GENERAL SAFETY AND HAZARDOUS MATERIAL INFORMATION

These methods should be used by laboratory personnel with experience in laboratory and chemical safety or students under the supervision of such trained personnel. The procedures, chemicals, and equipment referenced in these methods are hazardous and can cause serious injury unless performed, handled, and used with care and in a manner consistent with safe laboratory practices. Students and researchers using the procedures in Cold Spring Harbor Protocols do so at their own risk. It is essential for your safety that you consult the appropriate Material Safety Data Sheets, the manufacturers’ manuals accompanying equipment, and your institution’s Environmental Health and Safety Office, as well as the General Safety and Disposal Cautions section below for proper handling of hazardous materials in these methods. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these articles and has no liability in connection with the use of these materials.

All registered trademarks, trade names, and brand names mentioned in Cold Spring Harbor Protocols are the property of the respective owners. Readers should please consult individual manufacturers and other resources for current and specific product information.

Users should always consult individual manufacturers, the manufacturers’ safety guidelines and other resources, including local safety offices, for current and specific product information and for guidance regarding the use and disposal of hazardous materials.

The primary safety information resources for laboratory personnel are the following:

Institutional Safety Office: The best source of toxicity, hazard, storage, and disposal information is your institutional safety office, which maintains and makes available the most current information. Always consult this office for proper use and disposal procedures.

Post the phone numbers for your local safety office, security office, poison control center, and laboratory emergency personnel in an obvious place in your laboratory.

Material Safety Data Sheets (MSDSs): The Occupational Safety and Health Administration (OSHA) requires that MSDSs accompany all hazardous products that are shipped. These data sheets contain detailed safety information. MSDSs should be filed in the laboratory in a central location as a reference guide.

General Safety and Disposal Cautions

The guidance offered here is intended to be generally applicable. However, proper waste disposal procedures vary among institutions; therefore, always consult your local safety office for specific instructions. All chemically constituted waste must be disposed of in a suitable container clearly labeled with the type of material it contains and the date the waste was initiated.

It is essential for laboratory workers to be familiar with the potential hazards of materials used in laboratory experiments and to follow recommended procedures for their use, handling, storage, and disposal.

The following general cautions should always be observed:

Before beginning the procedure, become completely familiar with the properties of substances to be used.
The absence of a warning does not necessarily mean that the material is safe, because information may not always be complete or available.
If exposed to toxic substances, contact your local safety office immediately for instructions.
Use proper disposal procedures for all chemical, biological, and radioactive waste.
For specific guidelines on appropriate gloves to use, consult your local safety office.
Handle concentrated acids and bases with great care. Wear goggles and appropriate gloves. A face shield should be worn when handling large quantities.

Do not mix strong acids with organic solvents because they may react.

Sulfuric acid and nitric acid especially may react highly exothermically and cause fires and explosions.

Do not mix strong bases with halogenated solvents because they may form reactive carbenes that can lead to explosions.

Handle and store pressurized gas containers with caution because they may contain flammable, toxic, or corrosive gases; asphyxiants; or oxidizers. For proper procedures, consult the Material Safety Data Sheet that is required to be provided by your vendor.
Never pipette solutions using mouth suction. This method is not sterile and can be dangerous. Always use a pipette aid or bulb.
Keep halogenated and nonhalogenated solvents separately (e.g., mixing chloroform and acetone can cause unexpected reactions in the presence of bases). Halogenated solvents are organic solvents such as chloroform, dichloromethane, trichlorotrifluoroethane, and dichloroethane. Nonhalogenated solvents include pentane, heptane, ethanol, methanol, benzene, toluene, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), and acetonitrile.
Laser radiation, visible or invisible, can cause severe damage to the eyes and skin. Take proper precautions to prevent exposure to direct and reflected beams. Always follow the manufacturer’s safety guidelines and consult your local safety office.
Flash lamps, because of their light intensity, can be harmful to the eyes. They also may explode on occasion. Wear appropriate eye protection and follow the manufacturer’s guidelines.
Photographic fixatives, developers, and photoresists also contain chemicals that can be harmful. Handle them with care and follow the manufacturer’s directions.
Power supplies and electrophoresis equipment pose serious fire hazard and electrical shock hazards if not used properly.
Microwave ovens and autoclaves in the laboratory require certain precautions. Accidents have occurred involving their use (e.g., when melting agar or Bacto Agar stored in bottles or when sterilizing). If the screw top is not completely removed and there is inadequate space for the steam to vent, the bottles can explode and cause severe injury when the containers are removed from the microwave or autoclave. Always completely remove bottle caps before microwaving or autoclaving. An alternative method for routine agarose gels that do not require sterile agar is to weigh out the agar and place the solution in a flask.
Ultrasonicators use high-frequency sound waves (16–100 kHz) for cell disruption and other purposes. This “ultrasound,” conducted through air, does not pose a direct hazard to humans, but the associated high volumes of audible sound can cause a variety of effects, including headache, nausea, and tinnitus. Direct contact of the body with high-intensity ultrasound (not medical imaging equipment) should be avoided. Use appropriate ear protection and display signs on the door(s) of laboratories where the units are used.
Use extreme caution when handling cutting devices, such as microtome blades, scalpels, razor blades, or needles. Microtome blades are extremely sharp! Use care when sectioning. If unfamiliar with their use, have an experienced user demonstrate proper procedures. For proper disposal, use the “sharps” disposal container in your laboratory. Discard used needles unshielded, with the syringe still attached. This prevents injuries and possible infections when manipulating used needles because many accidents occur while trying to replace the needle shield. Injuries may also be caused by broken pasteur pipettes, coverslips, or slides.
Procedures for the humane treatment of animals must be observed at all times. Consult your local animal facility for guidelines. Animals, such as rats, are known to induce allergies that can increase in intensity with repeated exposure. Always wear a lab coat and gloves when handling these animals. If allergies to dander or saliva are known, wear a mask.

Disposal of Laboratory Waste

There are specific regulatory requirements for the disposal of all medical waste and biological samples mandated by the U.S. Environmental Protection Agency and regulated by the individual states and territories. Medical and biological samples that require special handling and disposal are generally termed Medical Pathological Waste (MPW), and medical, veterinary, and biological facilities will have programs for the collection of MPW and its disposal. Restrictions on how radioactive waste can be disposed of as regulated by the U.S. Nuclear Regulatory Commission can be found in 10 CFR 20.2001, general requirements for waste disposal or the individual Agreement States. The preferred method for the disposal of radioactively contaminated MPW is decay-in-storage.

Waste and any materials contaminated with biohazardous materials must be decontaminated and disposed of as regulated medical waste. No harmful substances should be released into the environment in an uncontrolled manner. This includes all tissue samples, needles, syringes, scalpels, etc. Be sure to contact your institution’s safety office concerning the proper practices associated with the handling and disposal of biohazardous waste.

Some basic rules are outlined below. For treatment of radioactive and biological waste, see sections on Radioactive Safety Procedures and Biological Safety Procedures.

In practice, only neutral aqueous solutions without heavy metal ions and without organic solvents can be poured down the drain (e.g., most buffers). Acid and basic aqueous solutions need to be neutralized cautiously before their disposal by this method.
For proper disposal of strong acids and bases, dilute them by placing the acid or base onto ice and neutralize them. Do not pour water into them. If the solution does not contain any other toxic compound, the salts can be flushed down the drain.
For disposal of other liquid waste, similar chemicals can be collected and disposed of together, whereas chemically different wastes should be collected separately. This avoids chemical reactions between components of the mixture (see above). Collect at least inorganic aqueous waste, nonhalogenated solvents, and halogenated solvents separately.
Waste from photo processing and automatic developers should be collected separately to recycle the silver traces found in it.

Radioactive Safety Procedures

In the United States and other countries, the access to radioactive substances is strictly controlled. You may be required to become a registered user (e.g., by attending a mandatory seminar and receiving a personal dosimeter). A convenient calculator is available to perform routine radioactivity calculations.

If you have never worked with radioactivity before, follow the steps below:

Try to avoid it! Many experiments that are traditionally performed with the help of radioactivity can now be done using alternatives based on fluorescence or chemiluminescence and colorimetric assays, including, for example, DNA sequencing, Southern and northern blots, and protein kinase assays. However, in other cases (e.g., metabolic labeling of cells), use of radioactivity cannot be avoided.
Be informed. While planning an experiment that involves the use of radioactivity, include the physicochemical properties of the isotope (half-life, emission type, and energy), the chemical form of the radioactivity, its radioactive concentration (specific activity), total amount, and its chemical concentration. Order and use only as much as is really needed.
Familiarize yourself with the designated working area. Perform a mental and practical dry run (replacing radioactivity with a colored solution) to make sure that all equipment needed is available and to get used to working behind a shield. Handle your samples as if sterility would be required to avoid contamination.
Always wear appropriate gloves, lab coat, and safety goggles when handling radioactive material.
Check the work area for contamination before, during, and after your experiment (including your lab coat, hands, and shoes).
Localize your radioactivity. Avoid formation of aerosols or contamination of large volumes of buffers.
Liquid scintillation cocktails are often used to quantitate radioactivity. They contain organic solvents and small amounts of organic compounds. Try to avoid contact with the skin. After use, they should be regarded as radioactive waste; the filled vials are usually collected in designated containers, separate from other (aqueous) liquid radioactive waste.
Dispose of radioactive waste only into designated, shielded containers (separated by isotope, physical form [dry/liquid], and chemical form [aqueous/organic solvent phase]). Always consult your safety office for further guidance in the appropriate disposal of radioactive materials.
Among the experiments requiring special precautions are those that use [³⁵S]methionine and ¹²⁵I, because of the dangers of airborne radioactivity. [³⁵S]methionine decomposes during storage into sulfoxide gases, which are released when the vial is opened. The isotope ¹²⁵I accumulates in the thyroid and is a potential health hazard. ¹²⁵I is used for the preparation of Bolton–Hunter reagent to radioiodinate proteins. Consult your local safety office for further guidance in the appropriate use and disposal of these radioactive materials before initiating any experiments. Wear appropriate gloves when handling potentially volatile radioactive substances, and work only in a radioiodine fume hood.

Biological Safety Procedures

Biological safety fulfills three purposes: to avoid contamination of your biological sample with other species; to avoid exposure of the researcher to the sample; and to avoid release of living material into the environment. Biological safety begins with the receipt of the living sample; continues with its storage, handling, and propagation; and ends only with the proper disposal of all contaminated materials. A catalog of operations known as “sterile handling” is usually employed in manipulating living matter. However, the actual manner of treatment largely depends on the actual sample, which can be quite diverse: Escherichia coli and other bacterial strains, yeasts, tissues of animal or plant origin, cultures of mammalian cells, or even derivatives from human blood are routinely handled in a biological laboratory. Two of these, bacteria and human blood products, are discussed in more detail below.

The Department of Health, Education, and Welfare (HEW) has classified various bacteria into different categories with regard to shipping requirements (see Sanderson and Zeigler, Methods Enzymol204: 248–264 [1991]). Nonpathogenic strains of E. coli (such as K12) and Bacillus subtilis are in Class 1 and are considered to present no or minimal hazard under normal shipping conditions. However, Salmonella, Haemophilus, and certain strains of Streptomyces and Pseudomonas are in Class 2. Class 2 bacteria are “Agents of ordinary potential hazard: agents which produce disease of varying degrees of severity...but which are contained by ordinary laboratory techniques.” Contact your institution’s safety office concerning shipping biological material.

Human blood, blood products, and tissues may contain occult infectious materials such as hepatitis B virus and human immunodeficiency virus (HIV) that may result in laboratory-acquired infections. Investigators working with lymphoblast cell lines transformed by Epstein–Barr virus (EBV) are also at risk of EBV infection. Any human blood, blood products, or tissues should be considered a biohazard and should be handled accordingly until proved otherwise. Wear appropriate disposable gloves, use mechanical pipetting devices, work in a biological safety cabinet, protect against the possibility of aerosol generation, and disinfect all waste materials before disposal. Autoclave contaminated plasticware before disposal; autoclave contaminated liquids or treat with bleach (10% [v/v] final concentration) for at least 30 minutes before disposal (this is valid also for used bacterial media).

Always consult your local institutional safety officer for specific handling and disposal procedures of your samples. Further information can be found in the Frequently Asked Questions of the ATCC homepage and is also available from the National Institute of Environmental Health and Human Services, Biological Safety.

General Properties of Common Hazardous Chemicals

The hazardous materials list can be summarized in the following categories:

Inorganic acids, such as hydrochloric, sulfuric, nitric, or phosphoric, are colorless liquids with stinging vapors. Avoid spills on skin or clothing. Spills should be diluted with large amounts of water. The concentrated forms of these acids can destroy paper, textiles, and skin and cause serious injury to the eyes.
Inorganic bases, such as sodium hydroxide, are white solids that dissolve in water and under heat development. Concentrated solutions will slowly dissolve skin and even fingernails.
Salts of heavy metals are usually colored, powdered solids that dissolve in water. Many of them are potent enzyme inhibitors and therefore toxic to humans and the environment (e.g., fish and algae).
Most organic solvents are flammable volatile liquids. Avoid breathing the vapors, which can cause nausea or dizziness. Also avoid skin contact.
Other organic compounds including organosulfur compounds, such as mercaptoethanol or organic amines, can have very unpleasant odors. Others are highly reactive and should be handled with appropriate care.
If improperly handled, dyes and their solutions can stain not only your sample but also your skin and clothing. Some are also mutagenic (e.g., ethidium bromide), carcinogenic, and toxic.
Nearly all names ending with “ase” (e.g., catalase, β-glucuronidase, or zymolyase) refer to enzymes. There are also other enzymes with nonsystematic names such as pepsin. Many of them are provided by manufacturers in preparations containing buffering substances, etc. Be aware of the individual properties of materials contained in these substances.
Toxic compounds are often used to manipulate cells. They can be dangerous and should be handled appropriately.
Be aware that several of the compounds listed have not been thoroughly studied with respect to their toxicological properties. Handle each chemical with appropriate respect. Although the toxic effects of a compound can be quantified (e.g., LD₅₀ values), this is not possible for carcinogens or mutagens where one single exposure can have an effect. Also realize that dangers related to a given compound may also depend on its physical state (fine powder vs. large crystals/diethyl ether vs. glycerol/dry ice vs. carbon dioxide under pressure in a gas bomb). Anticipate under which circumstances during an experiment exposure is most likely to occur and how best to protect yourself and your environment.

Cold Spring Harbor Laboratory Press (CSHLP) has used its best efforts in collecting and preparing the material contained herein but does not assume, and hereby disclaims, any liability for any loss or damage caused by errors and omissions in the publication, whether such errors and omissions result from negligence, accident, or any other cause. CSHLP does not assume responsibility for the user’s failure to consult more complete information regarding the hazardous substances listed in this publication.

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