Controlling Microbial Growth in the Environment

Many types of chemical and physical microbial controls

Modes of action fall into two basic categoriesAlteration of cell walls or cytoplasmic membranes

Interference with protein and nucleic acid structure

Action of Antimicrobial Agents

Ideally, agents should be:Inexpensive

Fast-acting

Stable during storage

Control all microbial growth while being harmless to humans, animals, and objects

Selection of Microbial Control Methods

Nature of site to be treated

Degree of susceptibility of microbes involvedEnvironmental conditions that pertain التابعة المتعلقة

Factors Affecting the Efficacy of Antimicrobial methods

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

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

Relative Susceptibility of Microorganisms

Environmental conditions that pertainIncreasing temperatures increases the efficacy of most chemical antimicrobialsThe converse of this statement is that relatively cold temperatures result in relatively poor disinfectionThe 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

Phenol Coefficient

Use-Dilution Test

Disk-Diffusion Method

In-Use Test

Methods for Evaluating Disinfectants and Antiseptics

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

Phenol Coefficient

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

Use-Dilution Test

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

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

In-Use Test

Exposure to extremes of heat

Exposure to extremes of cold

Desiccation

Filtration

Osmotic pressure

Radiation

Physical Methods of Microbial Control

Effects of high temperaturesDenaturation 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

Heat-Related Methods

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 heatBoiling

Autoclaving

Pasteurization

Ultrahigh-Temperature Sterilization

Moist Heat

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

Boiling

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

Autoclaving

Autoclave

Sterile Services DepartmentA 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 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

Pasteurization

140ºC for 1 second, then rapid cooling

Treated liquids can be stored at room temperature

Chemical degradation

Ultrahigh-Temperature Sterilization

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 المطاف نهاية means of sterilizationفيLoops (1500°C), carcasses, dressings

Dry Heat

Refrigeration is a great short term solution القصير المدى على عظيم , حلit 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

Refrigeration and FreezingFor 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

Drying inhibits growth due to removal of water; only microbiostatic. Desiccation prevents organism metabolism, and is a good means of preventing organism growthDesiccation 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 crystalsFreeze-drying involves freezing something and then evacuating air so that boiling occurs at low temperatures; this desiccates material thereby preventing deterioration and spoilageLiquid nitrogen or frozen carbon dioxide (dry ice)

Desiccation and Lyophilization (Freeze-drying)

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

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

Osmotic Pressure

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

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

Nonionizing Radiation