Control of Microorganisms: Disinfection and Sterilization
Formaldehyde
- 37% aqueous solution or gas - Carcinogen and irritant - Non-tuberculocidal - Not recommended on a routine basis
Glutaraldehyde
- Alkylation of RNA and DNA - Effective against bacteria, fungi, and viruses (10 minute exposure at 20-30°C) - Does not penetrate organic material - Noncorrosive
Alcohols
- Broad-spectrum - Inactivated by organic material - Work by denaturing proteins, dehydrating, and dissolving lipids (60-90%)
Functions of Chemosterilizers
- Causes cell leakage and death - Denatures cellular proteins - Inactivates enzymes - Damages RNA and DNA
Modes of Infection in the Microbiology Lab
- Direct contact - Inhalation - Ingestion - Needle stick
Chlorohexidine gluconate (0.5-4%)
- Disrupts cell membrane and precipitates cell contents - Broad-spectrum - Not effective against endospores and nonenveloped viruses - Severe skin reactions in infants - Optimal pH range: 5.5-7.0
Recommendations for Laboratory Safety Improvements
- Do not remove items from the lab. - Always wear lab coats over personal clothing. - Always wash hands before leaving the laboratory.
Hexachlorophene (3%)
- Effective against gram-positive bacteria - Interrupts bacterial electron transport - Low concentration: inhibits membrane-bound enzymes - High concentration: ruptures bacterial membrane - Quick (15-30 sec) - Prescription only, due to toxic effects
Chloroxylenol (0.5-4%)
- Effective against gram-positive bacteria - Used in skin applications - Unaffected by organic material - Neutralized by nonionic surfactants and PEG
Factors That Influence the Degree of Killing
- Endospores - Mycobacterial cell walls - Biofilms - Microbial load - Concentration of disinfecting agents - Presence of organic material - Nature of surface being disinfected - Contact time - Temperature - pH of agent
Filtration
- Filters with various pore sizes - Retains bacteria and yeast with pore sizes larger than 0.45-0.80 µm
When working with actively growing cultures, it's important to:
- Frequently wash hands to avoid exposure. - Wear appropriate PPE. - Cover wounds. - Prevent exposure when determining a microbial odor. - Use appropriate engineering controls. - Wear a splash guard when appropriate. - Avoid having open flames in burners. - Seal agar plates growing fungus; only work under a biohazard hood.
Standard precautions address the following:
- Handwashing must be done after touching blood, body fluids, secretions, excretions, and any items considered contaminated. - Gloves should be worn when handling blood, body fluids, secretions, excretions, and any items considered contaminated. - Mask, eye protection, or face shield must be worn anytime there is a potential for splashes or sprays of blood, body fluids, secretions, and excretions. - Laboratory coats must be worn to protect skin and clothing when contact with blood, body fluids, secretions, and excretions could occur. - Appropriate sharps disposal must be implemented with care to prevent injuries with sharps, needles, and scalpels. - Environmental control must include procedures for routine care, cleaning, and disinfection of environmental surfaces.
Centers for Disease Control (CDC) and Prevention Guidelines for Safe Work Practices
- Identify hazards associated with infectious agents/materials. - Identify activities that might cause exposures to the agents/materials. - Consider competencies and experience of laboratory personnel. - Evaluate and prioritize risks. - Implement and evaluate controls to minimize risk for exposure.
Advantages of Sodium Hypochlorite (Bleach)
- Inexpensive - Broad-spectrum killing
Disadvantages of Sodium Hypochlorite (Bleach)
- Long exposure time - Corrosive and pH-sensitive - Inactivated by organic matter - Rapidly degrades
Heat
- Moist heat (121°C for 15 minutes) - Dry heat - Boiling - Pasteurization
Ethylene oxide
- Sterilization - Plastics and heat-sensitive materials - 450-700 mg/L at 55-60°C for 2 hours, humidity at 30% - Alkylation of nucleic acids
Classes of Fire Extinguishers: - A: __________________ - B: __________________ - C: __________________
- for paper, wood, upholstery, rags, or other types of trash fires - for flammable liquids or gas fires - electrical fires
Categories of Medical Materials
1. Critical materials 2. Semi-critical materials 3. Non-critical materials
Different Types of Organisms and Their Resistance to Killing Agents
1. Prions 2. Bacterial spores 3. Mycobacteria 4. Non-lipid viruses 5. Fungi 6. Bacteria 7. Lipid viruses
The EPA has also made recommendations for hazardous waste reduction through the following methods:
1. Substitute less hazardous chemicals when possible. 2. Develop procedures that use less of a hazardous chemical. 3. Recycle chemicals when possible. 4. Segregate infectious wastes from uncontaminated trash. 5. Substitute micromethodology in antimicrobial susceptibility testing and identification of organisms to reduce the volume of chemical reagents, as well as infectious waste.
Disinfectant
A chemical substance that kills harmful bacteria or viruses
Antiseptic
A substance that prevents infection
Biological safety cabinets (BSCs)
A type of containment barrier that protects the worker from the aerosolized transmission of organisms
Infectious agents that would be classified as requiring _____________________ containment are agents that are well classified and are not known to cause disease consistently in healthy adults. Examples of organisms include Bacillus subtilis and Enterobacter aerogenes.
BSL-1
Infectious agents that require ____________________ containment and practices are agents that pose a moderate potential hazard for the employees and the environment. Examples of organisms include HBV, HIV, Salmonella spp., and Toxoplasma gondii.
BSL-2
_____________________ containment and practices are required for infectious agents that are either indigenous or exotic. These agents have the potential for aerosol transmission, and diseases with these agents may have serious lethal consequences. Examples of organisms include M. tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii.
BSL-3
____________________ containment and practices are required when working with agents that are dangerous and exotic. These agents have a high risk of causing life-threatening infections, can be transmitted by aerosols, or have an unknown risk of transmission. There are two types of laboratories, cabinet and suit. In the _____________________ laboratory, all work is performed within a class III BSC. In a _____________________ laboratory, personnel wear a positive pressure protective suit to perform all work.
BSL-4; cabinet; suit
The potential infectious agents of bioterrorism are divided into the following categories: ____________________ pose the greatest public health threat because they are easily transmitted and are highly infectious. Examples include agents that cause smallpox, anthrax, and tularemia. ____________________ have moderate morbidity and low mortality and are not as easily transmitted as category A agents. Examples include agents that cause Q fever, melioidosis, and typhus fever. ____________________ are classified as emerging pathogens. Examples include Nipah virus and hantavirus.
Category A agents; Category B agents; Category C agents
The _____________________, 2nd edition, by the Clinical Laboratory and Standards Institute, provides information for laboratory managers on the regulations governing hazardous wastes and addresses the following types of waste: _____________________. The guideline also emphasizes methods to reduce waste generation and methods to reduce the volume of and toxicity of unavoidable wastes.
Clinical Laboratory Waste Management Approved Guideline; chemical, infectious, radioactive, sharps, multi-hazardous, and nonhazardous
Biofilms
Colonies of bacteria that adhere together and adhere to environmental surfaces
Synergy
Combined action or operation
Semi-critical materials
Come into contact with mucous membranes (high-level disinfectants) and intact skin (intermediate to low-level disinfectants)
The CDC has developed a program that identifies how health care workers and other public health officials should respond to an event. This program (____________________) addresses biological agents and diseases, laboratory information, training, preparedness and planning, and surveillance.
Emergency Preparedness and Response Program
The CDC developed the ____________________ in 1999. This program developed a network of laboratories that could respond quickly and effectively to biological and chemical terrorism. The LRN developed three levels of laboratories: (1) ____________________, (2) ____________________, and (3) ____________________.
Laboratory Response Network (LRN); sentinel laboratories; reference laboratories; national laboratories
All chemicals in the workplace must be identified and clearly labeled with the ______________________ 704 ______________________, stating risk for flammability, reactivity, and health.
National Fire Protection Association (NFPA); hazard-rating diamond
The mission of the ______________________ is to protect workers within the United States. In 1991, the OSHA created and released the ______________________ to protect healthcare workers. This standard was revised in 2001 in conformance with Public Law 106-430, the ______________________.
OSHA; Bloodborne Pathogens Final Standard; Needlestick Safety and Prevention Act
Non-critical materials
Require intermediate to low-level disinfection before contact with intact skin
R: ____________________ A: ____________________ C: ____________________ E: ____________________
Rescue; Remove anyone who is in danger. Alarm; Know where the nearest fire pull box or alarm station is located and the number to call to report the fire. Contain; Close doors to contain fire and smoke. Extinguish; Use the properly rated fire extinguisher on small fires.
_____________________ are provided by the manufacturer or distributor for hazardous chemicals. These documents should be kept on file and made available to every employee.
Safety data sheets (SDSs)
____________________ constitute most hospital-based microbiology laboratories and are divided into two levels. ___________________ function at the frontlines and have the most capability, recognize possible bioterrorism agents and perform basic testing to rule out these agents. _____________________ have fewer analytical capabilities, but may handle suspect samples and would refer these specimens to an LRN reference laboratory.
Sentinel laboratories; Advanced sentinel clinical laboratories; Basic sentinel clinical laboratories
Sterilization
The process that completely destroys all microbial life, including spores
Disinfection
The process that eliminates many or all microorganisms, with the exception of bacterial spores, from inanimate objects
Critical materials
Those that enter sterile tissues or enter the vascular system. Most likely to produce an infection if contaminated and requires sterilization.
Iodine can be used as a disinfectant in one of two forms: tincture or iodophor. ____________________ are alcohol and iodine solutions used mainly as antiseptics. An ____________________ is a combination of iodine and a neutral polymer carrier that increases the solubility of the agent. This combination allows the slow release of iodine. When iodophors are used as skin preparation agents for sites where blood is to be drawn for blood cultures, it is critical that there is the proper amount of contact time, which is generally more than ____________________ seconds. All iodine tinctures and iodophors must be completely removed from the skin to avoid ____________________.
Tinctures; iodophor; 30; irritation
_________________________ are added precautions that are used when the patient is known or suspected to be infected or colonized with an infectious agent that requires extra measures to prevent spread or transmission of the agent. The categories of these precautions are contact precautions, droplet precautions, and airborne precautions. __________________________ are used to stop the spread of infectious agents that may be transmitted through direct or indirect contact with the patient or with the patient's environment. __________________________ are used to stop the spread of infectious agents that can be transmitted by close respiratory contact or by exposure of mucous membranes to respiratory secretions. The final category is _________________________. These precautions are used for infectious agents that can remain airborne and infectious over long distances.
Transmission-based precautions; Contact precautions; Droplet precautions; airborne precautions
Antagony
Two inactivate each other
Chemicals should never be stored _____________________. Chemicals must be stored according to established rules of _____________________ to prevent contact between reactive substances. Do not store alkali metals (e.g., sodium, potassium) with carbon dioxide, chlorinated hydrocarbons, or water. Acids and bases should never be stored together. Halogens are incompatible with ammonia, acetylene, and hydrocarbons. Flammable chemicals should be stored in a flammable storage cabinet.
alphabetically; compatibility
Flammable and nonflammable gas cylinders in use in the laboratory must always be properly restrained and stored in bulk and secured in vented areas. In the laboratory, cylinders must be located ____________________ from open flames and other heat sources. Because a leaking pressurized gas cylinder is a potential "_____________________," care must be taken to avoid accidental breakage or removal of the pressure valve on top. The metal cap that protects this valve on top of the cylinder must be kept in place during transportation and when the cylinder is not in use.
away; missile
The ______________________ uses an exhaust fan to move air inward through the open front. The air is circulated within the safety hood, passing through a high-efficiency particulate air (HEPA) filter before reaching the environment outside the hood.
class I biological safety cabinet (hood)
The ______________________ is the most common in microbiology laboratories. Air is pulled inward and downward by a blower and passed up through the air flow plenum, where it passes through a HEPA filter before reaching the work surface.
class II biological safety hood
The ____________________ is a self-contained ventilated system for highly infectious microorganisms or materials and provides the highest level of personal protection. The closed front contains attached gloves for manipulation on the work surface.
class III biological safety hood
All clinical laboratory personnel should have a thorough working knowledge of the hazards of the chemicals with which they come into contact, or _____________________.
employee right-to-know
Employers are required to have an ____________________, which must be reviewed and updated annually. This plan must be available to all employees and should include the following: - A determination of tasks and procedures that may result in an occupational hazard - A plan to investigate all exposure incidents and a plan to prevent these from reoccurring - Methods of compliance for standard precautions - Engineering and work practice controls - Personal protective equipment (PPE) - Guidelines for ensuring that the work site is maintained in a clean and sanitary manner - Guidelines for the handling and disposal of regulated waste - A training program for all employees
exposure control plan
All laboratory electrical equipment must conform to national electrical safety standards and codes. Each instrument must undergo regular preventive maintenance to ensure that it is functioning properly and in the best repair. Electrical cords should be checked for fraying. All cords should have _____________________ (three-pronged) plugs. Electrical equipment should never be placed near _____________________ because of the risk of electrocution.
grounded; safety showers
For infection to occur, you need ______________________. Biological hazards come from 2 major sources: ______________________ and ______________________.
infectious agent, mode of transmission, high concentration of infectious agent; processing of patient specimens; handling of actively-growing cultures
Laboratory personnel who are involved in the packaging and shipping of __________________ materials must be trained and certified. Personnel must be ___________________ on a regular basis.
infectious; retrained
Specialized clothing or equipment that is worn by an employee for protection is defined by the OSHA as _________________________. It must be provided and maintained by the employer; examples include gloves, laboratory coats, masks, respirators, face shields, and safety glasses. The equipment must be accessible to the employee and must be worn whenever there is the potential for exposure to infectious material; it must be removed before leaving the work area and must be placed in an area designated for PPE.
personnel protective equipment (PPE)
The role of ___________________ and __________________ in the LRN is to perform confirmatory testing. At the present time, more than 100 laboratories are members of the reference laboratory category. The national laboratories in the LRN are the ___________________, the _____________________, and the ____________________.
reference laboratories; national laboratories; CDC; U.S. Army Medical Research Institute of Infectious Diseases; Naval Medical Research Center
The combination of H₂O₂ and peracetic acid vapors is used in the pharmaceutical and medical device manufacturing industries. The major advantage to the use of the combination over each of its individual components is a ____________________.
shorter contact time
Acid and base or flammable ____________________ should be kept in areas where such substances are used. Equipment such as protective clothing, scoops and dust pans, forceps for picking up glass, and buckets should be kept in a designated area. In the event of a large spill, the ____________________ should be called for assistance.
spill kits; Environmental Health and Safety Department
Use of long ___________________ that extend to the shoulder is recommended when reaching into autoclaves or hot-air ovens. Burns may also come from extremely low temperatures found with use of liquid nitrogen or freezers that maintain temperatures less than __________________.
thermal gloves; −70° C
In 1985, the CDC instituted safety guidelines for the handling of blood and body fluids. These guidelines, called _____________________, were intended to protect hospital personnel from bloodborne infections. In 1996, these guidelines were updated and renamed. These new guidelines, ______________________, are still in effect; they require that blood and body fluids from all patients be considered infectious and capable of transmitting disease. Blood and all body fluids, including secretions and excretions, except _______________________, regardless of whether visible blood is present, are considered infectious. Standard precautions also include nonintact skin and mucous membranes.
universal precautions; standard precautions; sweat
Altering the manner in which a task is performed to reduce the likelihood of exposure to infectious agents is defined by the OSHA as _______________________. Examples include the following: - No mouth pipetting - No eating, drinking, smoking, or applying cosmetics in the laboratory - Disinfection of workstations at the end of each shift and after any spill of infectious material
work practice controls
