Controlling Microbial Growth in the Environment

Action of Antimicrobial Agents Many types of chemical and physical microbial controls Modes of action fall into two basic categories Alteration of cell walls or cytoplasmic membranes Interference with protein and nucleic acid structure

Selection of Microbial Control Methods Ideally, agents should be: Inexpensive Fast-acting Stable during storage Control all microbial growth while being harmless to humans, animals, and objects

Factors Affecting the Efficacy of Antimicrobial methods Nature of site to be treated Degree of susceptibility of microbes involved Environmental conditions that pertainالمتعلقة التابعة

Organismal differences Microorganisms differ in their susceptibility to antimicrobial agents." Often what fails to be killed by a disinfectant are endospores though certain viruses and some vegetative bacteria are also highly resistant to disinfection Also, the same organism may differ in susceptibility depending on growth phase with actively growing organisms typically more susceptible to disinfection than not-growing cultures

Relative Susceptibility of Microorganisms

Relative Susceptibility of Microorganisms Effectiveness of germicides classified as high, intermediate, or low High-level kill all pathogens, including endospores Intermediate-level kill fungal spores, protozoan cysts, viruses and pathogenic bacteria Low-level germicides kill vegetative bacteria, fungi, protozoa, and some viruses

Environmental conditions that pertain Increasing temperatures increases the efficacy of most chemical antimicrobials The converse of this statement is that relatively cold temperatures result in relatively poor disinfection The fewer organisms present, the shorter the time needed to achieve sterility. Thoroughly cleaning objects before attempting to sterilize them is a practical application of this principle. Clearing objects of tissue debris and blood is also important because such organic matter impairs the effectiveness of many chemical agents.

Environmental conditions that pertain (cont.) Concentration effects: Generally, the use of more disinfectant provides better killing than the use of less disinfectant

Environmental Conditions

Methods for Evaluating Disinfectants and Antiseptics Phenol Coefficient Use-Dilution Test Disk-Diffusion Method In-Use Test

Phenol Coefficient Evaluating the efficacy of disinfectants and antiseptics by determining the ratio of agent’s ability to control microbes to that of phenol Greater than 1.0 indicates that agent is more effective than phenol Has been replaced by newer methods

Use-Dilution Test Especially useful for determining the ability of disinfectants to kill microorganisms dried onto a typical clinical surface (stainless steel) Metal cylinders dipped into broth cultures of bacteria and dried Contaminated cylinder immersed into dilution of disinfectant for 10 minutes Cylinders removed, washed, and placed into tube of medium for 48 h Most effective agent entirely prevents growth at highest dilution

Disk-Diffusion Method A method that requires less manipulation to judge the efficacy of disinfectants Here filter paper is soaked with disinfectant and then simply placed on the agar surface of a petri dish that has been inoculated with a lawn of test organism The clear area around the disk following incubation is used as an indication of disinfectant efficacy

Disk-Diffusion Method

In-Use Test Swabs taken from objects before and after application of disinfectant or antiseptic Swabs inoculated into growth medium and incubated Medium monitored for growth Accurate determination of proper strength and application procedure for each specific situation

Physical Methods of Microbial Control Exposure to extremes of heat Exposure to extremes of cold Desiccation Filtration Osmotic pressure Radiation

Heat-Related Methods Effects of high temperatures Denaturation of proteins Interference with integrity of cytoplasmic membrane and cell walls Disruption of structure and function of nucleic acids Thermal death point – lowest temperature that kills all cells in broth in 10 minutes Thermal death time – time to sterilize volume of liquid at set temperature

Moist Heat Used to disinfect, sanitize, and sterilize Kills by denaturing proteins and destroying cytoplasmic membranes More effective than dry heat; water better conductor of heat than air. Moist heat is also more penetrating than dry heat Methods of microbial control using moist heat Boiling Autoclaving Pasteurization Ultrahigh-Temperature Sterilization

Boiling Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses within 10 minutes at sea level Temperature cannot exceed 100ºC at sea level; steam carries some heat away Boiling time is critical Water boils at lower temperatures at higher elevations; requires longer boiling time Endospores, protozoan cysts, and some viruses can survive boiling

Autoclaving Pressure applied to boiling water prevents steam from escaping Boiling temperature increases as pressure increases Autoclave conditions – 121ºC, 15 psi, 15 minutes Volume Contact Wrapping Testing

Autoclave

Sterile Services Department A sterile services department (SSD) is vital for an effective infection program. Dirty, recyclable equipment should be collected from the wards عنابرand transferred to the SSD, where it is washed, inspected, sterilized, packed, and dispatched back to the wards.

Pasteurization Pasteur’s method Pasteurization is the application of moist heat of less-than boiling temperatures to foods to prevent the growth of food-spoiling organisms as well as various heat-labile pathogens Pasteur’s method Today, also used for milk, ice cream, yogurt, and fruit juices Not sterilization; heat-tolerant and heat-loving microbes survive These do not cause spoilage prior to consumption These are generally not pathogenic

Ultrahigh-Temperature Sterilization 140ºC for 1 second, then rapid cooling Treated liquids can be stored at room temperature Chemical degradation

Dry Heat Used for materials that cannot be sterilized with or are damaged by moist heat Denatures proteins and oxidizes metabolic and structural chemicals Requires higher temperatures or longer time than moist heat Nevertheless, application of dry heat is cheap and easy Typically one bakes materials in an oven at (i)   171ºC for at least one hour (ii)  160ºC for at least two hours (iii) 121ºC for at least 16 hours Incineration – ultimate في نهاية المطافmeans of sterilization Loops (1500°C), carcasses, dressings

Refrigeration and Freezing Refrigeration is a great short term solutionحل عظيم على المدى القصير , it decreases microbial metabolism, growth, and reproduction i.e. it merely slows the growth of organisms rather than preventing it Chemical reactions occur slower at low temperatures Liquid water not available Psychrophilic microbes can multiply in refrigerated foods Refrigeration halts growth of most pathogens (Listeria, Yersinia) Slow freezing more effective than quick freezing Organisms vary in susceptibility to freezing

Refrigeration and Freezing For organisms that survive the freezing process, freezing constitutes a reasonably good long-term preservation method with prevention of deterioration increasing as temperatures are lowered Lower temperatures result in greater long-term storage: (i)  -20ºC is the temperature of a standard freezer (ii) -80ºC is a good temperature for long-term storage of microorganisms (iii)-180ºC is the temperature of liquid nitrogen, a common medium for long-term storage of microorganisms

Desiccation and Lyophilization (Freeze-drying) Drying inhibits growth due to removal of water; only microbiostatic. Desiccation prevents organism metabolism, and is a good means of preventing organism growth Desiccation is an effective means of organism storage for those organisms capable of remaining viable upon desiccation Lyophilization used for long term preservation of microbial cultures Prevents formation of damaging ice crystals Freeze-drying involves freezing something and then evacuating air so that boiling occurs at low temperatures; this desiccates material thereby preventing deterioration and spoilage Liquid nitrogen or frozen carbon dioxide (dry ice)

Filtration Used to sterilize ophthalmic للعيون solutions, antibiotics, vaccines, liquid vitamins, enzymes, culture media eg, tissue culture Membrane filters (nitrocellulose) with pore sizes 25µm to less than 0.01µm High-efficiency particulate air (HEPA) filters are used to filter the air flowing into aseptic environments ( e.g. operating rooms) and out of potentially contaminated ones (e.g., containment facilities)

Filtration

Filtration

Osmotic Pressure High concentrations of salt or sugar in foods to inhibit growth (jam, jerky, pickled food, salted fish) Water in cell is drawn out by osmosis; cell desiccates Fungi have greater ability than bacteria to survive hypertonic environments

Radiation Electromagnetic radiation (enrgy without mass travelling in waves at the speed of light, 3x105km/sec) Shorter wavelength equals more energy and greater penetration Radiation described as ionizing or nonionizing according to effects on cellular chemicals

Nonionizing Radiation Wavelengths greater than 1 nm Excites electrons and causes them to make new covalent bonds Affects 3-D structure of proteins and nucleic acids UV light causes thymine dimers in DNA UV light does not penetrate well Suitable for disinfecting air, transparent fluids, and surfaces of objects