chamber heated to 150°C to 180°C for 2 to 4 hours Materials that can withstand hot temperatures and dehydration; glassware, metals, powders, oils. Poor choice for liquids, rubber, plastics; time-consuming  Pyrogens in vials or glass components may be De-pyrogenation destroyed by dry heat sterilization at high temperatures. A recommended condition for de-pyrogenation of glassware and equipment is. heating at 250 ° C for 45 minutes. Pyrogens are also destroyed at 650 ° C in 1 minute or at 180 ° C in 4 hours. The British Pharmacopoeia (2010) states that dry heat at temperatures above 220 ° C may be used for the de-pyrogenation of glassware. Incineration Is treating of an objects to heating over 250° C until become black or turn into ashes Done for used equipment, disposal of biohazards Temperature range 800–6500-degree C°.  Below 100 ºC Pasteurization Moist Heat At 100 ºC Boiling Water Non-Pressurized Steam Above 100 ºC Steam Under Pressure Temperature Below 100 °C Pasteurization: Flash method works at 71.6°C for 15 seconds; ultrahigh temperature (UHT) uses 134°C for a few seconds. UHT can sterilize for longer shelf life. Disinfection of milk and dairy products to destroy milk borne pathogens; Temperature at 100 °C Unpressurized steam (Tyndallization) Arnold’s Sterilizer—100°C for 30 minutes on 3 consecutives days; items are incubated between treatments to allow endospores to germinate. Limited uses in sterilizing some media and foods that cannot be autoclaved; can be used to disinfect but not sterilize some medical supplies Boiling water Water bath or pan heated to 100°C; items may be placed in water for 30 minutes to kill vegetative pathogens. Limited to disinfection and sanitization of heat-resistant household objects such as eating utensils, clothing, sick room supplies, baby supplies, bedding, and water in emergencies; problem with recontamination Steam Under Pressure (Above 100 °C) (Autoclaving) Moist heat is more effective than dry heat at a given temperature or length of exposure. It has more penetrating power than dry heat Make complete killing of bacteria, their spores, fungi and their spores, parasites and viruses including Envelop and non- Enveloped virus. By far the most commonly employed method in Pharmaceutical industry. Temperature/time cycles for bottled fluids is are 121 ° C for 15 minutes porous loads (e.g., surgical dressings) 134 ° C for 3 minutes.  1-Air removal and steam admission Stages of 2-Heatin up and exposure operation 3-Drying or cooling  The principal benefit of cold treatment is to slow the growth of cultures and microbes in food and other perishable materials during processing and storage. It must be emphasized that cold merely retards the activities of most microbes. Although it is true that some sensitive microbes are Effect of low killed by cold temperatures, most are not adversely affected by gradual cooling, long-term refrigeration, or deep-freezing. In fact, freezing temperature temperatures, ranging from −70°C to −135°C, provide an environment that can preserve cultures of bacteria, viruses, and fungi for long periods. (cold, Freezing) Most vegetative cells directly exposed to normal room air gradually and desiccation become dehydrated or desiccated.* Delicate pathogens such as Streptococcus pneumoniae, the spirochete of syphilis, and Neisseria gonorrhoeae can die after a few hours of air-drying, but many others are not killed, and some are even preserved. Staphylococci and streptococci in dried secretions and the tubercle bacillus surrounded by sputum can remain viable in air and dust for lengthy periods. The combination of freezing and drying— lyophilization*—is a common method of preserving microorganisms and other cells in a viable state for many years Sterilization Processes Radiation  Radiation Ionizing Nonionizing X-Rays High speed UV radiation Gamma Rays Electrons  Several types of radiation find a sterilizing application in the manufacture of pharmaceutical and medical products, principal among which are accelerated electrons (particulate radiation), gamma rays and UV light (both electromagnetic radiations). Radiation The major target for these radiations is believed to be microbial DNA, with damage occurring as a consequence of ionization and free – radical production (gamma rays and electrons) or excitation (UV light). This latter process is less damaging and less lethal than ionization, and so UV irradiation is not as efficient a sterilization method as electron or gamma irradiation. Gamma Rays X-Rays UV radiation Ultraviolet irradiation The optimum wavelength for UV sterilization is around 260 nm. A suitable source for UV light is a mercury lamp giving peak emission levels at 254 nm. These sources are generally wall - or ceiling - mounted for air disinfection or fixed to vessels for water treatment. Operators present in an irradiated room should wear appropriate protective clothing and eye shields UV light, with its much lower energy, causes less damage to microbial DNA. This, coupled with its poor penetrability of normal packaging materials, renders UV light unsuitable for sterilization of pharmaceutical dosage forms. It does find applications, however, in the sterilization of air, for the surface sterilization of aseptic work areas, and for the treatment of manufacturing - grade water  Gamma ray's sterilizers 60 Gamma rays for sterilization are usually derived from a cobalt - 60 ( Co) source (caesium - 137 may also be used), For safety reasons, this source is housed within a reinforced concrete building with walls some 2 m thick, and it is raised from a sunken water - filled tank only when required for use. The primary application of gamma radiation is for medical devices, ranging from sterile dressings, tubes, catheters, syringes, infusions assemblies and implants; and single- use disposable technologies, such as bags for holding products or devices for making aseptic connections. The main reason why gamma radiation is selected as a sterilization method is due to its relatively high penetrability and as there is only a small temperature rise (typically