SAFETY IN THE CLINICAL MICROBIOLOGY LABORATORY

Sterilization

destruction of all forms of life including bacterial

spores.

Autoclave

pressure saturated steam at 121ᵒC for 15-20 mins

Disinfectant

A disinfectant is a chemical or physical agent that is

applied to inanimate objects to kill microbes

Antiseptic

a chemical agent that is applied to living tissue to kill

microbes

(Disinfection) (Quaternary ammonium compounds (Quats) (Bactericidal) (Bacteriostatic) (Chemical disinfection) (Physical disinfection) (Gas sterilization) (Blood-borne pathogens) (Material Safety Data Sheets (MSDS) (Personal Protective Equipment) (Standard Precautions) (Occupational Safety and Health Administration) (Biological Safety Cabinets (BSC)

(_________)	reducing the number of viable microorganisms present in a sample
(__________)	a type of disinfectant that is a cationic detergent
(_________)	an antimicrobial that kills a microorganism ex. Formalin, UV rays

(_______)	a condition where the multiplication of the bacteria is inhibited without killing them.
(________)	use of chemicals to kill or inhibit the growth of microorganisms
(________)	use of methods such as heat, UV light, and filtration to kill or inhibit organisms

(_______)	uses ethylene oxide gas; used to sterilize objects that can’t be autoclaved like plastic products. A relative humidity of 30% can destroy spores
(_______)	are microorganisms in the blood or other body fluids that can cause illness and disease in people
(_______)	a form with data regarding the properties of a particular substance. It may include instructions for the safe use and potential hazards associated with a particular material or product

(________)	includes all types of equipment used to increase individual safety while performing potentially hazardous tasks.
(________)	treat all blood and body fluids as potentially infectious and capable of transmitting HIV or other blood bornediseases.
(________)	a US federal agency charged with the enforcement of safety and health legislation

	these are hoods that are a type of containment barrier that protects the medical laboratory scientists from aerosol transmissions of microorganisms; handling of microbiological specimens should be performed in a BSC

(Types of organisms) (Number of organisms/microbial load/bioburden) (Concentration of Disinfecting Agent) (Presence of Organic Material) (Nature of Surface to be Disinfected) (Contact Time) (Temperature) (Biofilms) (Compatibility of Disinfectants)

Factors that Influence the Degree of Killing Organisms:

	spore-forming bacteria are highly resistant to disinfectants because of the thick protein coat of spores
	higher numbers of organisms require longer exposure to disinfectants
	Higher concentration, better disinfectan

	blood, mucus, and pus inactivates disinfecting agents and it prevents full contact between object to be killed and disinfectant. It should be removed first before disinfecting.
	certain materials require certain methods of disinfection
	the length of time needed for a disinfectant to be in contact with the microorganism to be destroyed Ex. Contact time for iodine and alcohol: 1-2 mins

	directly proportional to the degree of disinfection
	a community of bacteria that forms a protective material over them that protects them from environmental factors Ex. In catheters, in water pipes
	when more than one disinfectant is used, their compatibility should be considered Ex. Bleach and quats negate each other

Heat

Physical Method of Disinfection

(______) the most reliable method of sterilization of articles that can

withstand heat. (1) (_____) acts by oxidative effects as well as denaturation

and coagulation of proteins.

(Hot air oven) (60 minutes at 160C, 40 minutes at 170C, 20 minutes at 180C)

DRY HEAT

(____________) Articles to be sterilized are exposed to high temperature in an electrically heated oven. Dry heat acts by protein denaturation, oxidative damage and toxic effects of elevated levels of electrolytes. Different temperature-time relations for holding time are:

(Moist heat) (Boiling water)

(_______) acts by coagulation and denaturation of proteins.

o Superior to dry heat

A. (________) (100C) kills most vegetative bacteria and viruses immediately. Ø Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Ø Some bacterial spores are resistant to boiling and survive; hence this is not a substitute for sterilization.

Ø Certain bacterial toxins such as Staphylococcal

enterotoxin are also heat resistant.

(Steam at 100C:)

Instead of keeping the articles in boiling water, they are subjected to free steam at 100oC for 90 mins. Ø autoclave can also serve the same purpose

(121 C at 1/15 psi) (132C) (penetrative power) (dry air and it moistens) (Drenching and wetting)

Temp of the autoclave:

o (_____________) for medical apparatus

o (____) for medical waste

Ø Advantages of steam: It has more (_____________)

than (______&_____) the spores (moisture is

essential for coagulation of proteins)

Ø Disadvantages: (_______&______) of articles may

occur; trapped air may reduce the efficacy and it takes long time to cool

RADIATION

"cold sterilization".

(NON-IONIZING RADIATION) (IONIZING RADIATION) (

2 TYPES OF RADIATION:

1. (__________________) are low energy rays with poor penetrative power

• Mode of action: inhibits DNA replication

• UV radiation (doesn’t kill spores)

2. (_________) are high-energy rays with good penetrative power.

• Mode of action: damages the nucleic acids of microorganisms

• Electron beams and gamma rays (Sporicidal)

(FILTRATION) (Membrane filters) (HEPA filters)

D. (________)- does not kill microbes, it separates them out.

Ø (__________) with pore sizes between 0.2-0.45 µm are

commonly used to remove particles from solutions that can't

be autoclaved.

Ø (_______) are at least 99.97% efficient for removing

particles >0.3 um in diameter.

(20,000)

E. SONIC AND ULTRASONIC VIBRATIONS

Ø Sound waves of frequency >(_____) cycle/second kills

bacteria by exposing them for one hour.

Ø This method is not reliable since many viruses are not

affected by these waves.

(ALCOHOLS:) (ALDEHYDES:) (PHENOL) (HALOGENS:) (HEAVY METALS:) (HYDROGEN PEROXIDE:) (ETHYLENE OXIDE (EO):

CHEMICAL DISINFECTANTS:

(___________)

Ø Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.

Ø Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol

(___________)

Ø Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and damages nucleic acids. It kills all microorganisms, including spores. Ø Examples: Formaldehyde, Glutaraldehyde

(_________)

Mode of action: Act by disruption of membranes, precipitation

of proteins and inactivation of enzymes.

Ø Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified

cresol), hexachlorophene, chlorhexidine, chloroxylenol

(Dettol)

(_________)

Ø Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfhydryl groups of enzymes.

Ø Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores)

(___________)

Ø Mode of action: precipitation of proteins and oxidation of sulfhydryl groups. They are bacteriostatic. Ø Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts

(____________)

Ø Mode of action: It acts on the microorganisms through its

release of oxygen. Hydrogen peroxide produces hydroxyl-free

radical that damages proteins and DNA.

(____________)

Ø Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups. Ø A chemisterilant

(PHYSIO-CHEMICAL METHOD OF STERILIZATION:)

Mode of action: A physio-chemical method adopts both physical and chemical method. Ø Use of steam formaldehyde

(Alcohols) (Aldehydes)

Chemical Methods

1. (________) – can’t kill spores and viruses; disinfects by denaturing proteins *for blood culture, cotton balls should be sterile and alcohol should be filtered through a 0.22 micr0meter filter

2. (_______)– inactivates DNA and RNA through alkylation of sulfhydryl groups ex. Glutaraldehyde and formaldehyde have irritating fumes

-2% glutaraldehyde is is germicidal in 10 mins and sporicidal in 3-10 hrs.

3. Halogens – commonly employed as surface disinfectants Ex. Chlorine(bleach) as 0.5-1% sodium hypochlorite, and iodine, and iodophor(iodine +neutral polymer) which increases the solubility

Ø for tabletops with blood spills, use 1:10 dilution of 5.25% sodium hypochlorite

4. Heavy Metals - ex. Eye drop solution with 1% silver nitrate (to treat the eyes of newborns to kill N . gonorrheae that may have been acquired through passage of the birth canal

5. Quaternary Ammonium Compounds – are detergents. They reduce surface tension of molecules in a liquid Pseudomonas aeruginosa is resistant to quats.

(Chemical Safety) (Fire Safety) (Electrical Safety

GENERAL LABORATORY SAFETY

1. (_________) – lab should have a chemical hygiene plan that includes guidelines on proper labeling of chemical containers, MSDSs, and chemical safety training.

Ø -use fume hoods (exhausts chemical vapors to the outside) when working with toxic chemicals

2. (_________) – labs should post a fire evacuation plan and conduct fire drills

3. (_________)- no extension cords should be used in the lab, wirings should be checked regularly.

(communication program) (common) (assess) (information) (Material Safety Data Sheets (MSDS) (1) (0.1) (request) (defaced or removed) (information and training) (exposure limit) (responsible)

Chemical Hazard Communications Plan

Ø Develop written hazard (_______).

Ø Maintain inventory of all chemicals with chemical and (_______) names, if appropriate.

Ø Manufacturer must (_____) and supply (_______) about chemical or physical hazards (flammability, explosive, aerosol, flashpoint, etc.).

Ø Employers must maintain (____________) in English.

Ø MSDS must list all ingredients of a substance greater than (__)%, except for known carcinogens if greater than (____)%.

Ø Employers must make MSDS available to employees upon (____).

Ø Employers must ensure that labels are not (_________) and must post appropriate warnings.

Ø Employers must provide (_______&_______) (“right-to know”).

Ø Employers must adhere to Occupational Safety and Health Administration permissible (_________), threshold limit, or other exposure limit value.

Ø Designate (__________) person(s) for the program.

METHODS OF ACQUIRING INFECTION IN THE LABORATORY:

1) Rubbing the eyes or nose with contaminated hands.

2) Inhaling aerosols.

3) Accidental ingestion of bacteria by putting pens or fingers in the mouth.

4) Needlesticks – accidental needle puncture

(Wash hands) (gloves) (full-body gowns) (face masks and eye protection) (sharp objects) (Environmental control)

UNIVERSAL/STANDARD PRECAUTIONS:

W -(_______) before and after each medical procedure.

W -Wear (______) whenever there is a possibility of coming in contact with blood or other potentially infectious materials (body fluids and tissues)

W -Wear (____________) whenever there is a possibility of blood splashing

W -Wear (_______&_________) whenever there is a possibility of blood splashing into the face

-Dispose of all contaminated (__________) in an appropriate

• (___________) should be adequate and provide procedures for routine care and cleaning of surfaces.

(autoclaved onsite) (2 leak-proof plastic) (rigid carbon)

DISPOSAL OF INFECTIOUS WASTE

• Infectious waste from microbiology laboratories is (_________) or sent for incineration before disposing.

• Infectious waste should be placed into (________) bags for sturdiness.

• Pipettes, swabs, other glass objects should be placed into (________) containers before disposal.

• Sharp objects, scalpels and needles

o are placed in a Sharps container

o which is autoclaved or incinerated

o when full.

EPA (Environmental Protection Agency) 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 antibiotic susceptibility testing and identification of organisms to reduce volume of chemical reagents as well as infectious waste.

(limited/restricted) (wash hands) (eating, drinking, smoking, handling contact lenses, applying cosmetics, or storing human food) (safety glasses) (cabinets/refrigerators) (Mechanical pipetting) (policies/practices) (splashes/aerosols) (

STANDARD MICROBIOLOGICAL PRACTICES

1. Lab access (________) when experiments or work with cultures/specimens are in progress.

2. Lab personnel should (__________) after handling viable materials, removing gloves, or leaving lab.

3. No (____________________________________________________________) in lab.

4. Contact lens users wear (__________), goggles or face shields.

5. Food stored outside lab in designated (____________).

6. (____________) devices are used (i.e., no mouth pipetting).

7. Sharps handling (__________) in place.

8. Procedures minimize (_____________).

(daily) (work) (spill/splash) (labeled) (Cultures/stocks/regulated wastes) (durable, leak-proof, closed containers) (entrance) (Insect/rodent control)

STANDARD MICROBIOLOGICAL PRACTICES

1. Work surfaces are decontaminated at least (_____) and/or at completion of (_____).

2. Work surfaces are decontaminated after any (__________) of viable material.

3. Disinfectants are (______) for agents being used.

4. (_____________________) are decontaminated by approved method (e.g., autoclaving) before disposal.

5. Materials decontaminated outside of lab are transported in (_____________).

6. Biohazard signage posted at lab (__________) when infectious agents are present (signage lists agents and Person in charge name/phone).

7. (_____________) program in effect.

(BSL1) (AGENT)

Not known to consistently cause diseases in immunocompetent adult humans

(BSL1) (PRACTICES)

Standard microbiological practices (refer to next page)

(BSL1) (SAFETY EQUIPMENTS)

None required

(BSL1) (FACILITIES)

Open bench top, sink required

(BSL 2) AGENTS

Associated with human disease. Hazard: percutaneous injury, mucous membrane exposure, ingestion

(BSL 2) (PRACTICES)

BSL 1 practices plus: . limited access ‣ biohazard warning signs . sharps precautions biosafety manual defining waste decontamination or medical surveillance

(BSL 2) (SAFETY EQUIPMENTS)

Primary barriers: Class Ior II biosafety cabinets or other physical containment devices used for manipulation of agents that cause splashes or aerosols of infectious materials; PPE: laboratory coats, gloves, face protection is needed

(BSL 2) (FACILITIES)

BSL 1plus: . non-fabric chairs and other furniture that's easy to clean • autoclave • eyewash readily available

(BSL 3) AGENTS

Indigenous or toxic agents with potential for aerosol transmission; disease may have serious or lethal consequences

(BSL 3) (PRACTICES)

BSL 2 practices plus: . controlled access . decontamination of all wastes decontamination of lab clothing before laundry

(BSL 3) (SAFETY EQUIPMENTS))

Same as BSL 2

(BSL 3) (FACILITIES)

BSL 2 plus:

• physical separation from access coridors • hands-free handwashing sink • self-closing double door access • exhaust fan • negative air flow into laboratory • eyewash readily available in lab

(BSL 4) (AGENTS)

Indigenous or toxic agents with potential for aerosol transmission; disease may have serious or lethal consequences

(BSL 4) (PRACTICES)

BSL 3 practices plus:

• clothing change before entering

•shower on exit

(BSL 4) (SAFETY EQUIPMENTS)

Primary barriers: All procedures conducted in Class I1 biosafety cabinets or Class I or Il biosafety

(BSL 4) (FACILITIES)

BSL 3 plus: separate building or isolated zone • dedicated supply/exhaust,

(BSL1-3) (Yes)

Personal

(Product) (No)

Product

(BSL1-3) (Yes)

Environment

BSL1-3

I

BSC Class

(BSL1-3) (Yes)

Personal

(BSL 1-3) (Yes)

Product

(BSL 1-3) (Yes)

Environment

I1(A1,A2,B1, B2)

BSC Class

(BSL4) (Yes)

Personal

(BSL4) (Yes)

Product

(BSL4) (Yes)

Environment

(BSL4) (Ill-when used in suitroom with suit)

BSC class