Controlling Microbial Growth in the Environment

Sterilization(a) Sterilization is the killing of all microorganisms in a material or on

the surface of an object

(b) A surface or an object is either sterile or it is not sterile, there are no gradations in sterility

(c) Typically the last things to die when one attempts sterilization is the highly heat- (and chemical-, etc.) resistant endospores

Aseptic

Degerming

Terminology of Microbial Control

Terminology of Microbial Control (cont.)

Disinfection/disinfectants(a) Disinfection means reducing the number of viable

microorganisms present in a sample(b) A disinfectant is a chemical or physical agent that is applied to

inanimate objects to kill microbes(c) Not all disinfectants are capable of sterilizing, but, of course, all

disinfectants are employed with the hope of disinfecting

Antisepsis/antiseptic Antiseptic(a) Typically an antiseptic is a chemical agent that is applied to

living tissue to kill microbes(b) Note that not all disinfectants are antiseptics because an

antiseptic additionally must not be so harsh that it damages living tissue

(c) With this constraint imposed on antiseptics, in general antiseptics are either not as cheap or not as effective at killing microbes as disinfectants

Sanitization Sanitization is the cleaning of pathogenic microorganisms from

public eating utensils and objects such as that done by the kitchen of a restaurant

PasteurizationSuffix –stasis/-static 

Bacteriostatic means that the antimicrobial inhibits bacterial growth but does not kill the bacteria; consequently, removal or dilution of the antimicrobial can result in a resurgence of bacterial growth

Suffix-cidal An antimicrobial that kills a microorganism (or, more specifically,

a bacterium) is said to be bactericidal

Terminology of Microbial Control (cont.)

Microbial Death Rates

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

Cell wall maintains integrity of cellWhen disrupted, cannot prevent cell from bursting due to osmotic effects

Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell

When damaged, cellular contents leak out

Viral envelope responsible for attachment of virus to target cell

Damage to envelope interrupts viral replication

Nonenveloped viruses have greater tolerance of harsh conditions

Alteration of Cell Walls and Membranes

Protein function depends on 3-D shapeExtreme heat or certain chemicals denature proteins

Chemicals, radiation, and heat can alter or destroy nucleic acids

Can produce fatal mutants

Can halt protein synthesis through action on RNA

Damage to Proteins and Nucleic Acids

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 involved

Environmental conditions that pertain

Factors Affecting the Efficacy of Antimicrobial methods

Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects

Method and level of microbial control based on site of medical procedure

Site to Be Treated

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

Decimal reduction time

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

Autoclave

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

Sterile Services Department (cont.)

Workflow of the SSD - in the ward, dirty re-usable instruments are collected and put into clearly labelled containers and delivered to the SSD. The dirty instruments are then received in the dirty area of the SSD. All equipment is first washed in hot water and detergent. The equipment is then inspected for cleanliness and damage. Instruments are then packed into individual trays for ward use and autoclaved and/or disinfected as required. The packaged trays are then inspected to ensure that they are dry and then sorted for ward collection. The sterile packs should be stored in well-ventilated rooms ready for dispatch to the wards. Collections should be regular and there should be a written record of receipt and delivery.

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

MilkBatch method – 30 minutes at 63ºC

Flash pasteurization – 72ºC for 15 seconds

Ultrahigh-temperature pasteurization – 134ºC for 1 second

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 means of sterilizationLoops (1500°C), carcasses, dressings

Dry Heat

Refrigeration is a great short term solution, 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

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 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 shorter than 1 nm – electron beams, gamma rays, and X rays

Ionizing radiation is radiation that ionizes water; this temporarily turns water into an oxidizing agent

Create ions by ejecting electrons from atoms they strike

Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxide ions; hydroxide ions denature other molecules (DNA)

One potential application of ionizing radiation is as an antimicrobial is in food preservation

Ionizing Radiation

Electron beams – effective at killing but do not penetrate well

Used to sterilize some microbiological plastic ware, and medical and dental supplies

Gamma rays – penetrate well but require hours to kill microbes

Used to sterilize some medical instruments

X-rays require too much time to be practical for growth control

Ionizing 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

Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA

Effect varies with temperature, length of exposure, and amount of organic matter

Also varies with pH, concentration, and age of chemical

Tend to be more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa

Chemical Methods of Microbial Control

General RulesCertain rules should be followed when using disinfectants. The manufacturer’s instructions should be followed. The expiry date should be checked and the optimum dilution used. Articles to be disinfected should be washed and cleaned before disinfection. The disinfectant container should not be refilled without sterilizing between each use and not be topped up. Disinfectants should be supplied ready for use from the pharmacy and empty containers should be returned to the pharmacy. Empty containers should not be discarded or used to store any other solutions as this is potentially dangerous. Open containers of disinfectant should not be tolerated as there is a serious risk of contamination with multiple antibiotic-resistant bacteria, such as Pseudomonas species and spores.

General RulesDisinfectants should be used according to instructions at the optimum dilution. Some disinfectants may be rapidly inactivated by organic matter, therefore any object that is to be disinfected should be cleaned thoroughly with warm water and detergent prior to disinfection. When disinfectants are indicated for use on surfaces, they should be applied by wiping rather than bathing as bathing wastes disinfectants. Sustained-action disinfectant should be used for hand hygiene by staff and for cleaning the skin and mucous membranes of patients. Alcohol preparations over 40% are not recommended because of the risk of fire when use in conjunction with diathermy. In general, hard surfaces do not require disinfectants - warm water with detergent is usually sufficient to remove all organic contamination. The exceptions are where persistence of potentially dangerous pathogens, such as HIV or HBV is suspected, when the surface should be wiped by a disinfectant afterwards.

Major CategoriesPhenols

Alcohols

Halogens

Oxidizing agents

Surfactants

Heavy Metals

Aldehydes

Gaseous Agents

Antimicrobics

Chemical Methods of Microbial Control

Intermediate- to low-level disinfectants

Denature proteins and disrupt cell membranes

Effective in presence of organic matter and remain active for prolonged time

Commonly used in health care settings, labs, and homes (Lysol, triclosan)

Have disagreeable odor and possible side effects

Phenol and Phenolics

Intermediate-level disinfectants

Denature proteins and disrupt cytoplasmic membranes

Evaporate rapidly – both advantageous and disadvantageous

Swabbing of skin with 70% ethanol prior to injection

Alcohols should not be applied to wounds since they can cause tissue damage

Tinctures

Alcohols

Intermediate-level antimicrobial chemicals

Believed that they damage enzymes via oxidation or by denaturing them

Iodine tablets, iodophores (Betadine®), chlorine treatment of drinking water, bleach, chloramines in wound dressings, and bromine disinfection of hot tubs

Halogens

Peroxides, ozone, and peracetic acid kill by oxidation of microbial enzymes

High-level disinfectants and antiseptics

Hydrogen peroxide can disinfect and sterilize surfaces of objects

Catalase neutralizes; not useful for treating open wounds

Ozone treatment of drinking water

Peracetic acid – effective sporocide used to sterilize equipment

Oxidizing Agents

Surfactants are substances that are soluble in water but are able to dissolve lipids ie. “Surface active” chemicals that reduce surface tension of solvents to make them more effective at dissolving solutes

Soaps and detergentsSoaps have hydrophilic and hydrophobic ends; good degerming agents but not antimicrobial

Detergents are positively charged organic surfactants

Quats – colorless, tasteless, harmless to humans, and antimicrobial; ideal for many medical and industrial application (benzalkonium chloride)

Low-level disinfectants

Pseudomonas

Surfactants

Ions are antimicrobial because they alter the 3-D shape of proteins, inhibiting or eliminating their function (cysteine)

Low-level bacteriostatic and fungistatic agents

1% silver nitrate to prevent blindness caused by N. gonorrhoeae

Thimerosal used to preserve vaccines

Copper controls algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks; interferes with chlorophyll

Heavy Metals

Compounds containing terminal –CHO groups

Cross-link with amino, hydroxyl, sulfhydryl, and carboxyl groups to denature proteins and inactivate nucleic acids

Only one liquid chemical, if properly used, is capable of rendering an item sterile. That chemical is glutaraldehyde. The item to be sterilized must be totally submerged in the glutaraldehyde solution for 10 hours. Before immersion, the item must be thoroughly cleansed and rinsed with sterile water or sterile normal saline. It should be noted that this chemical is extremely caustic to skin, mucous membranes, and other tissues i.e.Glutaraldehyde both disinfects (short exposure) and sterilizes (long exposure)

Formalin used in embalming and disinfection of rooms and instruments

Aldehydes

Ethylene oxide, propylene oxide, and beta-propiolactone used in closed chambers to sterilize itemsDenature proteins and DNA by

cross-linking functional groupsUsed in hospitals and dental offices

Can be hazardous to people, often highly explosive, extremely poisonous, and are potentially carcinogenicThe most effective method of gas chemical sterilization presently available is the use of ethylene oxide (ETO) gas. ETO gas sterilization should be used only for material and supplies that will not withstand sterilization by steam under pressure.

Gaseous Agents

Little evidence that extensive use of products containing antiseptic and disinfecting chemicals adds to human or animal health

The use of such products promotes the development of resistant microbes

Development of Resistant Microbes

Waste disposalAll hospital waste should be disposed of so that it presents no risk of injury or contamination. Clinical waste is generated during routine patent care and surgery. It is potentially dangerous and should be clearly labelled as high risk. Examples of clinical waste include dressings, body fluids, pathology waste, IV needles and syringes, drainage bags etc. Laboratory waste should also come under the high risk category and should be autoclaved before leaving the department and labelled biohazard. Other hospital waste include non-clinical waste and kitchen waste. The latter should be disposed of properly because it is a potential source of pests and vermin and thus may pose an indirect threat to the hospital. Provision must also be made for the safe disposal of radioactive waste. A simple colour-coding system should be used to separate waste so that the different components can be treated safely. In general, non-clinical waste is taken to a compactor and clinical waste to an incinerator or if not possible, to a lime-pit. Under no circumstances should clinical waste, needles or syringes be sent to the municipal dump. All staff handling clinical waste should be adequately trained especially with regards for the protocol of action in case of accidents. All staff must be provided with adequate protective clothing and replacement garments. Hepatitis B immunization should be offered to all staff and proper records kept for such immunization.

Waste disposalIt is totally unjustified to recycle needles and syringes. Broken glass should be included in the sharps category. To dispose safely of broken glass, thick gloves should be worn and the pieces collected with a newspaper. The glass is then wrapped securely in the paper and either disposed of into the sharps container or if not practicable, into a cardbox box which is then marked and sealed. Sharps containers must be leak and puncture-proof. Have a handle that allows lifting, a non-reopenable lid, and carry a biohazard sign. They should be sealed and replaced when it is no more than two-thirds full. Sharps containers are treated as clinical waste and should be put into clinical waste bags before being incinerated. In the event of an inoculation or contamination accident, it must be documented by the senior manager and reported to the Infection Control Team so that appropriate action can be taken. There must be clearly defined policy on the action to be taken after the injury and all staff should be aware of the policy.

General Use ArticlesBedpans and Urinals - gloves should be worn to empty the bedpan and its contents directly into the bedpan disinfector. Alternatively, the contents may be flushed down the sluice or toilet and then washed thoroughly with warm water and detergent to remove all signs of organic contamination and dried. The bedpan disinfector is the preferred method and it functions at no less than 80oC for 1 minute. Washing bowls - these must be washed thoroughly between each patient and stored and inverted to dry. Fresh water and towels should be used for each patient. Towels, soaps, hairbrushes, shaving brushes etc. - all these items should be for individual use only and should never be shared. Crockery and cutlery - each patient should have an individual set. the crockery and cutlery should be washed in very hot water (>60oC) and detergent. Disposable crockery is only necessary in cases of strict isolation such as rabies. Mattresses and pillows - they are a major source of cross-infection. Wet mattresses must be changed. Contaminated mattresses should be washed with warm water and detergent. Mattresses should be covered with an impervious layer so that they can be cleaned thoroughly between patients. Damaged mattresses should be discarded as they may easily trap microorganisms. Thermometers - they should be washed in warm water and dried before being wiped over with a swab soaked in 70% isopropyl alcohol. They should never be soaked in disinfectants. Trolley tops - they should be washed with warm water and detergent  

General Use ArticlesEndoscopy Unit Fibre-optic endoscopes are usually heat-labile and therefore require chemical disinfection. 2% glutaraldehyde should be used under strict controlled conditions as required by the COSHH regulations. Protoscopes and sigmoidoscopes may be disposable or reusable. The latter must be cleaned, sterilized, or disinfected by heat or 2% glutaraldehyde.Disposable Equipment Syringes and needles - disposable syringes and needles should be used. Under no circumstances should fine-bore needles be recycled because they cannot be cleaned. If syringes are to be recycled, it must be well controlled. After thorough cleaning, the syringes must be autoclaved or processed in ethylene oxide. Administration sets - administration sets for IV fluids must be disposable Urinary catheters and drainage bags - these should be single-use and disposable

ICUs, Operating Theatres Ventilatory circuits - multiple-use circuits must be heat-disinfected for at least 80oC for 3 minutes or sterilized by autoclave or ethylene oxide between each patient. Filters may be used between circuits and if properly maintained, a ventilated patient may use the same circuit for 4-5 days before disinfection. Humidifiers - humidifiers are a common source of viruses and Gram-negative bacilli and should be emptied daily and refilled with distilled water. They should be disinfected when contaminated or when the respiratory circuit is changed. Routine heat disinfection is essential after each patient use and if humidification is required for a prolonged period, the humidifier should be cleaned thoroughly, dried, and filled daily with distilled water. Endotracheal suction catheters - they are usually disposable but may be used up to 24 hours on the same patient. Endotracheal tubes - these may be recycled after thorough cleaning and autoclaving. Disposable endotracheal tubes are available but are more expensive. Ambu-bags - they are extremely difficult to disinfect and become contaminated very quickly. Heat is the most reliable method and 2% glutaraldehyde is a less acceptable alternative. Oxygen-delivery face masks - these can be disposable or reusable. If reused, they should be washed thoroughly and wiped over with 70% isopropyl alcohol. Suction and drainage bottles - these are usually disposable, with a self-dealing inner container held in a clear outer plastic container. The outer container should be heat-disinfected or autoclaved after each use. Non-disposable bottles must be changed every 24 hours or sooner if full. They must then be washed and autoclaved. Recyclable connector tubing should be cleaned thoroughly and sterilized. The system must be closed and risk to staff from body fluids should be minimal.