Control Of Microbial Growth PDF

**LESSON 11 Control of Microbial Growth** **COURSE MATERIAL** Control of microorganisms can be achieved by a variety of chemical and physical methods. ***Antiseptic*** is a chemical agent of disinfection that is mild enough to be used on human skin. Sterilization is generally achieved by [physical...

**LESSON 11 Control of Microbial Growth** **COURSE MATERIAL** Control of microorganisms can be achieved by a variety of chemical and physical methods. ***Antiseptic*** is a chemical agent of disinfection that is mild enough to be used on human skin. Sterilization is generally achieved by [physical] means such as heat, radiation, and filtration. Agents which **destroy** bacteria are said to be ***bactericidal**.* Chemical methods, whilst effective at *disinfection,* are generally not reliable for achieving total sterility. Agents which **inhibit** the growth and reproduction of bacteria without bringing about their destruction are described as ***bacteriostatic**.* **STERILIZATION** is the process by which all microorganisms present on or in an object are destroyed or removed. [Heating] is the most preferred means of sterilization if it may not damage the material. Boiling at 100 ^0^C for 10 minutes is usually enough to achieve sterility, if organisms are not present in high concentrations; in fact, most bacteria are killed about 70 ^0^C. If, however, endospores of some bacteria (notably *Bacillus* and *Clostridium*) are present, they can resist boiling, sometimes for several hours. To destroy the heat resistant endospores, heating beyond 100 ^0^C is required and this is achieved by heating under pressure in an **autoclave**. A typical laboratory treatment is 15 minutes at a pressure of 103 kPa (15psi), raising temperature of steam to 121 ^0^C. An effect like that of autoclaving can be obtained by a method called [intermittent steaming] or ***tyndallization*** (after the physicist John Tyndall, who was one of the first to demonstrate the existence of heat-resistant microbial forms). This is used for those substances or materials that might be damaged by high temperatures used in autoclaving. The materials heated between 90 and 100 ^0^C for about minutes on each of three successive days and left at 37 ^0^C in the intervening periods. Vegetative cells are killed off during the heating period, and during the 37 ^0^C incubation, any endospores that have survived will germinate. Once these have grown into more vegetative cells, they too are killed in the next round of steam treatment. High temperatures can cause damage to the taste, texture, and nutritional value of many food substances and in such instances, it is sufficient to destroy the vegetative cells by the process of ***pasteurization*** (after Louis Pasteur who demonstrated that the microbial spoilage of wines could be prevented by short periods of heating). Milk was traditionally pasteurized by heating large volumes at 63 ^0^C for 30 minutes, but the method employed nowadays is to pass it over a heat exchanger at 72 ^0^C for 15 seconds (HTST -- high temperature, short time). This is not sterilization as such, but it ensures the destruction of disease-causing organisms such as *Brucella abortus* and *Mycobacterium tuberculosis*, which at one time were frequently found in milk, as well as significantly reducing the organisms that cause food spoilage, thus prolonging the time the milk can be kept. One type of milk on sale in the shops is subjected to more extreme heating regimes; this is 'UHT' milk, which can be kept for several weeks without refrigeration, though many find that this is achieved at some cost to its palatability! It is heated to *ultra-high t*emperatures (150 ◦C) for a couple of seconds using superheated steam. The product is often referred to as being 'sterilized', but this is not true in the strictest sense. Milk is not the only foodstuff to be pasteurized; others such as beer, fruit juices and ice cream each has its own time/temperature combination. **Sterilization by irradiation** Certain types of irradiations are used to control the growth of microorganisms. These include both [ionizing] and [non-ionizing radiation.] The most widely used form of **non-ionizing** radiation is ultraviolet (UV) light. Wavelengths around 260nm are used because these are absorbed by the purine and pyrimidine components of nucleic acids, as well as certain aromatic amino acids in proteins. The absorbed energy causes a rupture of the chemical bonds, so that normal cellular function is impaired. Although many bacteria can repair this damage by enzyme-mediated photo-reactivation, viruses are much more susceptible. UV lamps are commonly found in food preparation areas, operating theatres, and specialist areas such as tissue culture facilities, where it is important to prevent contamination. Because they are also harmful to humans (particularly the skin and eyes), UV lamps can only be operated in such areas when people are not present. UV radiation has very poor penetrating powers; a thin layer of glass, paper or fabric is able to impede the passage of the rays. The chief application is therefore in the sterilization of work surfaces and the surrounding air, although it is increasingly finding an application in the treatment of water supplies. **Ionizing radiation has** a shorter wavelength and much higher energy, giving them greater penetrating powers. The effect of ionizing radiation is due to the production of highly reactive free radicals, which disrupt the structure of macromolecules such as DNA and proteins. Surgical supplies such as syringes, catheters and rubber gloves are commonly sterilized employing gamma (*γ*) rays from the isotope cobalt 60 (^60^Co). Gamma radiation has been approved for use in over 40 countries for the preservation of food, which it does not only by killing pathogens and spoilage organisms but also by inhibiting processes that lead to sprouting and ripening. The practice has aroused a lot of controversy, largely due to concerns about health and safety, although the first patent applications for its use date back nearly a hundred years! Although the irradiated product does not become radioactive, there is a general suspicion on the part of the public about anything to do with radiation, which has led to its use on food being only very gradually accepted by consumers. Gamma radiation is used in situations where heat sterilization would be inappropriate, because of undesirable effects on the texture, taste or appearance of the product. This mainly relates to fresh produce such as meat, poultry, fruit and vegetables. Irradiation is not suitable for some foodstuffs, such as those with a high fat content, where unpleasant tastes and odors result. Ionizing radiation has the great advantage over other methods of sterilization that they can penetrate packaging. **FILTRATION** is an alternative approach rather than killing the microorganisms, it simply isolates them. This can be done for liquids and gases by passing them through filters of an appropriate pore size. Filters used to be made from materials such as asbestos and sintered glass, but have been largely replaced by membrane filters, commonly made of nitrocellulose or polycarbonate. These can be purchased ready-sterilized, and the liquid passed through by means of pressure or suction. Supplies of air or other gases can also be filter-sterilized in this way. A pore size of 0.22*μ*m is commonly used; this will remove bacteria plus, of course, anything bigger, such as yeasts; however, mycoplasma and viruses are able to pass through pores of this size. With a pore size 10 times smaller than this, only the smallest of viruses can pass through, so it is important that an appropriate pore size is chosen for any given task. A drawback with all filters, but especially those of a small pore size, is that they can become clogged easily. Filters in general are relatively expensive and are not the preferred choice if alternative methods are available. High efficiency particulate air (HEPA) filters create clean atmospheres in areas such as operating theatres and laboratory laminar-flow hoods. **STERILIZATION using ethylene oxide.** Generally, chemical methods achieve only disinfection; the use of the gas ethylene oxide, however, is effective against bacteria, their spores, and viruses. It is used for sterilizing large items of medical equipment, and materials such as plastics that would be damaged by heat treatment. Ethylene oxide is particularly effective in sterilizing items such as dressings and mattresses, due to its great powers of penetration. In the food industry, it is used as an antifungal fumigant, for the treatment of dried fruit, nuts and spices. The materials to be treated are placed in a special chamber which is sealed and filled with the gas in a humid atmosphere at 40--50 ◦C for several hours. Ethylene oxide is highly explosive, so it must be used with great caution; its use is rendered safer by administering it in admixture (10 per cent) with a non-flammable gas such as carbon dioxide. It is also highly toxic, so all items must be thoroughly flushed with sterile air following treatment to remove any trace of it. Ethylene oxide is an alkylating agent; it denatures proteins by replacing labile hydrogens such as those on sulfhydryl groups with a hydroxyl ethyl radical. **DISINFECTION** *Disinfection* is the elimination or inhibition of pathogenic microorganisms in or on an object so that they no longer pose a threat. But it allows the possibility that some organisms may survive, with the potential to resume growth when conditions become more favorable. A d*isinfectant* is a chemical agent used to disinfect inanimate objects such as work surfaces and floors. In the food and catering industry, the term *sanitization* is used to describe a combination of cleaning and disinfection. Disinfectants are incapable of killing spores within a reasonable time period and are generally effective against a narrower range of organisms than physical means. *Decontamination* is a term sometimes used interchangeably with disinfection, but its scope is wider, encompassing the removal or inactivation of microbial products such as toxins as well as the organisms themselves. The lethal action of disinfectants is mainly due to their ability to react with microbial proteins, and therefore enzymes. Consequently, any chemical agent that can coagulate, or in any other way denature, proteins will act as a disinfectant, and compounds belonging to several groups are able to do this. **Alcohols** The antimicrobial properties of ethanol have been known for over a century. It was soon realized that it worked more effectively as a disinfectant at less than 100 per cent concentration, that is, when there was some water present. This is because denaturation of proteins proceeds much more effectively in the presence of water. It is important, however, not to overdo the dilution, as at low percentages some organisms can utilize ethanol as a nutrient. [Ethanol] and [isopropanol] are most used at a concentration of per cent. As well as denaturing proteins, alcohols may act by dissolving lipids, and thus have a disruptive effect on membranes, and on the envelope of certain viruses. Both bacteria and fungi are killed by alcohol treatment, but spores are often resistant because of problems in rehydrating them; there are records of anthrax spores surviving in ethanol for 20 years. The use of alcohols is further limited to those materials that can withstand their solvent action. Alcohol may also serve as solvents for certain other chemical disinfectants. The effectiveness of iodine for example, can be enhanced by being dissolved in ethanol. **Halogens** *Chlorine* is an effective disinfectant as a free gas, and as a component of chlorine-releasing compounds such as hypochlorite and chloramines. Chlorine gas, in compressed form, is used in the disinfection of municipal water supplies, swimming pools and the dairy industry. *Sodium hypochlorite* (household bleach) oxidizes sulfhydryl (−SH) and disulphide (S−S) bonds in proteins. Like chlorine, hypochlorite is inactivated by the presence of organic material. Chloramines are more stable than hypochlorite or free chlorine and are less affected by organic matter. They are also less toxic and have the additional benefit of releasing their chlorine slowly over a period, giving them a prolonged bactericidal effect. *Iodine* acts by combining with the tyrosine residues on proteins; its effect is enhanced by being dissolved in ethanol (1% Iodine in 70% ethanol) as tincture of iodine, an effective skin disinfectant. Its use is being superseded by iodophores (Betadine, Isodine), in which iodine is combined with an organic molecule, usually a detergent, to combat bacteria, viruses and fungi, but not spores. Iodine **Phenolics** The germicidal properties of phenol (carbolic acid) were first demonstrated by Lister in the middle of the 19th century. Since it is highly toxic, phenol's use in the disinfection of wounds has long since been discontinued, but derivatives such as *cresols* and *xylenols* continue to be used as disinfectants and antiseptics. These are both less toxic to humans and more effective against bacteria than the parent compound. *Phenol* is still used, however, as a benchmark against which the effectiveness of related disinfectants can be measured. Phenolic acts by combining with and denaturing proteins, as well as disrupting cell membranes. Their advantages include the retention of activity in the presence of organic substances and detergents, and their ability to remain active for some time after application; hence their effect increases with repeated use. Familiar disinfectants such as Dettol, Lysol and chlorhexidine are all phenol derivatives. Hexachlorophene is very effective against Gram-positive bacteria such as staphylococci and streptococci, and used to be a component of certain soaps, surgical scrubs, shampoos, and deodorants. Its use is now confined to specialist applications in hospitals since the finding that in some cases, prolonged application can lead to brain damage. **Surfactants** Surfactants reduce the tension between two molecules at an interface. Surface active agents or surfactants, such as soaps and detergents, could orientate themselves between two interfaces to bring them into closer contact. The value of soap has less to do with its disinfectant properties than with the ability to facilitate the mechanical removal of dirt and microorganisms. It does this by emulsifying oil secretions, allowing the debris to be rinsed away. Detergents may be *anionic* (negatively charged), *cationic* (positively charged) or nonionic. Cationic detergents such as quaternary ammonium compounds act by combining with phospholipids to disrupt cell membranes and affect cellular permeability. **Other Methods of Control** **Desiccation** or dehydration is a method used to preserve foods such as raisins, prunes and meat jerky. Although drying controls microbial growth, it might not kill all the microbes or their endospores, which may start to regrow when conditions are more favorable and water content is restored. Some foods are sun dried while others are freeze dried, the process is known as **lyophilization.** In lyophilization, the item is rapidly frozen and placed under vacuum so that water is lost by sublimation. It combines both exposure to cold temperatures and desiccation, making it a very effective process in controlling microbial growth. Water activity or the water content of food and materials can be lowered by adding solutes such as salt and sugars. High concentrations of salts or sugars dramatically reduce the water from the cells due to osmosis. Many microorganisms do not survive conditions of high osmotic pressure. However, there are microbes that are tolerant to desiccation and high osmotic pressure, such as molds and yeast, thus they may still contaminate these types of food. **Chemical food preservatives** Commonly used chemical preservatives include sorbic acid, benzoic acid, and propionic acid and their soluble salts potassium sorbate, sodium benzoate, and calcium propionate, all which control the growth of molds in acidic foods. Each of these preservatives is nontoxic and readily metabolized by humans. They are also flavorless, so they do not compromise the flavor of the food they preserve. Sorbic and Benzoic acids exhibit increased efficacy as the pH decreases. **Sonication** Sonication is the use of high-frequency ultrasound waves to disrupt cell structures. The ultrasound can cause rapid changes in pressure within the cell structures and eventually cause the cell to lyse or collapse. Sonication is used for cleaning surgical instruments, lenses, and a variety of objects such as coins, tools, and musical instruments. **Heavy metals** Heavy metals kill microbes by binding to proteins, thus inhibiting enzymatic activity. Heavy metals are oligodynamic, meaning that very small concentrations slow significant antimicrobial activity. Ions of heavy metals bind to sulfur-containing amino acids strongly and bioaccumulate within the cells, allowing these metals to reach high localized concentrations. This cause proteins to denature. Mercury is an example of a heavy metal that has been used for many years to control microbial growth. Various forms of mercury bind to sulfur-containing amino acids within proteins, inhibiting their functions. Topical antiseptics such as mercurochrome, which contains very low concentrations of mercury, and Merthiolate, a tincture was once commonly used. However, because of concerns of mercury toxicity to central nervous, digestive, and renal system, at high concentrations and has negative effects to the environment including biomagnification in fish, the use of the compounds has diminished. Silver has long been used as an antiseptic. Silver nitrate drops were once routinely applied to the eyes of newborns to protect against ophthalmia neonatorum, eye infections that can occur due to exposure to pathogens in the birth canal. Copper, Nickel, and Zinc Copper sulfate is common algicide used to control algal growth in swimming pools and fish tanks. Copper linings in incubators help reduce contamination of cell cultures. The use of copper pots for water storage in underdeveloped countries is being investigated to combat diarrheal diseases. Copper coatings are also becoming popular for frequently handled objects such as doorknobs, cabinet hardware, and other fixtures in health-care facilities to reduce the spread of microbes. Zinc chloride is quite safe for humans and is commonly found in mouthwashes. Zinc oxide is found in a variety of products, including topical antiseptic creams such as calamine lotion, diaper ointments, baby powder and dandruff shampoos. **Kinetics of cell death** Microorganisms do not die instantly when exposed to the mentioned process or chemicals. At a given time, a portion of them will be killed, it depends on how dense the population of the microbes are present. A portion of the surviving population is killed per unit time, it follows that the more cells you have, the longer it will take to eliminate them. As a matter of fact, the microbial population is a mixed of organisms with different susceptibility thus, that may require different treatment. The Tables below shows the summary of the Common protocols for the control of microbial growth. Source: Parker, Nina, et.al. 2018. ***Microbiology***. Openstax, Rice University, Houston, Texas Source: Parker, Nina, et.al. 2018. ***Microbiology***. Openstax, Rice University, Houston, Texas Source: Parker, Nina, et.al. 2018. ***Microbiology***. Openstax, Rice University, Houston, Texas **Readings:** Hogg, Stuart. 2005. ***Essential Microbiology***. John Wiley & Sons, Ltd. England. Parker, Nina, et.al. 2018. ***Microbiology***. Openstax, Rice University, Houston, Texas Control of Microbial Growth. Retrieved from: http://textbookofbacteriology.net/control.html\#:\~:text=Control%20of%20growth%20usually%20involves,referred%20to%20as%20static%20agents.